Tobacco Documents Online

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..._ .. ~_._ ~---x.~:._....... TO H ACCO Experimental and Clinical Studies A Comprehensive Account of the World Literature SUPIPIlement I P. S. LARSON, Ph.D. Professor of Phardnacolof;y H. SILVETTE, Ph.D. Visiting Professor of Pharmacology Medical College of Virginia, Richmond, Yirginia / BAtimore, The Williams & Wilkins Company 6 8 ~901 19 ~~ Gotsnc~l foV produ~e~ ~~'~ '~ Tobacco \esca;cli-US~, Inc.

Copyrigbc ©, lsss Tes Wn.tawats & Wvstnrs CompAn Made in Me United States of Ameriaa ISbtnry of Congress Catelog Card Number BI-5057 p~otluced bv Th~ Cauncillnc1 ubacco ReScarch USA, Composed and printed at tbe WAVERLY PItESB, INC. salt9more, Maryland 21202, U. s. a 0002905

1 -----r.- E..~........._.~~_. DEDICATED TO THE MEMO@Y OF Harvey BCT7EhNETdt Haag (190D-1961) produced b Tt~~ Cot' hcil fior Tobacco ReSearcil-USr1, Ince 0002906

V:. PREFACE The information gathered between the covers of this first Supplement to Tobaaco (1961) has been compiled from more than 4000 articles published through the year 1964 for the entire SWpplement, but, in the clinicull chapters (mainly, 15, 10-21), with considriable ed- ditions from the yearr 1965 and 1966, tailing off in number into 1967. A considerable number of pre-1959 articles, which had either escaped our notice or our [1959J deadline for the preparatimn of Tobacco for the press, have now been included in this 1959-196;i/1966 Supplement; and a similgr situation prex~ails with re- spect to the Supplement, since we have in hand a store of some 1000 abstracts of 196~1967 articles already_ prepared for once-anticipated incltlsion in Supplement I. However, certain exigencies and inelnsticities of Time have made it inexpedient, indeed attdeasible, to hold up publication to permit the necessary slow labor of com- pletely updating all of this Supplement to the last possible minute or even year, and for this deficiency we can only express our regrets to the charitable reader. In this connection, we must also poiAit out that the nota- tions, "No new data" or "No new reports", in nnmer- ous placeas throughout Supplement I often means simply that relevant new data or reports did not make it, so to speak, under the wire, although existing in the pub- lished Gt.era.tune and in our files. In partial recompense to the reader, we have supplied in this Supplement what were, unfortunately and for much the same reasons, lacking ijt Tobacco: a compre- hensive index and a complete system of cross-reference within the text.. Indeed, our present index compribes entries from Tobacco Ithe pagr-numbers of which are enclosed within angle-brackets, thus: pj aR.rell as from this Supplement; and pagmoted cross-references to Tobacco in the present text are ctlro invariably angle- . bracketed while cross•references to Supplement I ma- terial are given in bold-face section• (not pagc-) num- bers. We trust that these contrivances will make both Tobacco and S'uppfement I more useful as reference ~. ork;. By far the greater part of the abstractsu•ere prepared by P. S. L.: a1td, as before, the text a•as written by H. S., except that special thattks are due to Dr. Herbert \1cKennis, Jr., Professor of Pharmaeologr in the Aledical College of Virginia, for organization and writing of all but a small portion of the material on nicotine metabalites presented in Chapter 1, Section 52. We gratefully acknowledge grant•in•aid support from the Council for Tobacco Research-U.S.A. (successor to the Tobacco Industry Research Comruittee) during the entire period of preparation of this Supplement. We deem it only proper again tu inform the reader that all responsibility for the data presented and viea-s ex- presced herein, either explicitly or implicitly, rests entirely upon the authors, the Council for Tobeuco Research-U.S.A. support having been in accordance with its established policy of leaving complete freedom of planning and action to grnnt•in-nid recipients. We also owe a special debt of gratitude to our manu- script secretary, llrs. Nancy M. Berreth who gave of her time, skill, and devotion far beyond the call of duty; and, finally, we are happy to express our deep apprecia- tion to the following ladies, who, over the years, have aseisted so generously wid impnrtantly in one or another phase of the development and preparntiorf of this Supplement: llnt. Colcene B. Alents; Aliss Andrea L. \`artnnian; Mrs. Evelyn li. Shearin; \1r3. Terr.- S. Casebeer; Mrs. Ge•endolyn G. Stables. P. S. LAtreoN Produced by Th2. Council for . Tobacco Research-USA, Inc. vi 0002907

INTRODUCTORY COMMENTS "18 THE LITERATURE WORTH $EEP[NCP" J. Maddox, i$ull. Atomic Scientiats 19:19, 1963 Vpn Proosdij (1960, p. 225) has most perspicaciously re-observed that "it is tobucco•smoke which has con- quered the world, not just ,eny kind of smoke, and hot air +;till less." bluch of the present book is about Tobacco, and opinions will differ as to how much of it is Hot Air. But, as in Tobrtcco (1961b, p. vii), we here printt the Hot Air in the same type-size as the stitff of richer body, so that it may be said of this Supplement as it was once written of a certain historical n•ork, that seldom has a "democratic equality of sources has been so rigorously enforced" (Spectator, 29 January 1965, p. 142). The ultimate of such "democracy" is, of course, a bibliography or catalogue raisonnd; and what s'epa- rat.u our monographs on Tobacco from mere omniitms- gatha'rum is not alone organization of material, but rather our use of that interstitisl and connective tissue called "commentary". For it is commentary, perhaps as much as organization, which transmutes an annotated and indexed bibliography into that organism called a book. Books are usually (almost invariably, in fr..;t) made by deliberate seleetion of material; but w•itt our politic pslicy of deliberate non-sdection of materialD we can make our books only by juxtaposition of material to form a pattern, and then highlighting the pattern by commentary. Commentary in puttinR together such a monograph as this is inevitable: if it is not explicit, it is ah+•aym, im- plicit. Consider, for example, the semantically "loaded" verbs, such as "alleged" (for the noncommittal "re- ported") or "claimed" (for the neutral "stated"). There is considerable difference in connotation between "Dr. A stated thus-and-so, while Dr. B alleged this•an¢-that" and ''B duted this-and-that, n•1We A olteged thus-and- so." The difference lies in the implicitt commitment of the writer. To take another instance, the choice of which published observation or comment to print bejore another one on the snme subject represents implicit commentary. If Dr. C says that smoking has a certain action or consequence, and Prof. D declares that it dow not, the psychological or even intellectual impact on the reader wyll quite probably be ditferent were C's words arranged to precede, or to follow, those of D.1ye may term this "commentary-by-simple-juxtaposition"; but obviously a;\tonograph containing thousands upon thousands of items by hundreds of ditferent. writers cannot be printed to any considerable extent with A vii foUon-iR.g B following C and so on, a-it h only the separa- tion of a full-stop or a paragraph indentation between the work or opinions described. For something has to come between, if only to make the text reasonably readable and logical; and +rith this we come to explicit commentary. It makes a difference, aud often an enormous one, whether we separate an 'sccount of A's work from B's with and or but, also or on tAe other hand. In context, all such conjunctive words cannot fail to have a critical or judgmatical, and even an emotive (as in the case of "alleged" vs "reported") quality or connotation; they represent. single-word commentaries which pass a valuP judgment as explicit tis any sentence- or paragraph•long comment. It is nonsense, therefore, to expect a monograph to be sine commentarfo; the reader must be guided through the maze of material if he is not to be hopelessly lost in it. The commentator (as we may then fairly term the fellow who puts together a monograph) has, of course, the inescapable duty to guide his reader past everything seeable-not to travel the broad clean boulevards and neglect the dirty back-alleys, nor yet to steer the reade: from alley to alley, only crossing the boulevards in passing. It is clear, however, that this guided tour of all the walkways must be made in some order of progress, of whatever sort-alley to boulevard or boulevard to alley, back and forth, until the City of Tobacco is entirely covered. The reader is, so to speak, durchkom- pioniert; his is a conducted point-to-point tour, not an over-all aeroplane view. The actuol path taken repre- sents another form of implicit commentary; and it is doubtful whether any two monogruphists, using the very same body of Tobacco Literature, would or could follow the same path; and the end-result would be two quite different books, even though neither monogrnpher had ever uttered a single +rca•d of his oani. More often, however, the simplest plan, the plain cotqunction-as- comment, is insufficient. In the metnphor we have been developing, it is as i!', here and there in the City of Tobacco, there are gaps between u-alk+vays filled only with muddy fields or morasses. Here it is the clear duty of the monographer (n•ho may be presumed to know the hidden pitfalls of his city better than the tripper to it) io hoist his reader on to his own back and carry him over the mud; or, more elegantly, he may emulate Sir 11'alter Raleigh and cast his own cloak over the muddy Produced by The Council for o 0 0 2 9 0 8 Tobacco Research-USA, Inc.

viii Ii\TRODLtCTORY COMMENTS f patch so that Queen Elizabeth (for the reader is always pattern. Another sort of pattern-untouched by Time Fnvorrlvn) mav 'rmm drvRhMl. These latter actions of because it is nart of a particular time-is apparent in the monographist-guide may be likened to sentences the bony skeleton of organization and the connective and paragraphs of commentaty based on his knowledge tissue of commentary. When these patterns emerge, we of the terrain. It is no longer possible to imitate the trust they will justify transference of our monographs medieval mapmakers, and simply erect a curlicued sign from the shelf of "Current Reference Works" to the reading: "Here Be 1lJud" instead of "Monsters". Vot cupboard of Medical Histor3•. Much that the scientific all readers of this Supplement may know that Tobacco man is forcea to read, J. NN. Langley told the Physiologi- is widely, and not unaffectionately, known as the cal Section of the British Associntion in his pre.sidential "Green Alonster".] address of 1899, "consists of recordy of defective experi- It is pertinent to recall that two or three respected ments, confused statements of results, weari.come de- reviewers of the Green Monster had objected to the scription of detail, and unnecessarily protracted discus- commentary (others, it is only fair to add, expressed sion of unnecessary hypotheses." If this was the ense in delight in it; but. we did not take a poll), one even 1899, how much more true is it of the va.=tly multiplied going so far as to assert that the commentary in the scientific literature of the 1960s. All that now remains sections on Lung Cancer "re9ects that of the tobacco of "such matter", as Langley termed it, is this great Industry,' while another implied as much. This was pharmacologist'scommentuponit' not the case, as we had carefully pointed out in the The inevitable pattern of our Monographs on To- Preface to Tobacco (page vi). If certain of our.tiews or bacco is imposed b3• (1) the several thmus.nnd individual comment prove to be agreeable to the Tobacco Indus- building-stones [abstracts of alrendv-pahli-lhed art.icles], try, this is scarcely surprising, for the subject is a vast and (2) the necessity of using ererJ single atone. The one; very probably, anyone in the spectrum from architect working to these requirements must needs Tobacco-Foes to Tobacco.Friends to Tobacco-Fiends possess a certain amount of ingenuity, butt the builder could find some comment to their liking-or disliking- carr3-ing out the plan requires something more: cement, in these monographs. which is the name in this particular metaphor (from Tobacco was pre-emittently a preclinical affair dealing 1,rity°' to ••building" is, after all, a natural association) largely with pharmacological and toxicological aspects for "commentan•". Obviously, some of the eiore friable of nicotine and smoking; the clinical material occupied stones require a substantial reinforcement with cement onlyabout 25% of the monograph, and, not surprisingly, to keep parts of the structure from collapsing. Just as most of the commentary was concentrated in the ehap- obviously, several thousand random•sized and irregu- ters dealing with Tobacco and Disease. This Stipple. larly-shaped rocks cannot be fitted together with the ment has turned out to be quite different in nature; it deals pre.eminently (about 60 per cent) n•ith clinical close precision of a brick outhouse; and we are not matters (in which is to be included the Tobacco Habit allowed to do even in metaphor ivhut the stonemason in its human aspects), and fully half of the text is does in practice: chip away at a rock until it does fit or devoted to Tobacco and Disease, by far the greater part shows a usable face. A number of Fizable interstices to the single subject of Lung Cancer. This change of between stones !.ud thus to be filled with sub- empha9is towards the clinical carries with it the neces- stantial plasterings of cement. In at least two in- sity for increased, rather than diminished, commentary; stances ]Drug Dependence (1126) and Causation in for Tobacco and Disease is a morass indeed. (Experi- Disease (1192-A)), the holes left between rocks were so mental t.icotinisation or "smoking" of animals or their large that they might be looked upon as n•indows-and tissues or organs usually speaks sufficiently for itself.) were thereupon Qttad with requisite oindow.displays. Considering that readers, even of inedico-pharmacologi- This Introduction may be looked upon, in part, as a cal monographs, are only human, no doubt there will chimney through which the Hot Air can find convenient again be a few objectors to the commentary contained exit .rithout blou•ing up the castle. And the words of in this Supplement. But writers (as contrasted to Edward Gibbon which grace our Introduction to cataloguers, who are in course of being supplanted by Tobacco ("1 cannot determine what I ought to t.rnns- computers, anyoray) are also only human. Their material cribe, till I am satisfied how much I ought to believe"j imposes a pattem on them, but they also impose, con- Ina3 tt.ill be regarded as the insurance•polic3• protecting sciously or unconsciously, a pattern on their material. both buildings. One cannot borrow Gibbon's genius, In a monograph of this sort, the obsolescence-rat.e of but one can share his irony. much of the source-material is unusually high (un- To the implied question of the present motto: "Is the fortunately, not every decade presents our subject with [Tobacco] literature worth keeping?", one can only a J. N. Langley or a Louis Lewin), and when the ma- reply: "Time will tell." Is this Tobacco Litcivturc worth terial itself eventually becomes obsolete, only the pat- witting aboutP is quite another question. The answer to tem r!mains. The passage of Time and the remorseless this is an unqunlifiPd Ycs. impersonal advance of Science will rectify our demor- racy of type-siee, and the result p-ill be one sort of H• 8nverre ~ Produced bv Th^ Council for ; Tobacco fieseaich-USA, Inc. 0002909 i i ~'v.~ :.~.,._ , .. . .~.

CONTENTS Preface .................................... vi Introductory Comments ................... vii 1. Absorption and Fate ... . ............... 1 Absorption .............................. 1 Ret¢nt'aon............................... 2 Membrane Permeability ................... 4 Distributdon .............~............... 5 Excret,ion................................ 6 Detoxication (Metabolism) of Nicbtine...... 7 Metabolism of Nicotine by jMicro-Organisms. 12 Metabolism of Nicotine by Insects .......... 14 Enzymatic Format.ion of Nicotine. .......... 14 Thiocyanate ............................. 14 2. Special Senees ............... . .......... 15 Vieion .................................. 15 Hearing ....................... ,.......... 19 Taste ................................... 19 $mell ................................... 21 Touch .................................. 22 Pain .................................... 22 Equilibrium ............................. 22 8. Nervous System ........................ 24 Spontaneous Activity ..................... 24 Behavior ................................ 25 Learning ................................ 26 Conditioned Reflexes and Conditioned Behavior .............................. 26 Visceral Pain ............................ 30 Convulsions ............................. 30 Autonomic Nervous 8y$tem ...... . ........ 35 Cerebral Cortex .................... . . . ... 43 Thalamus ............................... 48 A Iedulla Oblongata; Pons .................. 48 Cerebelium .............................. 48 Spinal Cord ............................. 49 Spinal Re9exes......................... ,. 49 Spinal Nerves and Nerve Endings......... 51 Cerebrospinal F1uid ....................... 52 Invertebrate letvous 8yatem .............. 52 4. Skeletal Aluscle ......................... 53 Intact Animals ........................... 53 Isolated hfuecle.......................... 56 Chemistry and lietabolism of Musele........ 59 Muscle Work............................ 60 I nvertebrate ll3 uscle ...................... 60 b. Blood .................................. 62 Erythrocytes ............................ 62 H:emoglobin ............................. 62 Carbosyhemoglobin ....................... 62 Blood Groups ............................ 63 Leucocytes .............................. 64 Thrombocytes ........................... 64 Coagulation of Blood ..................... 65 Blood Proteins ........................... 67 Fibrinolysis ......... .......... 67 Fluorescent Substances in Blood ........... 68 6. Cardiovascular System .................. 69 AnimsJs:................................ 69 Heart.Rate............................ 69 ElectnElectrocardiogram and ltocaogram. 72 Cardiac Output, Etc.................... 74 Epinephrine and Norepinephrine Liberation and Uptake by Cardiac Tissues....••-• 74 Blood Pressum......................... 81 Blood Vessels .......................... 91 Special Circulations .................... 91 Nan: .............................. 99 Pulse-Rate............................ 99 Electrocardiogram and Ballistocardiogram'. 104 Coronary Blood-Flow ................... 106 Blood Pressure........................ ~~ 107 Peripheral Circulation ................... 109 Special Circulations .................... 114 7. Respiratory System ..................... 116 RespiratJon.............................. 116 Intrapleural Pressure and Resistance in Animals............................... 123 Pulmonary Function in I1Ian............... 123 Smooth \Iuscle of the Respiratory Tract. ... 133 Cilian• Activity (Trechea) ................. 134 Gas Exchange in the Lungs ................ 136 Histopathology ..... ............. ..... 13S Oxygen Uptake by Lungs ................ 148 Pulmonary Diffusing Capacity ............. 148 Pulmonary Clearance ..................... 149 Pulmonary Edema ..................... .. 150 Mucus Secretion ......................... 1 50 Alveolar Surface Tension .................. 150 Exoerimental Respirator.%• Infeetions........ 150 8. Urinary Traat .......................... 152 RenalBecretion .......................... 152 Histopathologs of the Kidneys ............. 153 Water Balance ........................... 154 Ureter and Urinary Bladder............... 154 9. Gastrointestinal Tract ..............••.. 155 Appetite ................................ 155 iac Produced by The Council for 0002910 Tobacco Res^arch•ltq. lnc.

x CONTEhTS Hunger and Hunger Contractions.......... 155 .: .:..- T':^ ... . .. 15:i Action on the Gastrointestinal Tract as a Whoie ................................ 155 Itlotmr Effects on the Alimentary Tract:..... 155 Es,iphagus............................ 15:i Ciliary Activity (Esophagus) . . . . . . . . . . . . . 156 Stomach .. .......................... 156 Small Intestine. . . . . . . . . . . . . . . . . . . . . . . . . 156 Lahge Intestu~e ... .. . . . . . . . . . . . .. .. . . . . . 167 ....... lfia Ileocolic Sphineter.. . . . . . . . . . . . .. 168 Oral Cavity .............................. 109 Teeth ................................. 169 Paiadentium and Gurhis ................. 169 Alveolar Bone Loss ..................... 169 Orial Alucosa ........................... 169 Bccal ?liucosa and Hard Palate ......... 169 Salivary-Gland Secretion ................. 169 Absoiption from Gastrointestinal Tract. .... 170 Gastric Secretion. .. .. . ; .................. 170 Histmpathology of the Stomach............ 171 Liver and Gall Bladder. . . . . . . .. . .. . . . . . . . 171 10. Metwbolism ............................ 173 Carbohydrate ............................ 173 Fat ..................................... 174 :Qitrdgen ................................ 179 Mineral ................................. 1S0 Vit.atbwi................................. 181 Serotonin (5-Hydroxytryptamine). ........ 182 Heat and Energy Excha'nge ................ 183 Enzyme Systems ......................... 184 Tissues and Organs. . . 186 Growth and Developmeint ................. 187 11. Endocrine Glands ... : .................. 194 Adrenal ?tie3ulla ......................... 194 Adrenal Cortet .......................... 200 Adenobypophysis (AnteA•ior Lobe) .......... 200 Pars Intermedia; Chroniaiophores.. . . ...... 200 Neurohypophysis (Posterior Labe) .......... 201 Thyroid ................................. 201 12. Reproductive Organs; Reproduction; l.actatimn ............................ 202 Male Sex Organs ......................... 202 Female Sex Organs ....................... 203 Reproduction ................... . ........ 2W Placenta; Placental Permeability ........... 20.i Litter Size and Dlortality .................. 205 Lactation ................................ 2Uti 13. Local Actions of Tobacco ................ 207 Antisept9e Action......................... 207 Irritant Effect of Tobsuco•Smoke........... 207 Experimental Carcinogenesis ............... 207 14. Toxicology ..... . ...... . ... ........... . 234 Acute Toxicology for Bacteria and Animals.. 234 Acute Toxicity for Atan ................... 242 Toxic Agents in Tobacco and Tobacco-Smoke. 244 Indnstrial Tobacco Poisoning .............. 2,18 Chronic Poisawig in 111an ................. 249 Chronic Poisoning in .4iwnals .............. 251 15. Hypersensitivity and Tolerance; the Tobacco Habit. . . : .................... 257 Hypersensitivity to Nicotine ............... ?a7 Tolerafice . . . . .......................... . 257 Tachyphyla&ia ...... .................... 254 ~ Experintental Tobacco Dependencc...... . . . 259 The Tobacco Habit.. : .................... 259 Medical Opinion Concerning Tobacco•L'se.. . 319 16. Immunology of Tobacco ....... . ........ 3x3 Immunulog.• of Tobacco and Tobacco•Smoke. 323 Effect i,#' \icotine, Tobacco, and Smoking on Lnmiiue 1{eactions ..................... 324 17. The Effect of Tobacco and Smoking on the Hunwaia Organism as a V1'hole....... 3% Longe%-i ty .......... .................... 3:X \latendell Smoking and Reproduction.. . .... . 328 Juvenile Smoking.. . . . .................... 330 Influernce of S6noking on Afental Ef6ciency. ... 340 Influence of Smoking on Physical Efficiency and Physiqne .......................... 342 Lay Opinion Concerning Smoking .......... 344 Action of Smoking on the Body: Review•s.... 345 18. Tobaceo and Disease; General Considera- tiona.... 346 Tobacco as a 6~ise of Disense. . .. .. . . . . . . 347 Tobacco in the Nfanagement of Disease.... . 362 Relationship of Animal Experiments to Clini- cal Conditions ......................... 363 Individual Resp-nnse to Smokoig........... 363 Definition of ,3`mokers,.Xon-Smokers, and Ex-Smokers ... .................. 363 \ l easutemen te of Smoi:ing ................. 364 Reviews of Tobacco and Disease ........... 366 Morbidity - Rates in Smokers and Non- Smckers................................ 366 Atortality-Raes in Smokers and Non-Smokers 367 Mortality Risk of Smoking ................ 37S 19. Tobaceo in Relation to Specific Diseases. Part I. .. ............................. 381 Eye..................................... 381 Hearing; Ear ............................ 385 Taste Sensations ......................... 383 Olfactory Senhatlons ...................... 385 T ervous Systom .......................... 385 Uuscular S.ysoem......................... 387 Blood and Reticuloendothelia! System. ..... 387 Respiratory Traet (Excluding Cancer)...... 38R Urinary Tract ............................ 410 Digest ive Tre'ct ................... . ...... 416 h t et abolism .............................. 4'34 Endocrine Glands ........................ 424 ,llale and Female Cionads; Reproduetion.... 41-1i AllergY .................................. 430 Skin .................................... 431 Produced b1i 'th4 Council for 0002911 Tobacco Research-USA, Inc.

-:~.. ..., . _..~..~._.~.-~~ __...~._ .__.. ~ CONTENTS Infectious Dieeases............. .......... 431 1)ivrw+voa Tlnn 1n 7ib. oia.1 4.mn1v 499 Diseases Due to Chemical Agenta .......... 434 20. To rt~in Relation to Specific Diseases. ................................ 435 Cardiovaocular Diseaees .........:......... 435 Hypertension .......................... ~ 438 Hypotdnsion .......................... 439 Diseases of the lIeart................... 439 Diseases of the Peripheral Vessels ........ 467 21. Tobacco in Relation to Specific Diseases. Part III ............................... 476 Neoplastic Disease ........................ 476 Oral Cavity (General) .................. 482 Cheek (Buceal Cancer) .................. 486 Gums ................................. 488 Tongue ............................... 488 Lip................................ .. 489 Palate ........ ........................ 489 Nose atnd Paranasal8inuses .............. 490 Pharynx, Fauces, and Tonsils............ . 491 Epiglottis ............................. 492 Digestive Tract........................ 492 KidneY,and U' nnary Bladder ............. 494 Skin; Ear ............................. 496 xi Brain ................................. 496 T~.. ~Mr Salivary Glands ........................ 496 Brea4t ................................ 496 Cervix ................................ 496 Prostate .............................. 496 Testes ................................ 496 Pancreas .............................. 497 Leukemia ............................. 497 Cancer of the Treehea .................. 497 Cancer of the Larynx ................... 497 Lung Cancer ............................ 499 22. Aiedical Uses of Tobacco................ 675 Case•Reports IRuatrat'aig the Medical Uses of Tobaeco............................... 675 Veterinary Uses of Tobacco and Nicotine..... 677 Nicotine as an Insecticide ................. 678 Appendix I. Biological and Chemical Methods for the Determination of Nicotine. . 679 Appendix II. Some Notes on the Pharma- cology of Certain Derivatives of Nicotine..... Bibliography .............................. Index ...................................... Produced h Th Co!}ncil for Tobacco Ccsca,cil-USA, Inc. 680 686 761 0002912

N. 1. ABSORPTION (1) 2. srTes (1) 8. Orul Caroity (1) 4. Reapirofery Tnoct (1) [No new data.J S. Gualrofnleattnai ~ract (1) 1 ABSORPTION AND FATE studied by Borzelleca. When doses of 10, 25, or 50 mg/kg (-)•nicotine ('m, 0.9% NaC) ot appropriate buffer) w•ere pinM.i into the hladder of male d©gs anesthetized with pento. barbitsl sodium,,with ureters and urethra suitably cannu- lated, systemic effects were noted usually in 2 ttun (Hor- zelleca, 1960). When the solutions were perfused through the bladder, the onseQt of effects seemed to be more rapid with the more alkaline sohutions. Wqthin the PH range investigated the rate of absorption of nicotine from the bladder of male and femrtile dogs appeare.d to be dependent upon the pH, absorption being more rapid at higher PH ()3orselleca, 1961). These resolts on dogs were contrary to the findings of Macbt (1918)(3b) in this species~ but supported the findings of Travell (1990e.,b)(3b) in,cata Contrary to Travell's find- ings, however, I3mrzelleca (1961) demonstrated that absorp. tion of nicotine from the bladder of cats did take piace at PH 5.3. at which 99.84% of nicotine exists in ionized form (nicotine buffered to maintain this pH). Damage to the bladder by pretreatment with turpentine delayed absorption of nicotine. Borzelleca (196'2) then sttedied nicotine absorption from the urinary tract in albino rats anesthetized with pentobarbital, in which a ureter, the urinary bladder, or the urethra was isolated, washed with w•arm saline, and the drug instilled and permitted to remain in contact with the tissues until cardiac arrest occurred. A 25% solution of nicotine alkaloid buffered at PH 5.3. 8.3, or 11.3 was used, and characteristic changes in cardiovascular and respitatory functions taken as indica- tions of drug absorption. Nioot6ne was absorbed from the ureter, bladder, and urethra, but more rapidly from the bladder, the rate of absorption being dependent upon the concentration of the solution (limrzelleca, 1982, 1963b). The absorption was found to be pH-dependent, for nicotine was absorbed more readily from the alkaline solutions than fttun the solution with the low PH (Bcrzelleca,1962). Since, at pH 11.3, 99.94% of nicotine is non-dissocieted, while at PH 3.3, 99.84% is dissociated, the authot concluded that both forms are absorbed from these sites (Boraelieca, 1983b). Lora1 damage of tne niucosa, produced by pretrestment with tur- pentine, caueed a delay in absorption at pIl 11.3, but an increase In absorption from all sites at pH 8.3 and 5.3. The inherent qiifficulties in in-situ lneparatiuus led Hor- selleca (1903a,1984a, I985a, b) to investigate drug absorption from ia+lated urinary bladders of adult male rabbits; the organs were mounted in a Mm1 bath of oxygenated Tyrode'e solution maintained at 37°C, and drugs in Tyrode's solution were added to the bladder in volume of 3 mllkp body-weight of the rabbit; pH of solutions were kept identical Inside and out, and were alsp osmotically balanced with sucrose. 8amples of bladder coutents and bath-cnedium were removed for analyses at 1-, 2•,, and 6-hour intetv aIK. l nitiall,v, the potential difJerence between the inside and the outside of the bladder Travell (1960) reported once mora that fatal absorption of nicotine took place from the ligated stordach of the eat when the PH of the gastric juice was alkaline, but not when it was acid (see'IYavell (1940a)(1b)). In contrast to nicotifte, lobeline was not absorbed into the blood following its oral administration to dogs (Schievelbein: Werle and Eckert,1983). 6. Skin (2) Diad•smann (1950) reported that nicotine in aqueous or benzeno solution permeated the skin of the rat's tail; permea- taon from alcoholic soltttinns was less, achd there was no per- meation from glycol solution. Travell (1980) applied nicotine in alkaliue and in acid solution to the clipped skin of cats, and found that all of the snimals treated wiLh the alkaline solution died, while none of those with the add solution succumbed; of the former atri- mals, all vomited, and only 19% J the latter; and the effect came on more quickly with the alkaline solution. INlalkinson (196D) has reviewed the subject of percutaneous absorption of toxic substances, including nicotine, in industry. 7. :Utaoue Membranes (3) 8: Peri.•ardium (3) 9. Bye (3) (Ato new data.) . 10. Newona Sysfem (3) Injection of 2 tng nicotine hydrogeA tartrate into the cerebral ventricle of dheep produced only transient effects (A. C. Palmer, 1969). 11. (ienito•urfnary Trod (3) TraveU (1980) injected buffered solutione of nicotine into the bladder of cats, and found the absorption and toxic effects ptogtesaively imereassd from PH 4.5-8.0 to PH 9 Isee Traveil (htoa,b)(3b)I• The absorption of nicotine from the urinary bladder under many and vruied experimental conditions has been especially I Produced by The Gom°'I for Tobacco Rtscarc1'1-USA, Inc. 0002913 i

2 TOBACC(}-EXPEjt1D'+lEtiTAL Ala) CLLVICAL STUDIES. SUPPLEMENT I Nab -4 + 1 rioV: after 12D min. -3 -k 1 mV (BorsepeM 1963a). Within the, range of nico• tine tested (1, 10, 100 rwlf), the movement of the drug from buffered solutions pla'ced within the bladder was not depend- ent upon the potential difference nor the rmnecmtration of the original solution; and the rate of movement vi•as not altered by refrigeration of the bladder for I week prior to testing. Pretreatment with the metabolic inhibitors, cyanide, aaide, and iodoacetate, did not markedly alter the tatal amount of drug transterred (Borzelleca. 1963a,1964a); nor,was the rate of movement altered by nitrogen or dinitrophenol (Borzelleca, 1963a). On the other hand, movement of nicotine was de- 1>endent upon the pH of the solution: more nicotine vres lost fmrn a solution of pH 9 than from one of pH b'.1; that is to say, drug movement was greatest from solutions in w•hich the ratio of non-dissociated to dissociated moieties was highest (l3ortelleca, 1963a, 1964a). The results of these experiments suggested that movement of nicotine and other drugs from the urinary biadder is primarily a passive procesc Further experiments by Borsellecxi (1965b), in which the profwrtion of nicotine and of salicylic aeid transferred acrop's the bladder wall was found to be relatively constant over a wide rettge of concentrations, also suggested that drugs move out of the bladder by a passive process, rather thwin by a saturable transport process. U1ten an admixture of nicotine and (-)- cotinine n•as placed in the bladder, each was tradnsferred inde- pendently of the pWnee of the other. '1'tpe concentrations of nicotinc placed in the bladder had no influence on the per- centage of nicotine transfermd front the murosal to theloerosal side (liorselleca, 1965a). Recording of potential differenre (1.D.) between the tuucasal snd serosal sttrfacet showed that exposure to nicotine usually caused a dei•rease in P.D., and modified the spontaqeous activity of the bladder. 1•;shosure of the bladder mumsa for 1 hour to metabolie inhibitor:q includ- ing sodium cyanide (10"' 11), 2.4-dinitrophenol (10-=.U), sodium aside (10'* 11), and to anaerohiasis (nitrogen At- mosphere) resulted in very low 1'.D.s, suggesting that the tissue wa3 not actively metaboliaing. 12. Subrataneovs (3) Absorption oi nicotine from subcutaneous tiucstte of cats was greater, and mortality higher, the higher the pH (Travell, 1960). 13. Viarrllonraua (3) /\o unclassifiable data) 14. nt:TEA'rION OF •d•OnALY'o-rdaoitt: COJiBTITtl1'tt'hi tX atA\ (4) Pol)'dorova (19A9) found an average retention of 80- of the inhaled smoke in medium-heavy smokPr.• smoking a cigarette. In another study of the retention of smoke iathe tvkpiratorv tract during ritqarette-amoking }'ieta,~ch (1961) reported that it required the exhaled smoke from about 25 cigarettes smoked with inhalation to equal that of I cigarette smoked without inhalation, that is, retention a•a~ 25 timeF grealer on inhaling than on Ina/Kng. R. 1. Mitchell (19fi•l) meaa.rrd the retention of rigarette-smoke ttu•a under N:rec different test-conditions: taking the smoke into the bucral cavity, and expelling immediately; inhaling the smoke, and e:qwlling immediatel,i ; and inhaGnR the smoke, and exl>rlling it after it had remained in the respiraton• tract for about 30 ser. 11'htm none of the smoke was inhaled, 211--50 7,, (average, 37.2 t 10.2) by weight of the smoke-parliclcs. was retained in the bureal cavitieP• retention was 82.1 * 7.1 !-, when the smoke was held in the lungs for about 6 see; and 97.4 f 1.7!; when toe atuune wna 4:.: :-. =..... T' : 41- atudies consisted of 7 men and 3 women. Despite the above, and also the considerable other literature containing quantita- tive obsen~ations to tke contt•ary, T. Sato, Suzuki and Fuku- yama (1962) stated that most of the ciRarette stnoke inhaled into the Wngs is seen to be exhaled, Rignifying that onl}• a small iwrtion of the visible part of the smoke is retained in the body. \ickel earbonyl and benzolaJpymne are both carcinoRenir constituents of tobactiro-smoke,and the fomaerhasbeen found to inhibit, th.e induction of bento/obcyrene hydroxylane in lung and Uver (F.11'. Suadertnan, Jr., ItMiBa, b)K by inhibiting benzu(aUta•rene detoaification, nickel tarbon.l may promote the retention of benanlalp~•rcme ip the lungs, and thereby facilitate pulmonary carcinogeneais (aundennan, 19fiGa). In w iew• of the faet that the highest mean concentrations of polunium-210 had been found at segmental bronchial bifurvn- tions, and also on anatomical and physiological grounds, Radford end Hunt (1f164b) expected that chronic retention of smoke-particles in the bronchial epithelium would probably be quite sharply loralised in the regiou of the bifurcation it-4-lf, in o<n area perhaps of only a few square nun. Using gol'o in tobacco•Fmoke as a tracer for particle deposition and movement in the lunp, Radford and colleagues (1964) con- cluded th'at perenchymal clearance wres slow, with marked regional wariations of depaaitinn or clearance of particles in the lungx: According to yfatsden (19fr1), a tnan inhaling the smoke of 25 average tiritinh rigarettesa day probably deposits on his IuaFs and trachea same 8 picocuriea per day of ixtlo- niutn. Foi a more detailed arcount of delmiition and retention of r'^Po in the lung:c, see below, 541. R. H. Holland and co•norker.c continued to study the de)w+itioro of tadinartive arsenic• atong the reRhiratory tract Moliand et al. (1958)(-ta)). l'ptakes of 4.5-8.8~', of the total radioactivity were found in 8 tenninal eanre• patients n•ho inhalcvl the smoke f:»m cigarettes impregnated for haif of their lenj•Ih with 205 mirrocurics of r'.1s (Holland, McCall and L,ana, 1959). .a1a=orynion-rate from the brwtchial tree ans ralbid for the first several days, then tapered off slowly. In anutF.er si cdy on 8 volunteers, r•ome of whom had lung ran!rr, ent h subject inhaled the Fnoke from a similarly radioaMive- an-enic 4•ir;arctte, and uptakes wete said to range from 2.2 to 8.61 of 1he total which had been injected into the cigarettes (Hollund et al, 1959). Itair ®nd Ililir,v (1f)65) .'ailed to detect attr signifivant influence of cigarette-stuoke inhalation on the pulmonary clcarancC of radioactive dusts of iron oxide (+tFeeOa) and i-hromiuo,i oxide (+tCr,Oa) from the lung+ of rals and dogs. ln rat.~. arute extxn:ures to cigarette-smoke did not markedly influencw the clearance of inhaled radioartitr partirle., from thc luna; and poiisibk effects of chronic ciKarette-smnking a•ere exsmined in dogA utilising a terhnique by a•hich beaklr- clup "smoked" 20 cigarettes a day. Uetmo:ithin of inhaled insoluble particles and subsequent pulmonary-rlearanm rslwlrilitie+ of the dogs nere tested 1>Priodirall,r by exKazing the aniitwlc to aerosols of °'FeaOt or "(Ye(1r ; pulmonary clearance u•a, measured using wholr-hody counting and kml;itudinnl-body Rcanning tcrhniques. No marked effert~ were obfen•ed in the dogs after ciltarette.gmoke inhalation 5 da•t•s: lu'r week for over a year. IThis paper wa.a pnsented at the Eercm+d International S~tntx~Nmt on Inhaled Partick+ and Va)wrs, held in Caunbridge in 1965; see C. \. Daviec (1(IR6)j. Produced by The Councfl for 0002914 Tobacco Researcil•USA, Inc.

ABajOltl'TION AN1) FATE Kershbaum and associates (1965b, 1968a) reported com- ... ....,r... .... ~ ...ti:~. . a.. ;. ....d r• . .... .,r.:..,. ..r , c•igarette or a 1-gm terminsl segment of a cigar containing d-glucose-r'C; less radioactivity was detected in the blood after rigar-smoking, and thcre was greater absorption of r'C (as CO and CO= from glucose pyrolysis) with cigarette- smoking. In dogs, however, blood-radioactivity curves were found to be similar after inhalation of smoke from cigarettes and cigars containing random-labeled r'C-glurose (Kersh- baum et al., 1965c). Since, in these studies on dogs, lhe degree of inhalation was kept constant, the differences found bet wtrtt human cigarette-smokers and cigar-smokers were presunwbly due to the presence or absence of inhaling. TDE h,l-dichloro-2,2•bis (pK•hlorophenyl)ethane) is an insecticide used on tobacco, a small fraction of any residue of which trar~~fers to the mainstream smoke. liowery and co- workers (1965) exfwsed New Zealand Red rabbits, selected for their tolerance to mainstream toltacco-smokc, to smoke from cigarettes containing 12 and 48 rem of r'C•labeled TDIi per cigarette in Holland smoking-boxes. The animals received smoke from 20 cigarettes per day for periods ranging from 2 weeks to 6 moutha, after which thcy w•rre sacrificed, and 20 tissues exanrined for total and organosoluble radioactivity. Deposition via inhalation was said to appear o follow that of oral ingestion a•itb accumulation of TDE in the fat, followed by slow metabolism and elimination. Spleen and pancreas also tended to show relatively high accumulations of 1cC• activity, as well as adrenal glands in the case of the high-level exposure. In studies on man, non-inhaling smokers apimared to exhale all TDE components of mainstream smoke, whereas inhaling smokers appeared to retain (for subsequent storage and metabolism) about 370 of the TDE contained in the ciga- rette. According to Runeckles (1961), the radioactivity of ciga- rette mainstream smoke is due almost entirely to its content of 1"K. The amounts taken into the lung, even by the heavy smoker, are minute compared to the bodily uptake from the diet, and are rapidly eliminated from the lung tissue. ISee W. A. 11'olff, iPurdom and Itenhower (1959b)(4a)•) 15. 11 irotine (4) Several brief reviews of the abrorpt ion of nicotine on stnck- ing have been published (Van Proosdij, 1960, pp. 18-20; Earle, 1961b; Schievelbein,1962); absorption of nicotine from cigarette-, pipe-, and cigar-smoking has been reviewed by Schleicher (1964), and absorption of nicotine under various conditions of tobacco-use has been reviewed by P. S. Larson (1980). 16. Nicotine in Ciyarefle Sntake (4) . SchroAhl, Consbruch and llrurkrey (1954) reported that 15-20~'a of the nicotine of cigarette-smoke was retained by the body on simple puffing, and about 85-&R 17, after iultala- tion of the smoke. The comparable figures reported by Nagylucskay (1980) were about 10% and about 90%, resper- tively. The transfer of nicotine from the tobacco to the main- stream smoke could be in8uenced to a great extent both by the properties of tobacco-productr= and the mode of smoking, for example, rate of puff (Nagyluc•xkay, 1980). Egri (1981) determined the average amount of nicotine deposited on the mucous membrane of the mouth as a result of tanoking in the folloa•ing way: Subjects aspirated smoke every 30 sec from an "Austria 3" brand of cigarette; and, after each 2-sec• puff, the mouth was rinved nith a•nter. After the subjects had smoked Produced by The Council for Tobacco Research-USA, Inc. 3 25 cigarettes, the combined aresFtitrga contained aa average of v in . . .a;•:... We may mention here that .krntilaRe and Milt'on (1965) found the mainstream nicotine content of a specially blended cigarette to overage 1.3 mg per cpgarette. Taking a s•aiae of 2 nut nicotine iabxorbed by a 70-kg man in 10 min of smoking, thii, was calcotlated to correspond to a doax of 3 µg/kg/min of nicotine absorbed through the lunl;s. ArtnitaQe (1965) de- scribed a method for introducing smoke from a cigarette, smoked under carefully controlled conditions, into tbe lungs of an artificially-ventilated cat, using a 2-sec durati~,n of pufl ead a puff-frequency of I per min, with the cigarette being smoked to a 20-nun butt-length. Increa.:ing the puff-volumc from 20 to 35 ml was found to increase the total nii:rotine per cigarette by about 51/1„ the nirntine jwr puff hy alTnut Rfl1,, , while the time required to snooke a cigarette to a fixed butt- IenRth u•a.s reducrd by 20!',.14'hen tested on catFanesthetized with chloralose, the effect on blood IireaY;ure of a single puff was found to be almost identical to the effect of half the amount of nicotine in the puff when injected i.v. These find- ings on cats were said to suggest that the amount of nicotine entering the blood of a smoker who inhales is likely~ to be in the range of 50-150 µg of nicotine (basel per puff, u•hi,•h alrprovmates 1-2 µg/kg body-w'eight. Allowing for speeic-.., differences, Arnritage considered that an i.v. dose ol 5-10 vglkg in a csit is a fair aplrroxintntion to a human smoking- dose of nicotine. Absorption of nicotine from eigarhtte-xmoking has been reviewed by 1'. S. Larson (1960) and Schleit•her (1964). 17. Nirotine in Cigar-Svirode (5) The absroryition of nicotine on cif~ar-.moking also haa been reviewed by Larson (1960) and Schleic•her (19&t). I(ershbaum and co-workers p9ti5a, 1966s) detemtined the comparative effect of cigar- and cignt•ette-amokinK on free fatty acid npobilisation and ratechtdlamine excretion (see below, 696-B and 758), and, from their trsults, conclt4ded that probably less nicotine is absorbed after smoking cipars. The preFence or airsence of inhaling wns taid to be reaponsible for these differences in response to ciuar- and cigareite sntok•int (I:^rshbaum et al., 1966a). It. \'ieoti.ne in C/rcuing-9b6attv. (6) Nicotine absorption from tobncco-a•heu•ing has been re- viewed by L>dtsvn (1960). 19. Wirntine in A njl (6) The abrorption of nicotine by snuff-users has also been reviewed by Larson (19t30). 20. A'itbttne from Inlwlatiort of to6ucco-Duat (6) I\o new dafa.) 21. Other Conatifaenta of 3'obarro,Srnoke (6) Preliminaryytests by llorbt~ly t17U65) tvere said to sliow that carbon monoxude is absnrln•d escltjeively in the deep reryrira- tory airways, gnd not in ttu• huccal cavicy. Thus, smokers who admitted inhaling cigarette-amokc were found to have high carboayhenoglobin levels, while nou-inhalers had normal levels. In eqreritnettts carried out by llaagen Cmit, Rtvoell and lfara (1969), when cigarette-smt~kc was inhaled into •:he lungs, and then exhaled, the cxh'al,-a smoke contained no 0002915

I 4 TOBACCO-EXPERh.111:NTAL AND CLINICAL STUDIES. SUPPLEMENT I detectable oxides of nitrogen. If the smoke was not inhaled into the lungs, the concentratron 01 nitrogen oxwaes m tue exhaled smoke was about one-third of the original concentra. tion. In studies on 3 subjects reported by Iidkhoven and Kiessen (1961), practically no retention of carbon monoxide or oxides of nitrogen (NO + r0s) was found on puffing cigarette-smoke, compared to about 80%a and about 95% retention, respectively, on smoking with inhalation. Shabad, Pylev and Kolesnichenko (1f)64) studied the kinetics of the elimination of benzo(aJpyreae from the pul- monary tissue of rats following introduction of different suspension-comlwsitionsand repeated intubatiotqs. When the vehicle was saline, cumulation failed to occur; but cumulation was achieved when introduction was tnade with black-ink puN.ler and Infusine. In ea•periments on male and femalc hamstP-A, Saffiotti and co-workers (1964) administered benao[a)pyrene intratracheally in fine particulate form at- tached to betnatite in saline. In the first experiment, the proportion of benso/a)pyrene to hematite remaitung in the lungs after a single dose of 5 and 45 mg, tespeetively, de- creased by a constant rate from entry-time to the end of the first week. Further tests with a single dose of 3.4 ing benzoiaJ. pyrene, and with 9 a-eekly administrations of 3 rng of the carcinogen, showed recovery-values consistently higher for the latter group of animals, but retention-rates were similar for both groups, indicating that no metabolic adaptation had occurred during 10 weeks. In another series of experiments with single doses of 33, 3.4, and 0.96 mg , betlzo(o)p)•rene, respectively, the higher the dose, the higher the rate of re- covery; after the second day, the retention-rale remained only slightly higher for the higher dore-groups. Wynder and Hoffmann (1960a) found that 80-t10 0 of cigarette-smoke irondensste was retained on deep inhalation. These workers reported amounts of smoke-condensate (and nicotine) for mainstream emoke of 10 popular brands of cigatettes; for plain 85-mm long cigarettes, the tutal con• densate varied from 3540 n:g per s•igarett.e; for plain 70-mm ones, about 30 mg; and for 85-mm filter-tip cigarettes, 18-26 mg. Nicotine values varied proportionately. However, as Van Proosdij (19B0, p. 27) noted, it is not easy to estimate the quantity of tobacco-tar absorbed by a smoker, because the percentage of tars in the smoke and the sntoking technique are liable to vary considerably. Average values may be meaningless in any particular case. For an airount of absorption and/or retention of naturally • oocureng or added radioactive substances (r'opo; r41s; •oK) in cigarette-moke, see above, 14, and beltn•, 541. 22. FM.'TOttS 1NFLtlE\CINa THF: AnSORhT10N or N/COTINE (6) 23. .4nimal Ezperiments (6) Dlassmnnn (1956) measured skin permeability of toxic substauirs by dipping a measulr.rl area of the tail of rata into the test-solution, the index of absorption being subsequent death of the animal. Nicotine was found to permeate from alcoholic solut ions, but less so than from aqueous ones; perme• ation from benzene corresponded to that from water; there was no permeation from glycol solution. In aqueous solution at pH 8.8, permeation was markedly greater at solution temperature of 39°C than at 29°. As other authors had demon. strated, permeation wa.° also dependent upon an alkaline pH. For e.um)/le, Travell (1960) noted that, when itdd or alkaline solutions of nicotine arre applied to the clipped skin of eats, or injected into the inguinal fold, the alkaline solutions were .n• ....w v lUC UIUIe laAlt', WUl'CUUal/4a /ul vVU/wa/oH- and 11, above. 24. Absorption in Smokiny (7) Nicotine absorption in smokers is influenced by depth of inhalation (Schm&hl, Conabruch and Druckrey, 1954. Nag.•- lucskay, 1960; Pietzsch, 1961; among others) and rate of smoking (1\aUluc8kay,1960; Desoil.e et al., 1963), as well as by the properties of the cigarette itself (length; filter-tip) (11'ynder and Ho$mann, 1960a). This subject has been dis- cussed in de:ail, and smnmariced in Tobacco (8). \laking use of a model system instead of actual smokers, Artbu and Greb (1964) studied the absorption of nicotine from ciga-+ette-smoke in a buffered liquid similar to saliva, and found that this artificial "saliva" retained a larger pro- portion of smoke when relatively alkaline (i.e. with high, rather than low, pH), and in accordance with the surface-area of coqtact with the smoke (equivalent to depth of inhalation) and the duration of contact. The butt-length was also said to play an important part in the experiments, since a considera- ble quantity of nicotine may be retained during the pa,ssage of smoke, and then redistilled when the combustion zone approaches. 23. Non-Pulmonary Absorption of 11'i.rotine in )Uan (8) (\o new data.) 26. Vembrane Pernteabitity (8) In their pharrnacological review of the effects of nicotine on varioros organs or organ-systems of the human body and on experimental animals, «'erte and Schievelbein (1966) made the initial observation that nicotine has a pronounced effect on biologiesl membranes, i.e., it increases the permeability of these membranes to certain phannacologically active sub. stances, such as nr,repinephrine, elrinephrine, dopamine, etc. This effect was thought to be probably non-sperific. 27. Arlificial Alembranes (8). (To new data.J 28. Tissues (9). For an account of placental permeability to nicotine, see below, 816. Radioaetive-calcism ('°Cs) uptake by isolated froF• sartorius murcle was increased 3-fold by nicotine (G. B. Weiss, 1966a, b, c). This increase was inhibited by lowering the pH of the Ringer's bath solution (N'eit,%, 1966a, c); also, the uptal;e of t'C-labeled nicotine into the muscle was frnind to be more than doubled by increasing the pH of the Ringer's solution (Weiss, 1966c). 29. Membrane Potentials (9). 13orselleca (1965a) showed that eaposmv of isolated bhuldcr of thc rabbit to nicotine usually caused a decrease in potential difference between the nturo=al and serosal surfaces; tee above, 11. According to Werle and Schievelbein (1f16u), the aggrega- tion of blood-platelets by nicotine Itme below, 2111 may be caused by a change of the electric chatgc of the tbrombocyte membrane, because there is strong evideurn that nhrotine may enhance the permeability of the platelet mtmbrane by chang- ing its charge. S0. Phase-Boundary Potentials (9). [No new data.) Produced by The Council for Tobacco f iCSOarch•USA, Inca 0002916 i

ABSORPTION AND FATE 31. FATE (10) 32. DtaTalatTrlON (10) 33. Elood (10) One hour after s.c. injection of 5 mg/kg nicotine in rabbits, blood•plasma concentration was 3.0 rrg/ml; 3 hours later, 1.0 µg/ml; and no nicotine was detectable in the plasma after 6 hours (1. Yamamoto, 1955). The corresponding plasma concentrations for dogs receiving the same dose of nicotine were 4.2, 2.0, and 1.0 pg/ml, respectiveiy. Within 5 ntin after i.m. or i.v. injection of (-)-nicotine• methyl-I1C into mice, all tissue toncetttrations of the radio- active compound were higher than the blood concentration (Sehmiterlow and $aasson, 1962; Hansson and Schmiterlow, 1962). (For the tissues examined, see below, 34.) Schievelbein and Schirren (1964) rutade i.v. injections of nicotine into dogs at rate of 0.66 mg/kg of free base per min, and drew blood immediately following respiratory arrest; the blood was then fractionated for analyses of nicotine content. The sum of the nicotine contents in erytbrm•ytes, leukocytes, platelets, and plasma ttmounted to about 10% of the iu;ected dose, the platelet content amounting to less than 1%. A method for determining the nicotine content in human blood was described by Desoille and co-workers (1963), who found wide differences in 20 smokers, and attributed this to varying rates of smoking. In several of 16 persons who had not smoked for 12 hours, measurable quantities of nicotine were detected, possibly due to "sneaking a smoke". Of 6 non-smokers, l showed traces of nicotine in the blood. The wide variation in smoking habits leads to every conceivable pattern of fiuctuating blood-levels of nicotine during the day (Smoking and Health, 1964, p. d9). 34. 7tissues (10) 1. Yaroamoto (1955) reported the distribution of nicotine in the tissues of rabbits and dogs 1, 3, and 6 hours after s.c. injection of 5 mg/kg of the drug. ln rabbits, the nicotine content (µg/gm of tissoe) was highest in the kidney (8.9) at the end of 1 hour, followed by pancreas (7.9), ileum (6.3), ventricular muscle (5.2), skeletal muscle (4.8), lung (4.2), spleen (4.0), cerebral cvrtex (3.7), omental fat (3.5), and liver (3.3); none of the tissues examined had detectable amounts of nicotine at 6 hours. In dngs, at 1 hour, nicotine content aas highest in thc kidney (14.3), foilon•ed by pancreas (13.9), brain (13.7), ileum (10.8), liver and omrotal fat (8.6), spleen (i1.0), heart (7.8), muscle (7.0), and lung (6.1). At 3 hours in this latter species, the relative concentrations were the same; at 6 hours, the highest concentrations were in the pancreas and skeletal muscle (4.1), followed b.• the kidney (3.9), and Gver and ventricular muscle (3.4). Nicotine det.erminations were made by a silicomolybdir-acid method and also by a cyanot,mn•bromide method (limits of sensith•ity, re.qpectivel.•, 0.1 ntg/nil and 0.5 pg/nil or gm). Hansson and Schmiterlow (1962; prelintinary report by Schmiterl3tr and Ilansson, 1902) injected mice i.m. or i.v. with (-)-nirotine-methyl-14C, and then studied its distribu- tion with an autoradiographic technique. Within 5 tnin after injection, high concentmtions of nicotine were found in brain, adrenal medulla, stomach wall, and kidney; lower conrentra- tions were found in liver, skeletal muscle, and blood; but all tissue concentrations were higher than the blood concentra- tinn. Kidneys showed a high activity from 8 min to 4 hours 5 after injection, with a maximum during the first hour. The o~ironol mwl.dln .ein nnn0oinnd w r.iA, o.»nn..* nr o..:..:~.. I and 4 hours after injection, but little or no activity was ob- served by 24 hours. A high content of radioactivity appeared in the walls of large blood vessels, and the activity remained high for the fust 30 mim. Bone marrow also contained a high amount of radioactivity by 30 min. Autoradiograms of brains of animals sacrificed 15 and 30 rain after injection showed that the radioactivity in this tissue disappeared relatively rapidly. (Metabolic investigations showed two radioactive compounds in the brain, one being unchanged nicotine, and the other presumably cotinine; and perhaps the reasun why the radioactivity dropped quickly is that the metabolic com- pound, cotinine, has not the same affinity for brain tissue as nicotine, and leaves the brain tissue easily.) IlvP min wfter injection, the radioactivity in the pituitary gland was higher than in the brain, and remained so until l hour Accumulation of radioactivity was observed in the salivaq• glands, probably reflecting an excretory pathway. f,1-hole-body autoradiogranns of mice and cats injected i.v. with (-)-nicotine•methyV'C showed an initial accumulation of radioactivity in tlre central nervous system; this high concentration of nicotine and or its metabolites in the brain largely disappeared within 30 min to 1 hour (Appelgren, Hansson and Scbmiterliia•, 1962). Chromatograms of chloroform extracts of brains of mice revealed only one nicotine metabolite, cotinine, which oras also isolated and identiSed. The rate of conversion of nicotine into cotinine in the brain was followed by quantitative radio- ttesays of the i(bnig-positive zones on chromatogran>b from brain extracts made froyn mice sacrificed at suctrssivr times after i.v. injection of W.; nicotine. Five, 15, 30, and 60 min after nicotine injection, the per-cent radioaetnity of the nicotine spot was, n.wpecttvely, 72.9, 80.9, 24.9, and 13.1; and of the cotinine spot, 27:1, 19.1, 75.1, and 86.9. Interpreting theste data, the authors suggested that nicotine receptors in the brain may not be able to serve as receptors for cotinine, thus e.plaiuing the more diffuse distribution of radioactivity in the bnain seen 3o min after injection of nicotine (sce also below, 148). llowman, Hanason, Turnbuil, A1clCennis and Schntiterlow (19frS) studied the disposition and tate of radio- active cotinine in the mouse, and thex workers disrussed the difference in di.tribution between nicotine euld rotinine in ternis of a possible relationship to the marked phamiacoluKical activity of nicotine and the contrasting lesser acti%ity of its n.etabolite, eo;iniae. Schmiterlbtiv and Hansson (1965) have reviewed their earlier studies on tissue distribution of MC-nicotine, to which they added the report of an experiment in which "C-nicot ine solution was applied directly into the trachea of mice. Auto- radiograms of the mouse sacrificed at 2 min showed a very high amount of radioactivity in the respiratory tract and lungs, and again demonstrated that nicotine enters the central nervous system rapidly. An autoradioQram of the mouse axcrifirrd at ]5 min showed a still high concentration of radioac•tivity in the lungs, while the brain now contained considerably leer activity than at 2 min, and the kidncys and stomach contained large amounts. In 8 rats given "C-randomly-labelled nicotine i.p., and sacrificed 3 hours later, the kidney contained the highest concentration of radioactive rompounds on a per-gm basis, followe d by t he lung, liver, brain, skelet al muscle, spleen, and heart (F'ishman,190.4). In I dog given radioartive nirotine i.v. and sacrificecl at 3 hours.l1C a•as prt-vnt in the stomach wall Produced bv The Council for 0002917 Tobacco Research-USA, Ine,

a TuRACC(1--EXPE1t1h1EItiTAL AND CLINICAL BTUUIES. SUPPLEAiENT I• c., l.,r o.patar cnncnmrnt wbn thnn in anv other ti+sue analvzed; liver was second. When pregnant rats were ltiven LI)a duera of nicotine s.c. I day before expected delivexy, nir•otine titers in fetal tissues were presumed not to have attained maternal tissue levels (J. E. King and ltecker, 196ti•). 35. I•ffcw Chronfr Tobacco .ldnrfntatrolfon (12) (;:o new data.) 36. In Fa(al To6acco or Xirotene Poisoning in llau (12) \Isehly and 13onnichren (1963) rreported 46 cases of fatal nicotine poisoning in Sweden in the years 1956 1963; these writers dc.acribed a method for analysis of nicotine in body• tissues, and presented data in 16of their cams on the nicotine content of blood, liver, kidney, and stomach. The case, reported by Ioanid and Hors (1960), of a young woman who died from drinking a 96 % nicotine insecticide preparation is of special interest here, for the body was ex• humed almost I month after burial; but, as death had occurred in the winter time, and the churchyard was sandy and dry, the corpse was well preserved; and the authors were able to make quantitative determinations of the nicotine content of various organs (see table given in the original article). Kico• tine was found in large amounts in the stomach contents, and, in decreasing order, in the following visc•era: stomach; lungs; intestine; liver; kidneys. In the caee of 2 small children who had died after presuma- bly snalloating cigarette-butts, the extremely small traces of nicotine found )wst mortem in the blood, brain, liver, and stomach, were said to have corresponded with the amount which would be found in cigarette-butts (R. Gupta, 1»0). 37. excnF:rtoN (12) 39. Urine: Anenwls (12) In 7 guinea pigs given i.p. injections of "C-randomly- labelled nicotine, urine u•aF collected at 18 hours, and ccn- tsined about 900 of the administered 11C (Fishmat4 I963). In 3 dogs giv.n radioactive nicotine i.v., the 24-hour urine contained 77-95 ~'o of the "C. Autoradiographie anal)sis of chrotnatograms of the dog urine indicated the presence of at least 10 t4C-containing comlwunds. In studies of the disposition of t'C-labelled nicotine in 3 rats, 5070 of the injected radioactivity was exereted in the urine within 2 hours, and 72.4% (range, B+B.S-'6.9) appeared in the 24-hour urine (Hatuzson and Schmitcrltin•, 1962). ln studies on 4 mice, the 24-hour urinary excretion was 68.2ck% of total dose (range, 59.2-72.1) (Schmiterl8w and Hansson, 1962; Hanagon and Sehmiterlow, 1962). Chromatographic separation of urine radioactivity revealed at least 8 nirotine metabolites, of which two alrywareci to be cotinine and hy- droxvcotinine. McKennis and co-workers (1964a) gave male rats i.p. injections (in neutral aqueous solution) of tadioactive (-)-cotinine methonium iodide and radioactive 8-Irvridyl- aertic acid methonium chloride-both nicotine metabolites-, and recovered 94.6-97.7 o and 62.9-67.9%, respectively, of t4C-artivity in the urine. 39. Urine: Man (13) As demonstra<ed by gas chromatography, acetonitrile (CIItCN) is present in ciRarette-smoke in the amount of about 1 mg per cigarette (J. K. Campbell, Rhodes and Grnss,1963). H. C. McKee and co-workers (1962) reported the pnesenre of acetonitrile in urine of smokers, and itn virtual absence from the unne ot non-snmsers tuic iugiircI eiutcic .n:uc :.•... :.. ~ non-smoker was 0.74 µF/100 ml uride). Among thom whu consixtent ly smoked 3 cigdrettea per da)• or more, the loweat value determined was 2.2 pg/100 ml; ann heavy smokers showed concentrations in exc•e,:~ of 20 brg/100 mi. However, a perfeet quantitative correlation was mot found; rather, thc data tended to scatter about a regression-lune indicating the best pc"ble fit, and this scatter cvres thought to be due tu variatiotu+ in the individual's smoking habits, such a:< depth of inhalation, length of cigarette left ultsmoked, and number of puffs per cigarette. The authors announced that work wag under way to attempt to contrul smtve of the variables, so that their effect on the quantitative,telationships could be determined, the th-mght IminK that urbnc-amtonitri)c content might provide a more accurate rncasune of the actual degree of exposure to tobairo•smoke than the number of cigarettes smoked or other similsr mmsures eomitmnlj• used (see below, 1194-C). Meliven and co•a•orkers (1965) also used a gas-chromato- graphic method for the deterntination of nicotine in smokers' urine, and they presented a table showing tng of nirotine found in 24-hr specimens from 6 cigarette-smokers, 2 cigar- smokers, and I pipe-smoker. Related to nuRnber of cigarettes smoked or gm of tobacco consumed (cigar and pipe), rather large variations appear; these were considered to be probably a reflection of the smoking habits of the individual along with other factors. 1'r,ing a gas-liquid chronuuographic method for the determination of nicotine, Beckeltr Rowland and'!'riggs (1965) showed that the rate of nicotine exkretion in urine is influenced by the pH of the fluid. Subiecti smoking 20 ciRa- rettes a day at fairly regular intervals escreted 0.1-4.3 (mean, 1.0) µR nicotine/ntin under normal conditions; 0.4-13.0 (mean, 5.0) NR nirotine/tnin when the nrise was acidic after oral administration of ammonium chloride; and less than 0.1 pg/min when the urine wns alkaline after oral administra- tion of sodium bicarbonrte. 40. F'eees (14) In 7 guinea pigs given i.p. injeMions of 1c(:-randoml,r- labelled nicotine, feces were collected at 18 houni, and found to contain about 2% of the administered 1113 (}ishman, 1953). In 2 dogs given radioactive nicotine i.v., the 24-hours feces contained 2.5 and 7.2cl,, respectively, of the radioactivity. Followittg Lp. injection (in neutral aqueous solution) of radioactive (-)-cotinine methonium iodide and radioactive 3-pyridylacetir add methonium chloride into male albino rats, 0.78-1.21 % and Q:c-0.56% of the '4C activity was recovered in the feces (NIeKennis et al., 1964a). 41. Bile (14) In studies on 3 tats, 2.00-3.49;", of the total radioactivity ol injected "C•labelled nicotine aas excreted in the bile in 6 hours (Hattwon and Srhmiterlotr, 1962). Autoradiogranu; of chrotnatogranu of biie showed two radioactive compounds with the same R, values ar nicvthtc and cotiuine. 42. Erpfred :1£r (14) In studies of the disposition of 14C-labelled nicotine in mirc, cumulative excretion of radioactivity in the exhaled carbon dioxide during 2.f hours ranged from 10.7 to 15.2% of the adminixtered dose of (-)-nicotim'-methyl•11C (Schtniterlow and Hanr.-on, 1962; Hanwmn aud rehmiterl&a, 1902). Following i.p. injectinn (in neutral aqueou- solution) of Produced bv The Council for o 0 0 2 918 Tobacco ReseacJi-USA, Inc.

ABSORPTIO1t AND FATE 7 radioactive (-)-cotinine methonium iodide sad radioactive o.,...;.i../....m:.. ...„.1 ....sl..,..;...,, ,•tiln.i.ln //.nfh nimfinn metabolites) into male albino rats,'fC activity recovered in respiratory carbon dioxide was, respectively, <0.01 °ie and 0.16-0.18'70 (hlcKennia et al., 1069a). 4S. Skin (15) "fhe review by Schievelbein (1962) on the excretion of nicotine by various routes includes excretion in sweat. 44. Saliva Accumulation of radioactivity, observed in the salivary glands of mice receiving i.m. or Lv. injection of f'C•labelled nirntine, probably reflects an excretory pathu•ay, since the concentration seemed to be high in the salivary ducts (Sehmiterl-on• and Hansson, 1962; Hansson and &hmitee'lbw, 1962). 45. (JoaG'i.cJuice V5ve min after injection of (-)-nicotioe-methyl-t'C in mice, radioactivity was observed in the mucosa of the fundus region of the stomach, but none, or at lesst very little, could be seen in the stomach contents (Schmiter(btv and Han'sson, 1962; Hantson and Schmiterlbw, ,19ti2). Autoradiograms taken 15 min after injection showed an appreciable amount now sec'reted into the stomach, and this was more evident 30 min after administration. Gastric excretion, of "C-nicotine was exi.rnined in mote detail by Andenrson, Hansson and Scllmiterliiw (1965) in mi-x, rats, and cats given i.v. injections of 2.5 µg (-)-nicotine-methyl•"C per gm body-aT'ight (corresponding to 0.10 mi•trocuries/gm), and sacrificed 5, 15, 30, 60, and 240 min aater the injection. A a•hole-body auto- radiogram of a mouse sacrifired 1 hour a(ter injection showed that nicotine and/or its metabolites became conrentreted in the stomach. From a second au:oradiogram of a mouse sacrificed 15 min after injection, it appeared obvious that, in this shorter interval, the amount of radioactivity was higher in the gastric mucosa than in the stomach contents, whereas, aftar 1 hour, a substantial amount had been excreted into the stoinach. From autoradiograms of stomachs opened and wiped free of content, it appeared that radioartivity occurred only in the glandular part of the stomach. P_xperiments on rats and cats were said to have given similar results. Studies on mice and rats of the amount of radioactivityevcreted into the stomach gave rather wide variations, 1he values obtained varying from 1.8 to 10% of the amount initially administeted. To study this, the stomach of rats was jmrlused in situ with solutions buffered to pHs *anging from I to 9; "C•labelled nicotine was given i.v., and the amount of acthity ajipesrinF in the perfusate was determined. Results showed that the highest excretion-in 30 min--occurred at pli 1(mean value, 4.4 0), and that excretion diminished ti~ the pH increered (down to 0.0',"o at pH 9). Chromatogtaphir analysis of the perfusate showed that, after 30 tnin, 657, of the radioactivity represented unchanged nicotine, the remainder being present in the only ntetabolite which could be deierted, namely, coti- nine. For the possible relationship of tjtese findings to the genesis of stomach ulcers in smokers, see below, 1312. 46. Milk (15) Excretion of nicotine in milk has been reviewed by Schiewel- hein (1962); and see below, 823. 47. Reoieros _ . r, .i .ti. . literature (mostly over the hrereding 10 yearx, but not limited to these) coverinr, excretion of nirotinr in the urine, feces, swerat, exhaled air, and milk. 48. DF.•TORICATION (uerenouas+) oF KtcanxE (15) 'f'he symposiuru on "Tobacco Alkalaids and Related Com- pounds", held in Stockholm in February, 1904, contains an extensive review by \IcKennis (1985, pp. 53-74) of the disposition and flate of nicotine in animals, based on 111 referenres, and also a review by I-lansson and ticl;imiterlbw (1965, pp. 87-97) of their own and other reported findintts on the metsbolism of nicotine in various ti,eucs. I'teviously, Schievelbein (1962) hhad presented a review of the newer literature (mostly over the preceding 10 years, but not limited to these) covering the metabolism of nicotine in the living orgunism, including nicotine detoxication by the animal organism and the nature of the transformation producta 49. Sites within the Animu/ Body (15) Some of the earliest experiments bearing on this subject have been cited by RothberFer and t1'intrrberg (1905a). Sehi(1' (Sur une nonodUe fonelion du foie ei 1•rlld de to figolure de fn aef.ne porfe. Arch. se. phys. nat., GfinAve, 1877) found that, following extirpation or t)inF off the liver, frogR were leac resistant to nicotine, while ligature of the renal-vein affereatry with consequent increase in blood-flow to the liver, (ncrea.aed resistance to the drug. Roger (A'ote aw le ro/e du foie dans fea inloru••alions, C. r*-nd. Sbc. biol., p. 63, 407, 1M) confinned Schitf's findings in all essential points. Heger (Ste !c pmaoir fi,rnlevr de oerfai»s organes pour lea olca(oidea ek., C. trnd. Acad. sc., 1880) found retention of nirorir.e ln the isolated, perfused liver. In experiments dealing specifically with nico- tine, Rothberger Ond \linterberg (1905a) themFCh•ea infused nicotine in dogs at a constant rate (0.17 or 0.25 mRlkg/min) into the femoral vein or arter.•, ileal artery, stomach vein, or sidehic vein, and deterinincd the duration of infdtsion re- quired to cause death. Death ensued quickrst when the nicotine was given via a femoral vein; arlerial injertion required a longer time; and injection into the sta,mach or splehic vein took longer still. From these, and results with other toxic mnteru0% these authors did not think it proper to designate the liver as the detoxication organ of the body. Significant detoxication also orcurnad from pexting, through other capillary beds, and the t}7e and manner of detdxication, it was concluded, cannot be the xame in all organs, but de- Ix•nds on such factors as dilution, fixation, diffusion, etc. In rats sacrificed I and 2 hours after s.c. injection of nico- thte, two main pyridine derivatives (identified as cotinine and dihvdrometanicotine) were isolated and purified from the liver (UeClereq and Truhaut, 1902). 50. Experinlenls on Tiasrce Br.ris, Slires, Homopenrttes, Extrorts (10) Schiff (18i c) found that trituration of a nicotine solution with liver brei reduced its toxicity, while kidney brei a•a.c ineffective; and Roger (1886) was said to have confirmed these fmdings (tre above, 49). I. Yamamoto (1955) prepared 25 ~-t, extracts or rabbit liver, lung, kidney, and .rrum with t{ citric acid; to thesr,,S mg/ml nicotine were added. the mixtures shaken in a bath at 37.5°C, and chan(ms with time and the amount of nicotine were Produced by The Council for lobacco _ Research-USA, Inc. 0002919

8 TOBACCO-EXPEIt111tENTAL AND CLINICAL STUDIES. gUPPLENiF.NT I measured using the 8ilicomolybdic-acid method. Changes in nieotirlP MlntPnt with IivPr Pvtrnrt wPro aAW tn }w mnro marked than with extracts of the other tissuea; however, with the cvanogen-bromid'e method of determining nicotine. the changes were not significant. This worker mbsidered it con- ceivable that detoxication of nicotine by the Gver extract involved changes in the pyrrolidine moiety of the molecule, which would not be; r+efiected in the deveka,inatiou with cyanogen bromide. Evidea<ce of some adsorption of nicotine by the liver extract vns noted. The detorica4ion of nicotine by liver extract was inhibited by addition of 1/1,000 mol potassium cyanide or sodium azide. Under anaerobic eondi- ebons, nicotine was dehydrogenated by liver extracts when a hydrogen acceptor (methylene blue) was present. Lobeline inhibited the ensymatic degradation of nicotine in liver slices from guinea pigs (Schievelbein and Werle, 19fi2b). Decker and Sammeck (1964a) reported that cotinine and nornicotiure were formed from nicotine in vivv, as well as by cell-free fractions of rabbit liver. In the latter system,10-21)% of the total degradation goes via nornicotine, which is then partially, dehydrogenated, yielding norcotieiine. No de- methylation of cotinine was observed. The furmation of nornicotine was accompanied by the liberatioh of free form- aldehyde, which was subsequently oxidized by the liver fraction to carbon dioxide. These same workers reported turnover of traCHrnicotine and ar.2'-1eC-alitotine by a oell-free extract of rabbit liver (Decker and Sammeck,19B4b); and they considered that they bad confinned the finding by Bucker, Gillette and Brodfe (1960) in cell-free extracts of a NADPH,; and Ordependentt formation of r.-cotinine, since, in their study, no cotinine was formed from nicotine in the presence of dichlorophenolindophenol. Under the esperi- mentsl cmnditions devised by Decker and Sammeck, incuba- tfon of nicotine for I hour in the cell-free extract tesulted in approximately 88% ootmine and 19% nornioot,ine + norcoti- nine. FSndiugs with n><nicotine showed that the enzymatic degradation in the liver was not stereospecific. 51. Nature ojfhe Ddoxication Process (17) See S0, above, and 52, below, for pertinent material. S2. Nature of the Producta of 1lletabotiron (19) Papadopotdos and Kintzios (1983) studied the metabolism of nicotine in vitro using a rabbit-liver-superaatant with added TPNH (reduced triphosphopyridine nucleotide (TPN)l and nicotine, including randomly-labelled14C-uicotine. Two. directionaE chromatography and autoradiography revealed 8 major nicotine metabcfites, 4 of which had the same mobilities 'ss nornicotine, chtinine, demethylcotinine, and dyridylacetic- acid standards. Noraicotine, cotinine, and dennetbylcotinine were isolated and identi6ed by physical and chemical methods. One of the other metaboliteF w-as found to co-chromatograph with nicotine-1'-oxide; the remaining 3 metabolites were not identified. Subsequently, Papadopoulos (19ti4a) reported that incubation of nicotine with an enzytittatic preparation from rabbit liver (a 9099 X g supernatant fraction of rabbit-liver homogenate). with ]'PNH and phosphate buHe.r of pH 7.4, resulted in the formation of nicotine-l'-Wde, which was then isolated by chromatographic proceoutes, and,characterised by melting-point of the dipicrate salt and ultra-violet, and inira-red absorption spectra in comparison with, an authentic sample, as a•ell as by radioisotopic methods. Nornieotine and formaldehyde were also formed, and incubation of nornicotine with the same test-system gave rise to deiriethylcotinine duomatograme of Gver extracts following i.v. administration of nornicotine to the rabbit. A'1so following i,v. injection of nkotine in the intact animal, nornicotine, cotinine, demethyl• ootinine, and nicotine•1'-oride were demonstrnible in ehro- matoYrnms from liver, kidney, sind urine extracta Incubation of cotinine with the in-vitro test-system furnished no evidence of the foranation of demethylcotanine; and in-vivo administra• tion, followed by chromatographic analysis of 6ver and kidney tissue, showed only traces of demethyleotinine in the liver. 17te following sequence was therefore proposed for the rabbit: Nicotine -» noroieotine + foraialdehyde -» demetbylcotit»ne I cotinine These and earlier findings have been summtlrized by Papadopoulos (1965). In experiments on mice and rars injected with "C-label)ed nicotine, ohromatographic separation of urine tadioacti.-ity revealed at least 8 nicotine metabolites, two of which had the same Rr values on paper chromatograms as i;otsinine and hydroxycotinine (SchmiterlSw and Itsnsson, 1962; Bant;son and Schmiterlbw, 1982). Autoradiographic autalysia by Ilsh- tnan (1963) of chromatograms of the urine of doga injected with t4C-rtwodomly-labelled nicotine indicated the ptesence of at least 10 "C-contaioing compounds. The metabolism of nicotine was investigated by Hansson, Hofftnnnn and Scbmiterlbw (1904) in tissue-sii~res of various organs of the mouse, using "C-labelled nicotine and Qhin-leyer chromatography. Liver, kidney, and lung were found to metabolize nicotine, while brain, diaphragm. sple~rn, stomach, tmtaU intestine, a.ad adrenal glands did not. Metabolic prod- ucts ideritified were cotinine (Over, kidney, lung), y-(3-pyridyl)-y-oxo-h'-nuthylbutyramide (li.7ee, kidney), hydroxycotinine (liver), and "CO, (liver); of theioe, rotinine was by far the most important quantitatively. Tmvo unidenti- fied products, fonned in liver, w 3re separated cheomdt+,graph. ically. Evidence for formation of inethylamine by liver was sought, but this compound could not be unequivocally detected. these and oti er findings have been reviewed by Hanaaon and Schtniterl6w (1985). 1. Yamamoto (1955) noted that, under anamobic condi- tions, nicotirle was dehydrogenated by rabbit-biver extracts when a hydrogen acreptor (methylene blue) was present; and he considered the possibility that this dehydrugenatian may occur in the course of nicotelline forrnation: The lorntation of cotSnine, norcotinine, and dtitnicotine in cell-free ext racta of rabbit liver (Decker and Sammeck, i994a, b) ha.- been described ebove, So. Attention may be drawn here to the review by Pailer (1985; 84 references) of the chemistry of nicotine and related aika- lo6b, including biocymthetic aspects. q'e are indebted to Dr. Herbert AlcKennis, Jr., Professor of Pharmacology, Medical College of Virginia, for the arcount that follows in this section, based in large measure on research conducted in his laboratory: A continuous flow of experimental data from many 1ab- oratories has added much to both the quantitative and qualitative aspects of nicotine raetabolism; although murh has been discovered, much more, as evinced by the pteaenre of missing intermediatea in the metabolic srquetare, leading to 3-py ridylacetir tu•id and beyond, rrmairn to he detetminrd. Subsequent to the initial observations (JicKeunis et a)., Produced b1f The Council for o 0 02 o'~ o ,Tobacco Research-USA, Inc.

I ABSORPTION AND FATE 1960) on the presence of a"ketoamide" as a urinary metab- olite of nicotine, a revised structure, ry-(B-pyridyl)-y-oxo- N-metbyibutyrsmtde, was presented (Mehennis et at., 1982L) aMrd it was considered (111cKennis, 1985) that this amphoteric compound could exist in equilibrium with a ring etructute. I r---~ ta. ~ 11 The open-chain structure I appears to be consistent with many prdperties of the compound, including its ability to form an exime. Synthesis of the ketoamide was accomplished through the following sequence of reactiobs: The synthetic compound (I or II) from the reaction of methyl-7-(3-pyridyl)-y-oxobutyrate (ItI) with metbyl- amine (V) tvas obtained in crystalline form from benzene. Ali of the properties of the product, including melting point (118°-12fl°),and infrared absorption spectra, were in agree- mmt with the metabolic compound. Upon admixture with the metabolic compound, the melting point was undepressed. Although the reaction of I with hydroxylam.ine to form an oxime and the mode of synthesis suggest that the keto- amide caq exist in the open-chain form, there is a coneid- erable body, of evidence (McKennis et al., 1966) to suggrst the plausibility of the cyclic structure II,1-methyl-b-hydroxy- &(2•pr'idyl)-2-pytrolidLrone. The evidence to favor this atmcture includes: an infrared absorption peak which cor- responds to hydroxyl nbsorption, ultraviolet absorption spectrum consonant with the absence of unaaturation in conjugatioL with the pyridine ring, and an exchangeable IowBeld proton in thL N.M.R. spect.rum In addition to satisfying a number of chemical and physical criteria, the postulated equilibrium affords some assistance In an understanding of the possible metabo)ic intermediates in the oxidstion of nicotine (VI) and cotinine (VII) to 7- (3-pyridyl)-9-oRobutyric acid (1I1). 9 If the hydroxylatiod of cotinine in the foregoing sequence is ensymaticaUy dikerted on a stereochemical basis, the eqturjanum oetwc~n tue cycac suu upnu-ciu~et iurws uuay afford an explanatiop for the abwence of optical rotation in the metabolite as isolated. Concurrent with the studies on the structure and synthesis of the ketoaatide it was found that a dibromo compound VIII could be catalytically hydrogenated to obtain the keto- amide in good yield. The intermediate dibromo compound was assigned a pravisional structure: The ultraviolet absorption spectrum of the compound sug. gested an a,0-uns6tihrated carbony) system and 3 moles of hydrogen were reqluired to give 1-methyl-5-hydroxy.B-(3- pyridyl)-2-pyrrolidinone (11). VIII appeared to correspond in all respects to the dibromoticoniae of P'mner (1893) who had considered the eompour.d to be a dione rather than a hydroxyamide. The dibromo intermediate proved to be more convenient than q-(3-pyridyq--y-oxobutyrate for the syn- thesis of 1-methyl-6-hydroxy-5-(3-pyridyl)-2 pyrrolidinone for metabolie etudies. In addition to revision of the structure of dibromot3conine brought about as a resuit of this work, it is notable that dibromocotanine, a'o optically active product which secom. panies the apparently racemic dibromoticonine in the bromi- nat3on reactaon, has also been assigned (Dw'fteld et a1.,1985) a revised structure. Although revisions appear to satfsfy the physical criteria, an explanation of the mechanism for the formation of degradation prcducte which led to Pbmer's original structures for the tw,) compounds has apparently confounded the modern investgaton:. Craig and Roy (1966) have studied the optical rotatory dispersion of a number of alkaloids, including (-)-nicotine and (-)-cotinine. The data from these studies, and the earlier chemical reduction of (-)-cotinine to (-).nicotine were viewed to be consistent with assignment of an t.ron- figuration to both of these compounds. Morselli et al. (1966) have described the total synthesis of 'r-(8-pyridyl)-y-methylaminobutyric acid-y-t4C MII) by a route which pro.ides a wide variety of nicotine metabolites, as well the intermediates for labeled cot3nine-1tC (VII) and nicotine-14C (VI). (See the following page for diegram.J Although nr.nicotine-r'C with the trC-label in position 2 of the pynolidine ring can be more directly prepared (Decker, 1964), the foregoing is preferred for many purposes. ln addition to providing metabolite XII1, the intermediate 111. itself a nicotine metabolite (&hwarts and McKennin, 1982, 1983), it yields y-(8-p)-ridyl)-y-hydroxybut, dc acid and b-3- pyridyltetrahydrofutanone-2, metabolites of nicotine (Mc- Kennis et al.,196ib). (Administration of the eyntbetic tura- Produced by Ths Council for o 0 o mn ' :Tobacco Reseaxh-US4 Inr.

TOBACCO-EXPERIAIE1vTA1. A41) CLINICAL BTUL•Il;.~. SUPPLEMENT 1 ^ C C Y ,. ...a 18 + Q .........i.y r ..rdODCrt1Ia ° X', A Cl)<1 0 VII Vi VI KenniP et al. (1903a) described the to•al t+ynthes'is of h.•droay Tu1luUlr ruurl.tKluuul~ w Ii1C Wl•WUV1i~ y~uuw ~. XIII XIV Xv U rone (Bowman, unpubli.dted) to the rat leads to the urinary ezcretion of 3-pyridylaretat, and y-(3-p1•t•idyl)-y-h}•dto.Y- butyrate.l The reductive amination of the keto arid 111 hko- videe y-(3•p,•ridylyy-aminobutyrie arid and demc•thylcotinidte, a•hich VPada et al. (1961) described, as metabolites of nor- nicotine in the dog in anticipation of inetabolic work of othpn: (Decker and Sammeck, 1964a; Papadoluoulos 1965). The chemical relurtion of optically artive deniethylcotiaine to give both optically active nornirotine and raremie nomir- otine has rJso been dirruP*ed by Wacia and ro•a•orkcm (19U1), and Scbu-ariz and T1rl:enni.a (19ii4) have dewribrd the metabolinm of (-)-demethylrotinine in the rat. In the sequence of the reactions IX through VI ach•antaRe was taken of the commercial availability of nirotinir arid- rarboryl•14C. 8inre the Pame sequence may be conducted with ethyl sucrinate (X) labeled in any or all carbonc, a de facto sixri6r labeling of all positions of the pyrrolidine ring of nicotine or norniroture has been arrompliqhed. The qnthraa of nicotirz-V-nuthyl•1/(' (\1cKennir, et al., 1982a), which >,•as originaily studied with the rat (.11rKenni= et al.. 1961a; TLrnbull et al., 1961) has a)ready become the basis for most of the 14C•nirotine that ia commerrially available toda}•. .1lthuugh the eramination of the urine of many animals after tuiministration of nicotine and of human smokers (\Ir- l:ennis and Bowman, 1961; lBowman and 1fe>;ennis, 198'1; 1•leKennis et al., 1903s) leads to eti•idence for the formation of hydrovrotinine, no Fnbstantial eridenre hns "t been lm.ented for the rorr"ponding demethyl romlmnnd (firh- a•artz and IIcl:ennis, 1962; 'Me>;enni< et al., 19ats). 11c- In this Keries, the intermediate a~rtamidoroqinine (XVII) was hydrolh:ed to the intermediate aminorutinine (XVIII), which was diazotized directly and without isolation. The product, hydroxi•cotinine (XlX), was obtained a° a mixture of two nL-pairn The.v were deKitimatrd (+, -)-hvdroav- cotininc, ir•omer I and (+, -)-hydrox}•rotininc, Lamer II. 'Che first, or higher mritina form a•a- r.wdveri with the aid of tartartir acid to obtain a dextrorotatory forni which melterl at 135°-137°, in contract to the natural dextrorotatnr•r product, p•hirh me1tY at 110°-111°. When stJi'irient quantities of the second i-amerie pair beramc available a re,cJution with tartaric arid was conducted to obtai>t a en•r.thetir de.etrorotatory iq•droa;vrotininr, m.p. 110°-Ill°, ja)b'° + 47.3°. This product rorre~iwnded in all rtape+•ati to the meta• bnlir compound. If it is a-nmrrl that the cot»etsion of aminorotinine to hydrmw•otinine dtle~ not involve a hydrav}'I migration, the mMabolite and P~ynthrtir compound bear a hydrm;•I t;nnqp at a I>o-ition a to the rarhorr.l Rroup of thc ring. .Uthcatfdl the definite tmAtimr and al"lute Kterm- ehemical configuration of the hydroxyl group were not ob• tained, it a•a>; twx-ible to obtain rvidrnrc for retent il.n of the absolute eonfilntrrtion of the rrmninina a+ymmetric pyrmli• done carlwn atom (adjacrnt to tee pyridine ripa) by a tvro• atep c-xvluenre. Metabolic hydro.x,vrotininr wa4 ronvertai to chlororotinineM reartion with thiluiy1 rldorkle:9'he re+ultant ehlororotinine na° converted to rotinine by h,rdroltenolvw4 in the presence of tinlladium on charroal. After iwlation a% a pirtir acid nalt. thr re-ullant vntininP na- converted to a cn•stalline hyYlmbl, alide; m.i.. Produced by The Council for 0002922 Tobacco Kesearch•USA, Inc.

ABNOR.1'T)ON AND FATE ---I. `NJ " ~NJ CN3 L~NJ CM2 J CM3 (-1-NankeOne 4)-Niconno -(3-Pynidyt).Y.mtlhyiaininobulyroid.bydP (c)•j•(lI-Py~idyq-Y-ewilMylandnobuqrk exid 1 . CMO 1` +i M2 L N Y -(s-Pycidyl). Y-incb.yrotdehyd. Y-t3-Pyridyl )-~-anineiwyfic acid (-1-Demedrylwtinine 3-I9-Pytidyllroaoihdrdaramne-2 Y-(9-Pyridyl).Y-Aydriaybuyric acid / fio.1-1. Schematic representation of some routes in the mammalian metaboliem of ( --nicotine. (From AfeKeanis et a1.,198Qa, reproduced by permiesion of the copyright owners). N il rio COOM ~ °I ~°" CC/DN 4.f3-Pi.idyq-3•buNneic acid Y-(3-Pyrldyl)boryrit acid 3-Pyridyloc.Nc ocld The physical constants of this salt were in substantial attree- meat with hydrobromic acid salts of both synthetic and metabolic (-)-co'inine. Consideration of the resultx of a variety of mammalian experiments, both in vivo and in vitro, have led ~icKendis et al. (1984a) to summarize some of the known inar.malinn metabolic alterationa in the pyrrolidine ring of nicotine as ahoa•ri in Figure 1-1. In addition to the comlmunde of this simplified acheme, in which some of the hypothetical inter- mediates are shovrn in brackets, there remain to be considered a number of additional metabolic rout~ including •methyl- stion (1'urnbull et al., 1960) and amine oxide formation, as well as a variety of pomible conjugates which may be too labile to aurvive the isolation procedureaof many of the e~ry~rri• meata ;,icKennia et ai. (1861b) observed that following adminir- tmtion of nicotine-methyl•"C to the dog that e~entialiy all (95 per cent) of the radioactivity of the urine n as retained on Domex 80-H"'. An exhaustive elutioa vcith 4to ammonia removed 65 i~ of the radioactivity on the column, and the remaining fraction a•na removed by strong hydrorhloric acid. This behavior served to suggest that approdinately 30% of the tadioaefioity of the parent comlmund thit ap~ared in the urine a•as in the form of quaternary ammonium compounda. In additional subsequent experiments AfcKennG et al. (1963h) found that the combined 27-hour urine of tao anesthetized male monKrel dop that had recei•red a total of 50.5 ing of (-)- nicotine•methA•1cC intravenously over an &hour peyiod contained taro Padiosotive com}wnentQ in ihe, quaternar, anunonium fraction. The chromatoRraphie propertica of the radioactive material rumexted the presence of nirotine iwo- methonium ion (XX) and rotinine tnetbonium ion (XXI). 0 Y t].Pyridyl)•Y.owboryric acid Xa XXI H. In a eub,,equent ea•peiiment, in ahich a male rhonarel dog (Ib kg) was given a total of 12 g of (-)-cotiniirc (100 mg per kg dail,v), a total of 5.85 g of (-).cotinine methiodide a•aR obtained from the procencd urine. In a similar eieperiment a human male (non-smoker) nreived a total of 16.$ g of (-)- cotinine over a 6•dat• period. Urine collected during this period aas processed to obtain a total of 375 mg of cotinine methiodide. That the an•mnietrir carbon atom of cotinine ~~'t T~~ l~uncil for 0002923 ~ ~~t8 FzS`U'drcf1-USA, lnc,'

ir= 12 TOBACCO-FJtPER1MENTAL AND CLINICAL S'TUDIFS. SOh'PLEMENT I metbiodide corresponded in absolute connguretiun w wai, ui ( -)-cotinine, and therefore ( -)•nirotine, was ascertained by converting the cotinine methonium ion to cotiaite by treatment with llrr Hr9O, in a realed quarts tube at 2811°- 290° for 15 hourn. The identity of resultant (-)-cotiaLte, la)m-20.6° was confirmed by conversion to a picrate. Chemical confirmation of the presence of nicotine me- thonium ion as a urinary metabolite of nicotine in dogs waa achieved by processing urine from five dogs that had received a total of 647 tng of (-)-nicotine. The yield of (-)-nicotine methonium iodide hydriodide not corrected for mechanical losses was 29.2 mg. As is the case in many other arca9 of nicotine metabolism, the full quantitative investigation of the role of quaternary- ammonium-compound formation in nicotine metabolisttt remains to be accomplished. Ciuss and Barbiroli (1963) have iuvestigated the wsymatic activity of a cattle lens extraet and the blood of catxle. It was concluded from their experi- ments that nicotine, 3.pyridylacetic acid, pyridine, and other compounds can serve as methyl acceptors. The relative stability or the A'-metbyl groups once they have become attached to the pyridine ring has been the subject of a pre- limiaary investigation (rlcKennis et al., 1964c). In addition to nicotine-l-oxide (nicotine-py-oxide), which has been encountcred in mammalian metabolic experiments and is partially discussed in this section, it is attractive to consider nicotine-pr.oxide. N" -» O CH, CII, -.O niootane-py.oxide niootine•pr-sadde Although nicotine-pr-oxide has never been encountered in mamma:ian studies, the formation of many tertiary amine oxides is so readily accomplished that the pr-oxide is atttactive to consider as an intermediary metabolite. A possible practical consequence of the presence of nicotine-piroxide in tobacco as described by Frenkenburg has been discussed by Pailer (1t165). Craig et al. (1984) treated nirotine•pr.oxide at pH 6.3 wit•b ferric ion and tarhate (a mixture which should supply ferrous ion). A gas chromatographic examination of the mix- ttue which was previously kept at 80° for 40 minuW led the authors to suggest the presence of unreacted nicotine, 7V- methylmyosomine, nornicotine, nirotyrine, cotinine and nornicotine. Regrettably, chemical identi5cation or more thorough physical identification (other than thin.layer and Eta.a chromatography) was not acromplighed in tpost iostances, so that the reader might critically evaluate the authors aspirations to a"simple unified mechanism capable of rationalising. the formation of all known metahnlites of nicotine". It is obvioux, however, that this interesting model falls far short of including all metabolites, and .1'-methyl (qunternary atntaonium) compounds in particular. Additional factors remaining to be explored include the po.,~ible intermediary role of dil>_vdrometanicotine (De Clerrq, and Truhaut, 1962) in the production of 3-pyridylacetate tmd sul:Fequent products. Di)wdrometanicotine could presumably provide (AlcKennis et al., 1964a) a source of y-(3•pyridyl)but)•ric acid which would y ield 3-pyridylaretate via a R-oxidatioa. With one posaible exception little or no evidence has been achieved to support the po&ibility of a complete reversal ui ituNUU.wuu. .:..y...:~.:... ::r m..o a boc oiorfat The conversion of nicotine to nomicotine in the rabbit and the demoiostation in vitro that S-adenosylmethionine (Axel- rod, 1962) can supply niethyl groups for the formation of nicotine arith norNcotihe as substrate is currently the only supporting evidence of teversibility. Morselli et al. (1967) administered cotinineAG to rats and could find no evidence for either nl.'otine or nornicotine in the urine of the anitmlf. Similarly, Bowman (unpublished results) administered b-(3-pyrk:yl)teLtshydrohusuoone-2 to tsts, and employing gas chromatographic procedures (Afefiennis et al., )967) applicablb' to a variety of nicotine met'abolites, could find no evidence for reve+eJ to the stages of cotinine, or ti•(3• pyridyl)-ry-oxobutyrate: The possibility,that cotinine it~eif may be ensynmtieali)• Eij•drolitcd to y(3•pyridyl) 7 me th I• aminobuty rle acid has been suggested on theoretical grounds and by one preliminary report of exper6mentation (Hueker and (lilletle, 1860). 53. Rali of DdarfratROn (24) From their studies of the effects of acute heavy doses of niiotine on mother rats and neonates, R.,F. Becker and King (1066a) were led to rotttider that their data did not support the idea of an extremt+lg• rapid elimpnatuon of nicotine from tientes when it Is prei-ent in near-lethal amounts. 84. 1/No6odibm by 1licrodlrpaitiema (24) A strain of P&-udomoatoa bacteria, iitolat,ed by Toczko (1tt48) from roil; was found to be able to grow on medium containing nli•otiae as a sole source of nitrogen and carbon. During growth, the content of nicotine in the culture diminished rapidly, ta•ith rimultaneous apixaranee ot new pyridine com- pounds,10of which were separated b%• paper chrotnatc+graphy, and 2 of thesc were identified as Y•heto.ry-(3•p)ridyl)•butyric arid and nicutinic arid! Hochstein and Rittenberg (1959b, b) fou,hd that crude cell-free extracts prelo.rcd from a soil bacterium, capable of growing at the expense of nicotine as the sole source of carbon and aitrbgen, degraded nicotine through a series of changes, and that the first oxidative product of nicotine cottrsponded iu properties to 6•hydroxynicotine (28b) . The second oaida- th•e product was shown to be 6-fio•drox)hiaeudooxynicotiue (Iiorhstein and Rittenberg, 1060), and the product of the third oxidative step was isolated and identified as a dihydror.y11'.meth,vbn,vosmine, probably the 2,ti•dihydroxy- compound (u. H. Richardson and R,ittenberF, 11t60). A com- pbund formed by the oxidation of either nicotine, e•hydroxy npcotine, or 8•hydrox,vpsettdno*•nhuIine, with the consumption of 1.8, 1.0, or 0.5 mirromoh+ of oxygen per ttticromol of substrate, rexl>et•tively, was e)nthteised en- $j•tnaticrally, isolated in crystalWte form, and idMitified as 2,8 dihydroxy-A'-me111)hnyurmine on the basis of itv ele• metttal analysiH and of its trorrcrpomletar in n.,-tml charac- teristics of its Naltlfirredut•ed derivative to 2.t4dil%%•tidol (jth•liarcL<on and liittcnhetg, IO61a). Tlie ey,miwund waw ttnt further nietabolized, ahid nas considered to be a aide-lu+udurt of the main pathway, of ah•uthte depqbilutiun by the batr- tetiutn employed (dexiltnated as etrriiu Il•3-/ a Itmm•nettatirc rod (26b)). Sulnequchtly, 2.&dih;vdrax~ryux~udi~ux~nirotior was identified as an active intermcdiate In the aim•ctiiou of 8•hydro:-,%•l.ReudnoM,wicotinc to 2.6-dibydroxypNCtuhwmy- nirotine (Richardsoa and Rittenherpt, (Ob1b). In the prenrurc of the irropcr enzyme fraction, thiH intchnedinte u•tw further us1 Produced bv The Council for 0002924 Tobacco researclrUSA, Inc.

ABSORPTION AND F:N7E metabolized; otherwit;e, it was coriverted non-oxidatively and _ n r 1•r uvuv.u..~u...wwi.,1 w ...e ._r s. :y ......... ~,W .....~ droxy-N-met hylmyosmine. 2, 6-Dihydraeypseudooxyn ieotine was shown to be clear•ed anaerobically by an enzyme fraction of Arlhrobacter n.rydana, with the accumulation of a paoduct having absorption maxima at 234 and 320 nw; in the presence of oxygen, this compound slowly disappeared w•ith the p'arellel forrndtion of the nicotine blue pigment (Gherna, Richardson and Rittenberg, 1965). 11'hole-cell and enzymatic metaholism of 6-hydroxypseudooxynicotine aL~o gave rise to the 234- and 32/-tnµ compound, which was crydtalliz'eI from an enzymatic reaction mixture and identified es 2.6-dihydroxypyl-idine. ry..ltethylxmriuobutyric acid, demonstmted chrontatoGt•aph- ically, was also shown to accumulate during the enzymatic rcacti'Tn; both it and 2,6-dihydroxypyridine were n~iclir.ed by cells and ext racts of A. ozydans grown on nieotine, but not of organisms grown on nutrient agat• Jitaleamic acid was also oxidized by extracts of cells grown on nicotine. These data were said to coafirm the identification of 2,6-dihydroxypseu- dooxynicotine and to have established additional intcrniedi- ates iir the pathway of nicotine degradation by the micro- otganusm, and to wdia+te the metabolic precursor of the nicotine blue pigment. A schematic diagram of the proposed pathway is inc•hided in the origin4l paper. Eberwein, Cries and Decker (1961) described the isolation of a soil micro-organism, ArWolracler ozydans, which, in the presence of trace-e)ements, can utilize 1-nicotiue as its sole source of carbon and nitrogen for growth. When grown on nicotine, this organism produces a typical extrracYllular blue pigment (conditions for growth and pigment productiqn are given in the original article). These authors supplied pre• liminary information on the type ot breakdown reactionr; and Decker, Gries and Brilhmttller (1~'i1) described a soluble (cell-free) enzyme extract from the san:e micro-organism which breaks dotrn nicotine in three oxidative ste;is. Decker, Eberwein, Gries and BrUhmUller (1960, showed that the degradation of (`-nicotine by the eell-frtee system frofn .1. otydana was aci»mplished hy the introduction of a hydroxyl group in the 6-livsition of the pyridino ring, opening of the pyrrolidine ring, and successive dehydrogeuation of the rksult- aat side-chain; the proposed stepy are schematic•ally given. The product of the first oxidation, for which artificial electron acceptors (such as 2,6-dichlorophenolindophenol, methylene blue, or ferricyanide plus catalytic amounts of quinonc) are emntial, is l-6-)iydroxynicotine (I'tocbstein and Rittenberg, 1959; Decker et al., 1960). This reaction tras studied by Decker and Cries (1961) using a partially purifizd eniyme, and was found non-stereospecific and ind:pendent of rnoleo- ular oxygen. Anabasine, nornicotine, meth3lmyosmine, but not nicotinic acid or pyvidiae, were hydroxylated in the 6-position by the same enzyme fraction. Further oxidation of 6hydroxynicotine was accompanied by an uptake of ivater, and cleavage of the pyrrolidine ring, yielding 3-(6-hydroxy- pyridyl)-y-N-methylnminopropylhetone. Contrary to the hydroxylation step, synthetic dyes were inhibitory to this teaction, oxypen being the onh• electron acceptor found active. Also, d-frh,rdroxynicotine was a potent inhibitor to the oxidation of the /-isomer. In the presence of one of the above- mentioned oxidants, the breakdown of nicotine ended with the formation of the compound, CtoHt:O:Nr (Decker et al., 191k1); a•hile incubation of the ketone with the cell-free extraet alune led to the fvrmation cd thoee deep blue pigments which are accumulated to a considerable extent during nicotine oxidation by intact bacteria. Decker, L•-bera•ein, Gries and 13 Briibm'.6ller (1961) reported again that cell-free extracts of .y......: .....,,.....o.• .::.:..........:.:.: :.... b .y......y' nicotine; and that, in the presence of ~arti6cial elect6on ac- ceptort, the same enzyme (which had been considerably purified) bydroxylated d-nicotine too; and thst, after bio- logical,separation of the enantiomorplw, 1-nkvtine yielded exclusicely 1-6•hydroxynicotine. An oxidati.•e re,aetion formed 3-(6-hy,droxypyridyl)-y-N-methylaminopropyl•lketone from 1-bydruxynieotine. This reaction was stercosiiec•iGi•, and d-hydroxyvicotine a strong inhibitor of it. ']'hc~e writers concluded that f-hyJroxynirotine fulfills the requirements for being a physiological intermedir.te in the !-nicotine degradation by this micro-organism. Grift, Decker and Briihmnller (1961) found that (ti-mcthylantiinoprop.l)-~6 hydrmypyridyl-('4),1.kPtnne waQ 1,rxinred in the -eidst6an of 1-6-hydroxynicotine by celldrec extracts of .1. yzydans. IThis substance had earlier been referred to as 6-b-vah»xr.3.(7. met.bylaminobutyryl)-pyridine (Decker et al., 1f1601, and as 6-hydroxypseudo-oxymicotine (Hochstein anc~ itittenberg, 1960).1 The mechanism of the oxidation of 1-6-bydroxynic•otine was discussed by the writers (Decker el al., 1961; Gries, Decker and Brilhm(lUer,1961), and the structure of a liost=ible enzyme-bound intermediate was suggested Decker and BrSlmtUller, 1961). Dehydrogcnation of the iuterauediate, y-methybmunopropyl-(6•hydroxypyridyl-(3)j-keiurte led to the formation of a labile compound, the structure of which could be elucidated'wdinrtly as being likely to lx identical to (y - methylaminopropeny1) - (6 - hydroxypy-ridyl - (3)J - ketone (Gries, Decker, Eberwein and 13ri11mtitller, 1rJ61). lt was found that this compound could be metaboli:cd in two ways: (1) if enzyTnstic degradation is blocked, a non-enzyntatic reaction led to compound V [see Decker et al. (1966)1; (2) enzymatic degradation led to the formation of the blue bacterial pigments. The reaction depended on tnolecular oxygen, which was catalyzed by a soluble systent, which in turn wa~ activated by heavy metals, particularly by (errous ions, and inhibited by the addition of cysteble, lwtaxittnt cyanide, and high concentration.< of redox dyes. DurinR the progress of the reaction, various intermediater could be demonstmted by their absorption spectra (Greis et aL, 19fi1; Decker, rreisK and Brillunilller, 1961). Decker and Bleeg (1965) reported that :Irlhrobartcr orydaas can be adapted to grow on n-nicotine as well as i.-nic•otine as its sole supply of carbon and nitroFpn, alt' ough t;rotvth is slower on the D-stereomen The eaz,rmes for the first two oxidath•e steps of nicotine degradation n-cre indwrd by the alka)oid and repressed by the simultaneous prrtxnce of a carbon (glucosc) and a nitrogen (ammonia) source. :1. oxydans grown on t.nicotine was found to synthesizo a non-atereo- specific nicotine oxidase and a highly stetroamciGe, t.-6- hydroxyuicoline o•ygeuasc, while u- and Dt.-uicotine also induced a n-specific oxygenam. lieotine oxidase, t,- and n•h,rdramyroirotine ox+ytena.cesa and ket-me oxidafe were reported to have been partially purified and separated on Seplucdex G•100 and DEAE-Sephadex. tr and tr6-hydroxy nicotine oxl•genarr were said to differ in moleculnr size, and both enrymes were inhibited competitit•ely by the 6-hydro)j.-- nicotine antipode which is nut a substrate. The Krn and lt;i values were found to be very timilar. G. D. Gritfith, llyerrum and Wood (1901) isolnted a gtum- negative, non-ntotile bacterium from tobacca-plant roots, which was identifitd as a member of the genus Artdrobortn. This organistn pcsatssex the ability to convert nicotine to 6-hydroxytiuc•otine; the maxhuwn concentration of 6-hydroxy. Produced by The Council for Tobacco researclj.JSA, Inc. 0002925

14 7!'OBACCO-EXPERIh1E\1TAL Ai\D CLIyICAL8TCIDIE.S. SUPPLEAfE%T I nicotine detectable in the culture-medium w•as appro9timately UU'.,, on a Lwlar uaisle, 01 artuai uMUUUr cuw'CaUaIiVY. To produce larger amounts of degradation 1?ralucts, liuffnrr, l:laushofer and, Kirchenmayer (1963) adapted their micro•organism to higher nicotine concentrations thiZn those used in earlier st udies (4 clc, compared to 0.1-1 cI ), , without thereby forming different produrts. licsideg nieotinoylpro- pionic acid and pseudo-ox)nicotine, the micro-cirganism produced a number of other substances containing the intact pytidine ring, among which these workers identified nicoty- ritie. After describing cultural characteristics of 6 bacterial isolates capable of rapid nicotine degradation and having the ability to use nicotine as qhesole source of carbon and ititrogen (Il,inz, 190.i), Ilins and Westenlu,efer (I9G6) compared their behavior with that of isolates furnished by other investigators, and these workers also described improved envirottmental conditions under which nirotine-degrading bacterial isolates (which did not form blue pigment) tolerated and metabolized more nicotine. 2,6•Dih,vdroeip)ridine was found to be osidized readily, while 2-h,rdrox)-pyridine was not; and the noh-pigment•forming isolates oxidized 6-hydroxynicotine at about the same rate as nicotinic acid. These findings were said to support those of Frankenburg and Vaitekunac I(1Q55b)(25, lFig. 1-2; 76a)1 and E. Wada 1(1957)(25a-b)1, who stated that hydroxylation of the pyridine ring at the 6-carbon position is involved in the metabolism of nicotine and nicotinic acid by nilwtine-drgrading bacteria. Schievelbein (1962) premnted a review of the newer litera- ture (mostly over the preceding 10 years, but not limited to these) covering nicotine deto.ocation by micro-organi:ms. The mi^robiologit•al degradation of nicotine was also reviewed by I:m'lne4 Klaushofer and Kircheninayer (1963); and it is again pertinent to mentitrn the review by Pailer (1965) on the chemistry of nicotine and related alkaloids, which includes biosynthetic aspects. 55. dfetobolism qf Nieotine by Inserts Metabolism of nicotine, by tobacco-feeding insects has been investigated by Self, Guthrie ar.,i Hodgsnn (1964). Actively feeding, immature stages of the tobacco budworm (fjeliotNia oirescena 1•'.), the cr.bba(ze looper (TricAoptusfo n4 Hubner), the tobacroreireaorm (Conodcrus trspertinus F.), and nymphs of the qrasshopper (3lefenophta diFrmntiolis Thom), arre collected on 8ue•cured tobacco (.\'icrtiana tabacum L.); broitght Into the laboratory; anti fed on alternate host plants for 2 days to prevent intehference of ingested alkaloids in later chemical analyeis. Larvae were then treated with 200 µg of nicotine free.base on the dorsum of the thorax, and killed for analyses after 10 hours. Similar e\heriments trere conducted atith cignrette-beet le larvae (La+si6drrnm serrirorne F.), which were treated with 10 pg nicotine, and with 2-day-old adult htntcc-8ie+, which welr 1'reatcd with fi pg nirotinP and hekl for 18 hours. The inserts and their fem were r•eparated, and aieotine and related melabolitcw were chromatoRral}hed on palmr, eluted, and subjerted to ultra•.iolet simctroscop.•. No alkaloids were apparent in untreated insects subjected to identical conditions. Results with bude•orons and cabbage IWtlk1. wuNpa'u uV r~~~•IMlY1 ,,,lau~~i.S... a nn, u.laN• tionalh•, when fed on tobacro, the four alkalnida apparent in the feces correFponded chromatopaphicadl,r and tmtr,ro- scopirally to the alkaloids in the tobaccw pklnt. The tobacco. trireerornr, cigarette-heetle, grasshopper, and house8c metab• olized nicotine to 1, 2, 4, and 3 other alkaloids, respectively; the principal one roreswnded to cofinine, and the other, were not idendified. 56. Entymalie Fonnafion of lCirotine Axelrod (1f10?e) reported that incubation of the soluble supernatant fcaction of rabbit lung with S-adcnoPylmethio. nine•methyl!cC and nornicotine res-ulted in the formation of meth)•1•1cC•nirotine. This enzyme i~olstcd from rabbit lung can 1'-metbylatte a variety of norntall.•-occurring cotipounds, as well as forrign rompoundr; takinR the rehative agtn•itY in methylating r•crotonin at 100, the activity in methylating nornirotiue falls at 22, and nicotine falU at zero (Axclrod, 1962b). [1)etails of the methodology are (t6ven by Axelrod (1962a).) 57. TetocrANATE (33i') 58. Egert of Smoking on TGioryanote (33i ) Serum-thiocyanate levels in 52 non-sniokers avet•aged 0.0797 mg r'e (range, 0.044-0.144), while the levels in I I smokers of 5-20 cigarettes daily racgcd from 0.235 to 0.490 mg 14 (D. Reitnrein and Liebermeister, 1961). 59. Solirary Excretion of T6iorg•anele (337) Vas'ko (1927, 1t129) found the pota*ium-4hioryatRate con- tent of the saliva of wnokera to be 2-3 timea higher than that of non•smokers. 60. t'rinary ):1crMion of TAior-yanotT (33i ) According to anahxes reported kv Djuric, Rairevir and Konstantinovir (1962a, b), the urine of non-smokers ron- tuined thiocyanate only sporadically and in trace aniounts, while the total 24-hour urinary cscretiont of 5 smokers of 20-M cigarettes a day varied between 21.5 and 3R.7 mg/bter; the 24-hour urine was cnllerted in S frartiona, each of ahich .%•a~ anahzed separately for thior)•at:ate, mnd analysis of variance>;how•cd that there p•ac no quantitative difference in the thiocyanate ronecntrations in the variouF fractibna The brand of cigarettes stnoked had considerable influence on the quantity of thwoayanate in the urine; with thc snme iuand of cigarette% urinary thioc}anate ronrctitratikm was propor- tional to the number of riqnrettef s,-moked. 61. Excretion of Tbiorganatr in qurdl (337) 62. ,Uechanisai of thc Ejlert of SraoAing on Thioryonutt (d.Y7) 63. Rclafion of Thioryanafe fo Dfarare (33R) I\a new data.j I Produced by The COuncil for Tobacco Research-USA, Inc. 0 0 0 2 92.6

I 0 v SPECIAL SENSES 1 64. VISION (7.') G5. Visual A cuity (27) Writing frorpi his own clinical experience, Mclanou-ski (1963) dlscribPd heavy cigarette-smokers and moderate drinkers, who, during periods of work in which they did not size of visual fields expanded 18-857o, the mean increase being 385~ (D. Af. Johnston, 1965). In 1 non-smoker who vol- nntecred to Fmoke (nithout inhaling) about a f-sck of ciFn- rettes a day for 2 weeks, the^ field decrer„rd 2i!',. Visual 6eldsof 2 non-smokersand 2 smokers who continued to tmoke remained relatively constant during this period. (It appeared to Johnston from these results that, during visual•ficld evarni- nationa, consideration should be given to the recency of smok- ing, which may account for changes in field-size of a paticnt on succes.cive examinations.) 69. Fusion Frequencg of Flicker (28) Simoneuin (1959) presented a general review of fusion frequency of flicker (FFF) as a criterion of central nervous system fadigue, during which lte mentioned the stimulating effect of srywking a cigarette on FFF. Contrary to the findiner of many of the preceding authors (26), Iiinonen, l'aarilainen and Peltonen (1961b) generally observed a loner FFF on smoking. An a series of 20 healthy conscripts, the nreasure- ment was made only once before, and once after, smoking 2-3 cigarei,tes, when a lowered FFF value was notirnd in 14, no change in 3, and an increased value in 3. 1i7 20 other subjects, the detcrmination was made before, and at 5-min intervals after, smoking I cigarette, anu' all showM a lower value after smoking. The mean nuudmum change of 4.2% appeared, on the average, 18 ntin (range, 8-36) fronr the start of smoking, and the control value was attained in 70 min (range, 38-106). Four suhject.a smoked a second eiga- rette at tlte stage when the effect of the first one began to diminish; lowered FFF values again resulted. Critical flicker fusion (CFF) thresholds were determined by f;VR,rnick and Ey:venck (1963) in groulwr of snrcd;err and non-s~nokers when nicotine was administered (a) through smoking a, cigarette, and (b) orally, 0.1 mg niiotin;e ah.orbed through the buccal membrane. (Plarebos were also used.) CFF thres'hold was raised after the administration of nhotine orally, and also after smoking I cigarette; however, there was no eharge after smoking for non-smokers 1ltreksumabl-v, because they failed to inhn/el, or for maokers ahu had not abst,ained )xforehand. This experiment was taken to support the hypotheitiF that nicotine is a stimulant drug (see also 1;yseak, 19ti5a, pp. 95-96). 70. Pupillary Changca FolfonE.yl A'irotinr (28) 71. Ilird (28). Nicotine in concentration of 10 µg/m) bath- fluid had no effect on ciliary nruscln from the chicken, whether it was dissected in an anteroposterior direction or in a frontal plane (Van Alphen et a1., 1982). \icotine also had no effect on choroidal st rips. 72. Frog (29). Xirotine had no effect on ciliarymusrle strips from the frog (Van Alphen et sl., 1982). fipplication lfi Produced W Tho Council for o 0 0 2 a z'. Tobacco Research-USA, Inc. eat, but smoked about 50 cigarettes, discovered almost suddenly that their vision was considerably defective; the acuity of vision was decreased to 0.1 and, frequently, even more. See also VisuPll Ficlds, below, 68. 66. Senaiti,ritp fo Lfght (27) Photuphobia,, according to Melanowski (1953), is one of the signs of "tobacco-alcohol amblyopia". 67. Dark Adaptation R. A. 'tlcFatkurd (1953; cited by N. Williams, 1964) is authority for the statement that relatively low blood carbon- nronoxide levels, such as those produced by smoking 3 ciga, rettes, result in a significant deterioration of night vision. Troemel, Davis and Hendley (1951) studied the effects of cigarette-s»toking and caffeine on dark adaptation in 9 sub- jects, all smokers; and they found that the course of dark adaptation seemed much faster under the conditions of smoking 1 inch of a standard-brand cigarette in 2 min (or 1 inch of 2 cigarettes smoked consecutively in 2 min each;, while caffeine cii;rate in light (3 gr) dosago at:emed to counter- act this effect. A larger (6 gr) dose of caffeine paradotically did not counteract this effect. Administration of caffeine alone had no effect on the course of dark adaptation. lmprove- ment of scoptir, visual adaptation was observed by I3ohnd (19112) with the aid of a Comberg nyctometer in 9 of 12 subjects after eigarette-sntoking, which led this worker to regard as quite, possible a genuine transitory improvement in dark adaptation in drivers of motor vehicles after smoking. Experiments by C. Johansson and Jansson (1965) led these workers to conclude that the effect of tobacco-smolcing on the ability to detect objects on the road is negligible from a practicnl point of view; the experiments were performed with 30 subjects aged '2"9 years, all cigarette-.mokers, under sitnulated night-driving conditions, to study the effect of smoking 2 standard cigaretais during 15 min on detection- time and redetectinn.time after glare, but no significant differences in eye action were found between the smoking sessions and control sessiom without smoking. 68. Scotonut (27); Visual Fielda In 11 subjects, examination of the peripheral visual field with a Goldmaun peritneter revealed no detectable changes after ciFarette-smoking (liohm?-, 1962). lu 4 habitual smokers who reduced their smoking, or abstained, for 2.eeeke, the

16 TOBACCO-EIiPERIAfENTAL AND CLINICAL STUDIPS. SUPPLEBfE1T I of Jo gg/ml mcotme tartrate pruuuuxi ,cu rutX' 0" :ur Z~ww• eter of the pupil of the isolated frog-(Rana nigrmnaculata nipronmcuWfa H.) eye (Deguehi et al., 1963). 73. RepHle,(29). Nicotine had no effect on ciliary-muscle strips from the aiiigator (Van Alphen et al., 1962). 74. Rat (29). [No new data.) 75. Guinea Pig (29). [No new dataJ 76. Rabbit (29). On ciliary-muscle strips from the rabbit, whether they were dissected in an anteroposterior direction or in a frontal plane, an effect from 10 µg nicotine per ml bath-9uid was either absent or so slight at to be quewtionable (Van Alphen at aL, 1962). 77. Cat (30). According to Van Alp)oen and co-erorkers (1962), nicotine frequently had no effect on cat longitudinal eiliary-muscle strips; however, in severRl experiments, 1 or 10 mg/mi nicotine produced a significant contraction, indic- ative of ganglionic stimulation; and this contraction was relaxed by hexamPthonium. Frequently, the contraction was iauaediately followed by relaxation. Schaeppi and Koepa (1964b) investigated isometric tension changes of radially oriented iris strips of the cat in vitro using a 90-ml bath continuously irrigated with fresh Tyrode solution at a rate of 20 ml/min. Nicotine (100 mg) in normal dUators yielded contractions of as much as 80 mg, but the same dose admin- istered within 30 min was ineffective. Nicotine did not produce contraction of chronically sympathetically-denervated mus- des. In cats,intracaratid injectiwt of 75 pg/kg nicotine caused ipsilateral mydriesis for 2 seconds, and 2 mg,/kg intravenously caused bilateral niydriasis for 20 min by paralysis of the ciliary rtang)ion (R. A. At w-mm-s and Long, 1961). 78. Dog (30). [No new data.) 79.Oth.er 11famiuale (Calf; Pig; Alonkey). On isolated sphincter-iris preparations of calf's and pig's eyes, nicotine bitartrate in minimum effective concentration (10-" mol/ Ilter) caused relaxation of the sphincter; with higher con- centrations, an increase in tonus frequently preceded the relaxation (Buteaandt, 1960). Neither dihydroergotataine nor hesametboniui- had any effect on this action of nicotine, leading to the conclusion that nicotine has a direct actio8 on this gtructute uruder these ea•perirnental conditions. On isolated iris-sphincter ringa from adult pigs, nicotine caused contraction, the threshold dose varying from 1 to 3 mg per 40-nil bath, while 100 mg elicited pronounced sphincter contraction (Scbaeppi and Koella, 1964a). The effect of nicotine decreased following previous administration of tbe drug. Oo preparations pre t-eatarl with either ?5 mg hoxamethonium ofi 10 µg atropine, nicotine was ine6ective. The authors concluded that nicotine t•eleases tuetylcholine from the nerve terminal of the neuro-effector junction. Subsequent experiments led Scltaeppi and Dodd (1966) to conclude that nicotine elicits contraction of the isolated pig- irie sphincter via a cholinergic mechanism. Nicotine produced contractions in narmal sphincters, but was ineffective in chronically parae)mpathetically-deaen•ated preparatires. In normal apbincters, the threshold dose varied between 0.3 and 3 mg/40-ml bath•volume; the contractions were always of shorter dttration than those elicited by acetyleholine. With repeated application of nicotine, at intervals of as long as I ~ _ `! ` .i.....r.n,-..,i Gn wmnlN,rrln rwrlirorkrlr• when large~ (30-100 mg) doses were administered. Nicotine- induced contractions were enhanced after pretreatment with neostigmine (1 mg) at diisopropylfluorophosphate (DFP[ (25 mg), and were abolished after pretreatment with atro. pine (0.01 mg)' or heramethonium (25 mg). Electricalh•- induced sphincter contractions were not changed by single or repeateb administration of nicotine in doses as high as 10 mg; 100 mg alight!y reduced the effect of electrical stimu- Iation. Contractions wete induced in isolated-iris sphincters from young pigd (" lb) by 50 mg nicotine (added to a 40-ml bath continuously irrigated with fresh Tyrode solution at a rate of 20 ail/min); but the drug had no effect on sphine• tPra frnm e%rc at snimalc mtbiectPd 2 weeks previously to unilateral ciGar) ginglionectomy (Srhaeppi, 1968). Nicotine in coacentretion of 10 yg/ml bath-fluid occasion. ally produced a'slight contraction of both longitudinal and circular cillary-nwsc)e strips from the monkey (Van Alphen et at., 1962). , 80. Dlan (30). [:Co new data.) 81. E,(led of New Stiaiulatioa .q/fer A'uotine (31) [No new data.) 92. Effect of New Setliora or Ganglion Esliriwtian (31) Cervical ganglione.ctomy did not significantly affect nico- tine mydriasis in rats (R. A. Atkinson and Lang, 1964). Combined ciliary and cervical ganglionectomy did not inhibit nicotine mydriesia although, in this situation, hexatne• thonium blocked it. (The authors noted that superioncervi• cal gaogliodiectom)• may out effect complete sympathetic de• nervation of the cat eye.) I4t`^otine did not produce contraction of chronically s}as- pathetically-denen•ated isolated iris of cats (Schaeppi and Koella, 1964b) or in chronically parasympathetically-dener- vated iEolated-iris of pigs (Scbaeppi and Dodd, 1965) or in isolated-iris sphincters from pigs previously subjected to unilateral iiliary gangIIonectomy (Schaeppi, 1966) [see above, 77 &,d 79). 83. Bffect of AdrenoleNn»,g (31) [No new dataJ 81. h4Ruence of Other Drugs (32) Nicotine m)•driagis in the cat was inhibitcd by various or-r+eceptor blocking agents: guanethidine, reserpine, Gducaine, tetrarnethy-larnmonium, hexamethonium, and hemicholinium, but was upaffected by atropine and neostigmine (R. A. At- kinson and L.ong,1g64). Nicotine in coneentrations of I or 10 Ng/ml bsth•9uid frequcrtly pr«lmrd a slight contredion of eRerinired cir- cular ciliary-muscle strips of the cat, and this was relaxed by atropine (Van Alphen et al., 1962). When nicotine produced a sustained contraction of longitudinal c'diarymu-ccle strips, he-mmethonium resulted in relaxation. 85. Uechaniam of the Pupiflarg EBerta of A'ieWine (33) R. A. Atkineon and Long (1064) concluded from their experiments (described above, 77, 79, gs) that mydriaeia from intrdcarotid in;ection of nicotine depends on trleaFe of catecbolarnines from s)mpathetic nerve•terminate in the iris. An underlying cholinergic mechanLKm did not appear to them duthors to be of importance, [althouRh Schaeppi Produced W The Council for 0 0 009.9IS Tobacco Research-USA, Inc.

L SPECIAL SE:LSF•B 79) that nicotine releases acetylcholine~from the nerve ter- minal of the neuro-effector junction). Ability of ganglionic blocking drugs to itthibit nicotine mydriasis after removal of known autonomic gGnglia suggested the presence of ram- ifying sympathetic ganglia beyond the superior cervical ganglion; and nicotine could produce mydriasis by stimu- Iation of these ganglia (see Butenandt (1900), above, 791. 86. E,ffut of Nicotine on the Ligh1 ReJtez (33) J\onert data.J 87. Retina (34) cc.e 1Fikller-Limmroth and Hieron.unuA (1962), below, 89. 88. Retinal Pignunl (34) iNo nPSt• data.) 89. Retinal Adion-Potenlials (34) \IiNler-lammrotb aqd Hieronymus (1962) studied the efiect of intramuscularly injected nicotine on the electro- retinogram (ERG) of u~anesthetized guinea pigs, and found a very interesting biphasic response. Small (1 mg/kg) doses resulted in an increase in the amplitude of the b•- and b-•ccaves; after 2 mg/kg, the ERG remained the same in size and form es before the injection; while 5 and 10 mg/kg caused a significant dinninution in the activity of the retina: the ERG .'attained with 10 mg/kg nicotine was scartely halF the height of the control ERG. The spectral sensitivity following 2, 5, and 10 mg/kg nicotine changed, the ERG being mort markedly reduced with a green-blue light stimulus of about 10-° meter wavelength. These workers also deter- mined the effect on the ERG in 5 human subjects on smoking 2 cigarctths. The results were not uniform, both increases and decreases in the ERG resulting; however, with green. blue light stimulus, a decrease in the spectral sensitivity occurred. 9D. Blood Vofu»ie of Chorofd and hdfno (34) Fellows and Hettman (1960) called attention to some of the difficulties in the experimental study of the intra-ocular bkad-volume; discussed their own earlier experiments (llettman gnd Fellows (1950)(34b)}; and remarked that the efiert of cigarette-smoking on the intra-ocular blood-volume of hunwns is not known. However, For what is known about the effect of nicotine and dgarette•smoking su retinal cir culation in man, see below, 480-C. 91. fliatopathofogg oj the Rdino, Optie Neroe, Curnea, Lens (34) In a study by anguera and Schwartz (1959) of $08 normal subjects, no influence of smoking on the incidence of ares- cular lesions in the fundus oculi was observed. Caffi and Paganoni (1900) made a histologic study of the optic nerves of rabbits given daily subcutaneous injections of 10 mg/kg nicotine sulfate for 80 days, and also of animals given 20-30 ml alcohol orally, and of animals treated simul- taneously with nicotine and alcohol. In all three groups, small, variously shaped zones, iatensely colored black by osmic acid, were demonstrable along the course of the optic. nerve fibers; this stainiag was said to represent products of disintegration of the myelinic sheath. In rabbits simulta. neously given intramuscular injections of 100 pg per day of vitamin B„ or one of its analogues, Cs, there was said to 17 ,:. .,.,.., a. . t.. ,». s .r . a r .a. t . having a therapeutic^action analagous to that of vitajnin 8,:, but di6ering from the group of cobalt amines to which Ba belongs by not giving rise to cyanocobalamine folloa•ing treatment with hydrogen ryanide.) The authors noted that cobalt accumulates, especially in the optic nerve and in the trigeminus, as demonstrated by Zim and I'ichler (cited, by Pichler, LYnen. IUin. 11'schr. 87:543, 1955) with radio-active cobaltocbloropbyll in the nervous system of the rabbit. Komcz}•nski (1958) noted that,,in white mice exposed to either light or heavy concentratiqns of cigarette-smoke for 10 min daily for periods up to 703 days, exophthalmia de- veloped in a large number of animals, the eyeball becoming opaque and whitish, and the mouse becoming blind. Histo- pathologicd.l etamination of the eyeballs nlwwed f,wal hynliuir degeneration of the sclera; changes in the blood cessels, with occasional degenerative changes of a fibrillar charagter in the walls, and proliferation of endothelial cells; degeneration of the spiral nerve cells of the retina; areas of focal h)per- trophy in Qhe cornea; irregttlar thickening of the lens; de- getremtive fibrillar changes in the blood vessels in the tunica propria. Petrova (1963) reported that rats exposed to methyl- alrnhol and nicotine vapors developed blepharoconjunctivitis and corneal opacification; for an account of these experiments, see below, 1083. 92. ACCOffireDATdON (35) (No new data.) 93. r.RfltiNstc Mnap.Ea oF THE Et F: (38) Exophthrilmia developed in a large number of white mice chronically,exposed to cigarette-sntoke (Komcs)nski, 195f3); see above, 91. In a study of effects of cholinotnimetics on cat superior- oblique muscle, sustained shortening was produced by intra-arteri0illy- and intravenously-administered nicotine (Sanghvi and Smith, 1966). 94. NtQr1TATlNO MEMnRANE (3g) J.11'. Thompfon (195g) has desr.rr,ibed a meth nd whereb,v an isolated preparation of the cat's nictitating membrane is obtained; this isolated muscle was contracted by nicotine, which the author e.y'ained by stimulation of fine postgan- glionic nerve fibers which lie among the smooth muscle, iand innervrete it. Large (0.1 or 0.2 mg) doses of nicotine injecaed into the lingual artery, and thus to the superior cervical ganglion, produred a transient contraction of the nictitating membrane of anesthetized cats, which was followed by complete block of responses to preganglionic nerve stimu- lation ('tfurayama and L'nna, 1963). Other workers have reported that lingual-artery ir'jec;tion of nicotine causrd strong contraction of the nictitating membrane (Ileraldo and Zanotto, 1980); but there may be no discrepancy here, since even a"transient" contraction may also be "strong" On the other hand, injection of nicotine into the diencephalbn, masencephalon, or medulla oblongits had no effect on con- tract3ons of the nictitating membrane; although inhibition of central autonomic reactions evoked by electrical atimu- lation did result (A. Schmitt and Hd1Ane Schmitt, 190,3). Intravenous injection of nicotine also inhibited the effects of electrical stimuli applied to the above structures. (For detaib of the Sdwitts' expedments, see below, 154.) In the spinal cat, a dose-response curve for responses of Produced bv The Gocincil for Tobacco Research-USA, Inc. 0002929 R.

16 TOBACCO-EXPElilrfE.\TAL Ati1) CLIICICAL :TUll)Et;. FUPPLE.1fE'.~T 1 the nictitating membrane caused uy uuau..: ..::; '^'^••- tions to the non-atrol»nized superior cervical ganglion of increasing amounts of nicotine was exhibited (A. Jones, 19B3). According to Armitage and Milton (1965), the mean threshold dose of nicotine for contraction of the nictitating membrane was 0.022 mq/kg f 0.005 (S.E.) in 20 exper)- meats; the observations were made on spinal eviscerated cats af either sex, the right superior cervical ganglion having been removed during an aseptic opcration 7-19 days pre- viously, and injectionsand infuwions were made into a femoral vein. In contrast to the effect of nicotine rapidly injected, a dose of 400 pg/rat could be given by alow• intravenous infusion over a 10-min period without contraction of the ntemin-nov; at,d a dose of 800 ag/cat, in an ec9m)ile ahow•n, csused only a small contraction. Results of this t)qreof study indicated that, given as a slow infuFion over a 10-min period, 10 to 20 timeq the amount of nicotine was required to cause a contrartion as w•hen given by rapid inject ion. Iloth arnmitage (1t')65) and Milton (1966) have separately published results of experiments on the nictitating membrane of chloralosed cats. In 2 experiments described by Armitage, the threshold dose for contraction of the innervated nictitating membrane wres 40 pg/kg; acute removal of the arolmrior cervical ganglion abolished the effect of this doxe and reduced the effect of 89 rug/kg, while the effect of t?0 and 120 µg/kg was abol6lted by adrennlectomy. This.vorker concluded that the eontraction caused by 40 µq/I:g was due to ganglionic stimulation, either directly or mediated centrally, and that caused by 80 µg/kt; was partly due to ganglionic stimulation and parth• to release of epinep!uine from the adrenal glands. At high d,~ses of nicotine (10 injections of 20-100 µfi/kg). Milton observed contraction of the denervated nictitating membrane in adrenaleetomised cat,-, presumably due to stimulation of 6anglia throughout the body resulting in release of norepi- nephrine. In comparison to the mean threshold i.v. dose of nicotine of 22 µg/kg required to contract th., dencn•ated nictitating membrane of the spinal eat (armitage and Milton, 1965), the threshold dose !or contraction of the denervated membrane of the cat anr3thetized n•ith chloralose was found to be $# 2 µg/kg (range, 2.5-17.5) (Annitage, 19&5). Arm• itage also described an eyieriment in which, in the normal preparation, the threshold dose of nicotine to contrart the nictitating membrane a•as about 2.5 pAlkt;; after cutting the spinal cord at the level of the srecond cervical vertebra, it was raised to slightly less than 10 pg/kg; and, after removing the adrenal glands, the dose was raised still higher to between 20 and 40 µg/kg. In this mqtcriment, the contraction of the membrane by doses of nicotine Iesa than 20 pg/kg was clearly due to release of catecholamines from the adrenal gland, since adrenalectomy abolished the response. I( the nictitating membrane w•ere first denert•ated, the actinn of nicotine w•as then very etnall (J. H. )iurn, Leach, Rand and `IYb,mpson, 1959). ('hromnthn cells were fuund in the normal membrane, and, in most casea, denervation reduced their number, and they appeared pyhnntic. (Tests were apparently performed on the isolated preparation ot the cat's nictitating membrane deccribed by J. tt'.'Chcmipson (1958) above.] High doses, for example, 0.6 mg nicotine per cat (in contrast to the threshold dose of 0.022 mg/kg) still caused contraction of the denervated membrane, and this could be blocked by luior injection of hexamethonium, although small doses of ephtcphrinc, for mmlde, $ pK/cat, atill rauRed a contraction (Armitage and \lihon, 1985). Schaeppi (1963) alsc found nicotine to be practically inef- tertire on the chronically denervated nictitating membrane (right supenor cel ki,n. nv/in)A1P/1 R-12 da4P )lre- viously) of spinal cats. In acute lawtK'anklionic dencrvatcq nictitating membranes (superior cervical tt:anl;lion removed on the day of the experiment), 0.3 mg 8tic•otine injected into the commnn carotid artery produced a contraction laetina 57 * li sec. \\'ith higher doses, the contraction wa<; followed by a period of relnsation, during which the iromctric tenrion of the membrane w•as smaller than the control level. 11•la•n doses of 0.',1 mg or more were given at intervals of 30 min or less, the evtttractions gradually became smaller. The nirnt inc contractions were unnltered by intrucarutid injection of atropine; were reduced by phentolatttine, and com),leteh• blocked by hezamethonium. The slope of the log-dose- reslanse curve for nirotiae tro3e steeply between O.t/3 and 0.3 mg. Follow•ing adrenalertomy, doses of nicotine w•hich had previously caused contractiun of thr i•at's nirtitatinF mem- brane were usually w•ithout effect (Annitage and Milton, 1965). If cats were given reserlunc beforehand, the actinn of nicotine on isolated preparatiort.ti of thh nictitatins membrane was then very smnll (J. H. llurn,l.eacli, Rand and Thompson, 1959). Reserpine treatment cawed the chromalfin cells in the membrane to diminish and to lose their granules, but, in contrast to the p.knotir chromatfin rells in nirotine-ireated denervated me,mbranes, they remained healthy in appest•ance. Hesamethmiium blocked the action of high doses of nic•o• tine on the chronically denervated nictitatinK membrenc of the cat (.lt•mitage and llihon, 1965). Intracarotid injection of 0.3 mg phentolamine reduced, and injection of 10 rug hexamethonium mmpletely blocked, nicotine contraction of the acutely denetvated rtictitating membrane in si>itmi rats (Schaeppi, 19fi3;. In the spinal cat, nicotine contraetions of the acutely denervated nictitating membrane were unaltered bv intm- carotid injection of 0.1 ntg atropine, a dose which almost completely inhibited acet•rlcholine contraction (&haeppi, 1903). In eats, Akineton Illiperiden; 3 piix•r)dino 1y,henyl-1- bicyrlo•hclgenyl•Lt,rohannll and its diastemomere tonn depressed the nictitating•membrane rerymttse to nicotine (H. Hass and iClavehn, 19555). The paragraphs immediateh• above describe tfr antntt- onistic action of other drup on nieotine cot+traction of the nictitating membrnne; the paragraphs i.nmediately folloK'ng describe the effect of drugs on the pre~•iou¢h•-nicotinited nirtitating membrane. Pilocarpine injected to the superior cervical ganglion via the lingunl artery produced coutrnction of the nic•titating membrane in anesthetized rats, and this action n•as completely abolished by nicotine (LC Trendelenburyt, 1953a; \luray'ama and ('mw, 1963). Histamine c,mtractuai of the nictitatinn rnentbrane could also be iubibited by niccrthm in Irernh•aing daar. (Trendelenburg, 1955a). Thestimulant action of ~•(n, ch)orophenrirarbanlmlosp)• 2•but,rnyltrimeth,vl•untmonium chloride (11r1.A-3.f3) on the nictitatinyt tnembrane of anesthetized cat.q w•as not blocked by large doses of nicotine (Uura)ema and l'nnn, 1962, 19ti3). Intra-arteriai uye-tions of Rmall amounts of McN-A-343 into the blood supply of norntal and denervated superinr cercical ganglia ot cats caused contraction of the nictitating ntcmbrane, which was abolished hy nirotine block; however, durinR the serond (late) phau of the niro: Produced by The Council for Tobacco Research-USA, Inc. 0002930

SPECIAL SENSES 19 i tine block. the p.anlelia n-eained their responsivcness to McN-A-343 (A. Jones and '1'rendetenburg, 1963). 1i hen the mui~carinic "ents-muscarine, McN-A-343 and AHR-602 (.Y-benzyl-3-pyrrolidyl aketate Ittethylbromidel-wena in- jected immediately after lingual-artery injection of 0.2-0.6 mg nicotine to the non-atropinirxd superior cervical gainglion of the, spinal cat, they fAiled to stimulate the ganglion, as judged by nihtitating-membrane contraction; full recovery of their action was observed 15-20 min later. Later studies showed that the block to the mUscarinic compounds R•as confilied to the depolarizing and not to the non-depolarizing phase of ganglionic block qy nicotine (_see also below, 1$7 •B). Pretreatmeqt of anesthetized cats with 100 or 200 µt: nicotiiue completely abolished contraction of the niaitating membrane produasd by 6ngual-artery injection ot 4 mg potassium chloride (Alurdyama and l`nna, 1963). Arnnitage and Milton (1965) concluded, from their e:xperi- rnents,a results described above, that the action of certain doses of nicotine in causing cohtraction of the nictitating mem- brane is due to release of epinephrine from the adrendl me- dulla. However, it would also appear that catecholamine release from nictitating-membrane chromaf6n cells is ai3o involved (see Rurn et al., 1959, above). M. F. Cuthbert (1964) has reported relative potencaes to nicotine for t}^ramine met.biodide, dopamine methobrornfde, and norepinephrine methochloride on cat nictitating pnem- brane. \icot'uhe. nornicotine, ethylnornicotine, allyinornic- otine, acetylnornicotine, beneoylnornicotine, and car- beminoylnoraiCotine all caused oontraction of the nictitating membrane of the anesthetized cat (Mattila and Vartiwinen, 1962). Quantitatively, injection of 1-5 gg nicotine into the lingual artery of cats immediately caused a contraction of the nictitating membrane; nornicotine and ethylnornicatine were about 10 times, allylnorniootine 100 times, aeetylnornico- tine 600 times, and carlra,minoylnomicotine 1200 times less active than nicotine (Mattila, 1963a, b). 95. tt.~ras-octn.ws raissnaE (37) [No new dath.) 96. svet,ros (38) Application nf nicotine lo tM ey~gs of guinea ligs and rats caused pain and blephanwpasm, which could be diminished oi inhibited by prior local application of ganglionic blocking agents (Jancsd, Jancsb-GSbor and TakAts, 1961). Blefpharoconaunctivitis was noted by Petrova (1963) in rats e1nposed to methyl-alcolwl aotd nicotine vapors (see below, lOg3). 97. LACRIMAT1OId 0$) . 99. HEARING (38) 99. HiswpaNwfogy oJ the .tuditorg Organs in C6ronic J'oisoning (3ti) Folloning a review of the liternture on damage caused by tobacco-smoke in the area of the inner ear, \IaHei and \fiani (1960) described histopathological changes in guinea pigs exposed to cigarette-smoke for varying periods of time. One group of 4 animals received relath•elv acute e~~po.cures, ranging from the smoke from 22 cigarettes in 18 hours to that of 65 cigarettes in 85 hoiurs, duration being related to the indiridual resistance of the animal to intoxication lap• parently, at least some of theae animals succumbed to the treatment). Injury of specific cochlear structures was not observed; circulatory disorders were found, with alterations both in the caliber of ti.e vew,eL and their wallr, and, in rare sections, rupture of the vessel with consequent hemor- rhage; the eustachian tube showed no abnormalities. A second group of 4 animals icris subarutely exposed to the smoke from 8 cigarettes in 4 hours daily for 2 months, and a third group was chronically e.ytoAed to the smoke from 7-12 cigarettes in 5 hours daily for 17 months. In these latter animals, in addition to vascular lesions, degenerative lesions of the neurosensorial epithelia and of other elements of the cochlea were observed. The eustarhian tubes showed the presence of abundant exudate in the lumen, and, in the chronically-exposed animalx, there was an indication of beginning mucous atrophy. 100. Ellect of S»wRing on ;4 uditorp Seneitivfly (39) S. L. Shapiro (1964) concluded that nicotine produces vasospasm of var59ng degree and duration in small-caliber blood-vessels, including vasospasm of the internal ear, and that, in a number of people, this va.roconstriction rsn and does produce lesions in the internal ear [\izetic and Altiere (1952) and De Gaspere a.yd AlaBei (1963)(39a)J. !n Shapiro's own clinical experience, the hearing impainnents in smokers have been irreversible, arrest of the condition being the most that could be hoped for, even after the complete abstention from smoking he rerommended in such eases. 101. TASTE (39) 102. Trta+e Rer.eptora (39) Ste 103, beloa•, for relevant data. 103. In,Ruerur orSneoLing on Tastc Threaliolda (39) The dimorphi.rn of the human race exhibited by the in- ability of about a quarter of the population to taste I'TC (phenylthiocarbamide; phenylthiourea) has been shown by L. H. Snyder and others to be dependent upon inheritance of asingle auarwmial recescive gene (Falroner, 1947; C. B. Thomas and Cohen, 1960). The data of the great majority of investigators have led them to the conclusion that the habit of Ivnoking does not nmodify the frequenry of PTC-non-tasting subjects or the mean taste thresholds lanalysis by Freire•\faia (1960)). Thus, comparison of a group of 217 male and 123 female smokers with a group of 173 male and 110 female non-smokers, all undergraduate students at London l'niversity, showed that smoking has clearly no influeluw on taste sensitivity to PTC (Fklconer, 1947). Al-w, no difference in the average taste thresholds for PTC nw found betwrcn 97 male non- Lscrimation induced in guinea pigs by local application of nicotine was niore or less inhibited by prior application to the eye of garpglion-blocking agents (.lancsiS, Janc-c:Gdlwr and Ttlkdts, 19hi1). Passey and Smith (1963) have pointed out that the human conjunctiva wichout a locil anesthetic can withstand expo- sure to t he full stream of smoke from a whole cignrette without experiencing any symptoms other than extreme lacrimetion and sopnc local discomfort, but no pain. Ilalik, Pavlova and Urbanova (195fs) found that the quan- tity of tears, and their glucaare content, did not change as a result of eigarett"moking. Thirty subjects amoked 2 ciga- rettes in 10-18 min, tears being callerted for 5 min hefov: and afdenvardz;. Produced by The Council for Tobacco Research-USA, Inc. 0002931 9

I 20 TOBAtxO-EXPERIAtE% TAL A1D CI:IrICAL STUDIES. l;UPPLE%fE\T I -vr•n AnA 197 smalre~ nn f.nsniards (Pons, 1955). Iht a group of 100 male and 100 female students, aged 19-25 years, at the University; of Lund, non-tasters of PTC were equally distributed between smokers and non.-amokers (Akesdon, 1959). Even when non-smokers were compared to heavy smokers (10 or more cigarettes per day, or an equivalent amount of tobaeco), no differences were observed. There was no diBerence in the taste response to PTC of male habitual cigarette-smokers, whether normal subjects or patients qith tuberculosis (Frgire-\Iaia and Queke-Selgado, 19M). The distribution of PTC tasters among the adult men of the Cape Coloured and European communities of Cape Town was similar in both the non-smoker and'saioker samples, and thur there did not appear to be a genetic inability among smokers to taste bitter (Bronte-Stewart; h;rut and Perrin, 19fi0; Krut, Perrin and Bronte-Sten•art, 1961). The khi- square test for smoking habit gave no significant result in a group of 225 males and 200 females tested with botl, 1?TC and quinine (IRguebe, 1960a, b). According to R. Fischer, Griffin and Kaplan (1993), the lack of correlation between smoking and quinine-tasting may have been due to Leguebe having tested PTC first, and then quinine, with con,ceqtient dul9ng of the taste-threshold for the latter substance. C, B. Thomas and Cohen (1960) also compared the ability of smokers and non-smokers to taste PTC; the subjkcts consisted of 59i white and 232 Negro male blood donors at the Johns Hopkins Blood Bank, all interviewed as to smoking habits. Contrary to other workers, however, these ind•es- tigatots found significant differences in ability to teste 17C between smokers and non-smokers, both white and Negro. Among white males. heavy cigarette-smokers showed a strikingly higher proportion of tasters (65.R o) than did non- smokers (42.7%). Fonner smokers, occasional smokers, and light smokers resembled the non-smoker as to the proportion of tasters. The significar-t findings were unrelated to age. In a sceond series of 408 white male blood-donors tested for their ability to taste PTC as related to smoking, the ss-ue trend appeared (B. H. Cohen and Thomas, 1962). ': hiinas and Cohen (1960) stated that, while the possibility that smuking directly afiects acuity of tastt• in respect to PTC must not be overlooked, it is Iw.wible that inborn differences involved in the ability to taste PTC may be part of a broad spectrum of genetically detennined biochemical differences which both predispese individuals to their particular smoking habits and influence their susceptibility~ to certain dismses. These stu~ies were the subjects of comment in Yrodftioner (188:442-443, 1962), which felt that the findings warranted further investigation. When taste thresholds of 127 subjects for quiniae sulfate were determitred, and the data analyzed in relation to the smoking habits of the same individuals, the proportion of smokers was found to be lower among sensitive tasters of quinine than among ineeruilive ta.Mers, and the converse relationship ures similarly true (R. Fischer, Griffin and Kaplan, 1963; Fischer and Griffin, 1964) A similar relation- ship was not found for 6-n-propylthioruracil (PROP-an odorless analog of phen•vithiourea) (Fischer, Griffin and Kaplan, 1963). Kaplan, Glanville and Fischer (1964) further reported on the association between the smoking habits of 31-50•yearold males and females and their taste thresholds for two bitter-tastinR compounds, quinine and PROP; the subjects consisted of 34 heavy smokers of eigarettea (20 or more per dqv) and 40 non-smokerc (also Kaplan and Fischer, 1965). Mean taste thresholds were significantly higher for both compounds in smokers as compared to non-smokers, and this was true for both males and fiernales• In the case of PltUY, ciassuicauon as W taaiero auu Jwu-wn,c1m significantly fewer tasters among the heavy smokers. The most significant statistical difference in taste threshold for quinine was observed between male smokers (higher) and u.ale non-smokers (Kaplan and Fischer, 1965). Female smokers were also less sensitiµe to quinine, although the difference was not quite as significant statisticallv as for the males. Taste sensiti.ity for PROP, like that of quinine, was fower in smokers than in non-sntokero; and these findings were taken to'confirm the e%istence of a relationship between smoking and bitter-taste thresholds. No significant age. related differences in taste sebsitiviby for quinine or for PROP were observed in a sample of 2ti,°t non-smoking subjects between the ages of 16 and 55, and no ses dirTeren•.e -4a, apparent (Kaplan, Glartt'ille and Fischer, 1965). In contrast, among 127 subjects who smoked 20 or more cigarettes lmr day, a significant detcrioration, in t:+s1e scnsitirit-r svas ap• parent with increasing age. Within the 16-to-20-year age- group, the sensitivity of non-smokera and heaty smokers did not differ, but from this age onward, a difference appeared which became increasingly more pronounced (It uas pointed out that sex difference sc^ietimes observed in population surveys seeme,d to be an artifact associated with differential smoking habits of men and women.) apparently. I:oplan and Fischer (19&fi) commented; aversion for foods and eit;a- rettes is anal'ogoualy related to taste acuity for bitternc.s only. Specificelly, low taste acuity for bittenrck, was said to be associated i.ith few or no food dislikes and with smoking; but these workers were unable to deterntine whether the congregation of insensitive tasters among heavy smokers indicates a eonstitutioual and genetic predidosition, or whether the taste-seruitit•ity difference is Imrtialw the result of diffelrnc•es in smoking habits. Fischer. Griffin and haplan (19M) regarded preciislwsitiuh for bitterness, in the form of genetically detemiined high taste thresholds for bitternem, as another fartor leading to "nicotine addiction" in the confinaed smoker. Bronte-Stetyart and his colleagues found no significant difference in taste th. ^.-holos of smokers and non-smokers with respect to salt (sodium chloride), sour (ritrk• a.cid), and sa•eel (sucrobr;; the perception of bitter (quinine) a•as, however, significantly woRe in smokers (lironte-fiteu•art• Krut ar.d Peo•rin, 19610; Krut,, Perritt and lironte-i+tewart, l9(il). The amoking of a cipstette had no acute effect on taste for any of the taste primaries in -mukers and non- stnokers (Krut, Perrin and Bronte-Stewart, 1961). The individuals subjected to the,e wtqng tests and to the PTC test described above-tamprised SO medical students (mean age, 20.5 years) and emrlnveec of an insurance eom. pany (mean age, 27.6 years), with similar re~sult~ being ob- tained in both sampics; they were questioncd aixmt their dict, and the fst rnntNnt of the:e.lir6 wa, anolyxrrl (Itronte• Sten•art, Krup and Perrin, 1969; I'errin, l:ntt and Hnunle. Stewart, 1901). The smokers consumed more iat than did the non-smokers, but the differente was emall; but, with mpect to the t•vles of food which constituted the over-all fat hitake, marked diffenhnces existed: heavy smokers consumed siqni8- cantly more meat and egpi, and non-Amokerr' con~umed more cakes, sa•eets; and chocolate. The authors concluded from their studies that, in these subja'ets at least, smoking affe-ted food preferences via the taste urechairism. (For the pos,iblc relatnroship of these findiw to scntm-eholesteml levels, see below, 696-B, and to coronar+• heart-direase, 1397.1 Arfmann and Chapanis (1962) studied the relative semri• i i , I 1 Produced bv Tfr Council for o 0 0 2 9 3 w Tobacco I-t;searcii-i;SA, Inc.

SPECIAL SENSES u tivities of taste and smell in 3 regular cigarette-emokers and 3 non-snmketn, nll frmale mllevP.atAiriPnts• thP ovnnori- ment consisted of judging varying concentrations of vanilla extract in relation to a constant odor of the same substance on a 7-point scale. As contrasted to the non-smokers, the smokers consistently rated the strength of the taste sensation lower, and the difference in relative taste acuity was said to be significant at the 0.05 level. The interaction of Groups x Concentrations was said to be significant at the 0.01 level of confidence. 104. Toste Prejerenres in Smokers (39) As we have seen in the preceding section, Bronte-Stewart and his associates have suggested that smoking sfected food preferences via the taste mechanism. The title of th(. paper by R- Fischer, Griffin and Kaplan (1963), also considered in the preceding section, is "Taste thresholds, cigarette smoking, and food dislikes". Apparently, these workers have emphasized, rather, a"Gking" for bitterness. See also Kaplan and Fischer (1965) on some biological and clinical implications of taste sensitivity for bitterness 1103). 105. Drug EBeets on the Ta&e ojSnmking De KSrSsy (1981) noted that, while the silver ion makes a cigarette distasteful, the effect of the gold ion on the taste of cigarettes was very much less pronounced. This effect of silver (as the nitrate, proteinate, or acetate) in rendering cigarettes unpalatable forms the basis of several methods of "curing the smoking habit" (for details, see below, (543a) and 1145-C). For example, it is aaid that a mouthwash of silver nitrate or 0.5% wpper sulfate, used 3 or 4 times daily, imparts a strongly undesirable taste to tobacco-smoke and is thus useful in helping patients give up the habit (L. Bern- stein, 1965). Newcombe (1965) reported that one of his patients being treated with salicylazosulphapyridine developed s strong aversion to cigarette-smoking, particularly to the smell of the tnba.cco-smoke, which led him to cease smoking alt.o- gether. During a 2-month interval when the patient was not taking the drug, he smoked again, and thA distaste for ciga• rettes was not present. It has also been remark.x) that, during bagassosis, ciga- rett:e-smoke takes on an unpleasant taste (Buechner et al., 1964). 106. SMELL (40) 107.OIjoefory Potenlinla (40) In the dog and monkey, electrical responses to all odors, including tabacco-smoke, were enhanced with increasing flows of oxygen when the flow of the odoriferous material was kept constant (Ueki and Domino, 1901). Injection of physiological saline or 0.001-0.05% nicotine solutions or other aqueous solutions into the nostril in the vicinity of the t ribriform plate all produced similar high-frequency electrical responses, making it seem likely that the::e were elicited by mechanical stimulation of the olfactory receptors, rather than by any chemical phenomenon. In unanesthetizctii cats immobilised with decamethonium bromide, in which the slow potential and electrcencephalographic (EEG) burst discharges of the olfactory bulb induced by olfactory stimu- latlon werrt simultaneously recorded, cigarette.emoke added to the air-flow into the nose caused continuous EEG burst discharges of high voltage and a marked positive shift (4-5 mV) of the sleady potential of the olfactory bulb, after which 21 the slow bulb potentiad became markedly depreseed; com- Tdnfe rP^lWPrv M"nNPd in RbnM ff rnin qftnr arnnlrn nwm ~Im (Ueki, Tanaka and Bupeo, 1909). Geateland, Lettvin and Pitts (1965) studied electrical activities in fibers of the olfactory nerve of the frog (Rana pipiens) in relation to responses to odota; and these workers stated that certain stimuli, such as tobacco-smoke in sufficient strength, would cause irteversible changes in the response of the mucosa. Criteria for the over-stimulated state were that the slow potentials became smaller and not easily evoked repetitively, and that the fibers became either unreaponsive or responsive to almost everl f.hing. 109. EBert oj Snm'ldny on Ol,fadion in A/an (40) Nfiani and Bottassi (1iJ59) employed the method of Elsbcrg (modified by that of Fortunato), to test the response of smokers and non-smokers to certain odorous materials (roasted and ground coffee; citral; vanilla; pyridine). The authors reported finding,a deficit in olfactory coefficient and minimum identifiable odor in smokers; the deficit was not very high in those smoking up to 20 cigarettee a day (20 subjects) or in those smoking 20-40 per day (20 subjects), but was sometimes remarkable in those smoking more than 40 cigarettes (10 subjects). A control group consisted of 20 persons who had never smoked. Several workers have used a phenol smelling test to deter- mine the influence of smokingand occupation on olfactory acuity. Using a simple olfaction test based on graded solutions of phenol and liquid paraffin, Fordyce (1961) tested 147 persons for the relation of smoking to acuity of smell; 90 of the subjects did not smoke, 40 smoked cigarettes only. 10 smoked a pipe, and 1 cmoked only cigars. There was no evidence of any difference between cigarette-smokers and non-smokers, but there dvss an indication of a possible de- crease w acuity among other smokers. These authors also examined the repeatability of their tests. Of 98 persons tested, 79 repeated their initial result to within -i: one grade on a second occasion; smoking habits did not appear to affect the degree of repeatability. Adams and Crabtree (1961) com- pared the sense of smell of 106 alkaline-battery workmen to that of a control group , of 84 men, matched for age. The battery workers reported significantly more anosmia than tbe controls (15 % to t:erb), and did lese a•ell bi the phenol- maelling test (27.3% to 4.8%). Since the controls smoked more than the battery workers, the authors did not comvAder that ihe anosnva could be attributed to smokiug, but, rather, to exposure to cadmium or nickel dust, or to a mixture of both. Joymer (1963, 1964) tested olfactory acuity of 500 employees of a petrocheniical plant, using 10 serial dilutions of phenol crystals in vials placed in random fashion in an open-top box; results of the tests were then correlated with previously-completed questionnaires covering age, smoking habits, history of nasal disease, craft, area of work, and other data. Comparative studios revealed statistically significant differences between smdkers and non•smokera, with di- minished olfactory acuity associated with smoking and the amount smoked. Matsker (1965) tested the sense of smell in over 1,000 individuals aged 10-90 years, and found that smokers had a significantly poorer sense than non•smokers in all age-groups over 15 years, regardless of the aromatic substance can- cemed. Ga.collne, coffee, peppermint, perfume, valerian, and anise were the test-matetials ured. The author stated that he diRerentiated only between "smokers" and "r.~on-smokers", and that "it may be expected that heavy smokers would Produced bv The Council for o 0 002 9 33 Tobacco Riweai-ch-llSA, Inc.

22 TOBACCO-EXPERIRfENTAL AND CL1N ICAL STUDM. SUPPLL%iF:4T I exhibit a clearly diminished olfactory ability with respect lu iigita nuwi.eta. The experiment reported by Arfmann and Chapanis (1962) in which cigarette-mnokers and non-smokers were made to judge varying concentrations of vanilla extract in relation to a constant odor of the same substance on a 7-point scale has been briefly described above, 103, in terms of "relative taste acuity". 109. TOUCH (40) Jarrett (1956) recorded action•potentials from the cutane• ous netves of English frogs, and found that nicotine had the same actions as acetylcholine; a concentration of 10-4 gm/ml excited a volley of impulses, and then rendered the skin unresponsive to any further stimulation, chemical or mechatd- cal, 110. P.4IN (40) Janrs6, Jancs6•GQbor and Takdts (1961) studied sensory stimulating effects, and their extinction, using chiefly the eyes of guinea pigs and rats; pain was measured by duration of blepharospasm, and inhibitory effects were manifested by complete absence or diminished duration of blepharo- spasm, or in considerable delay in the appearance of the reaction. In experiments on guinea pigs, one eye was first treated with a ganglionic blocking agent, followed by a solution of nicotine, and the response noted. Somewhat later, a control experiment was performed on the other eye, to which saline was applied prior to nicotine. Ganglion-blocking agents reduced or abolished, without previous stimulation, the sensitivity of the corneo-conjunctival pain-endings to the excitatory action of nicotine; blepharospasm, lacrimation, and scratching failed to occur or were diminished and/or delayed. Tetaaethylammonium bromide ares effective only at high concentrations and for a short time. \iethonium compounds were far more potent, and, in the case of tmpane derivatives, their action on nerve-endings imitated the gan- glionic effect quantitativelv ss well. Follawing its primary stimulating action, nicotine also desensitized the eye to its subsequent further application. The inhibition with these compounds was specific for nicotine and trompgunds with tricotinic aeion, nr, inhibition occurring to capsa:cine (N•(4- hydroxy-3-r.tethoxybensyl) -t3.metbylnon-tmtts•6-enamide], piperidine, dipyridyl, fonnnlin, or chlomcetophenone. The ganglionic blocking agents were also ineffective against mechanical stimulation, the corneal re8es being easily elicited by touch; this could be particularly well obrerved on rats. The nicotitwlytic, antiparkinsonian dntgs, caramiphea, 1-pi- peridirno•3-pheny6propsn-3-on HCI, and 1•piperidino•3(5,6, 7,8-tet.mhydro4-naphthyl)-propan-3-on, at low concentra- tions, all desensitized the eyre against nicotine; this effect occurred at concentrations below those exerting a true local ane.thetir e6ect., and the slxk•ifirity did not extend to caprwi- cine or piperidine, and did not ilJiuence the corneal reflex. Loral application to the eye of guinea pipc of eserine, coniine, pelletierine, sparteine, hordenine, strychnine, brucine, and quinidine also protected against nicotinic imin. The authors concluded from these and other esperintents that the lxiin• producing action of nicotinic stimulants was mediated by a distinct neural mechanism, which could br selectively blocked by gatt(tlion-blocking and nieothmlytir substances, wheteas, in other respects, sensibility rrmained unimlmired. ln addi• tion, a second neural mMhanism took part in the mediation of chmniral pain, which could he elimhnattd by a dcvnsitisinft capsaicine treatment. Deaensitization of rats and guinea pigs .,••,, . , ,,.... ...t,a. ....., ,.w ,........ .... .... .,..... and Tween 80, capsaicine was made into an injectable aqueous solution. Rats were given 4, 8, and finally 15 mg of capzuacinc subcutaneously under the donsal skin over a period of 1-3 days; and animals so treated became insensitive to chemichlly- induced pain, the re(ractoriness lasting 1-3 months. Thc refractory state produced by capsaicitie in guinea pigs might last for years. Rats or guinea pigs desensitized to nicbtine and other chemical stimulants by capnaicine in this tnabwer retained normal responses to physical stimulants. Anah-su of action-potentials of the involved sensory nerves showed typical trolleys from tactile stimuli, but none from chemical stimuli. M n!tils, Ahtec and Vartisinen (1965) mmqulrtl the possible analgetic action of nornicotine and several derivatives by means of the hot-plate test (Elddy and Leimbach, J. Pharni. Eap. Ther. 107.387, 1053). Nicotine in dosage of 2.5 tnu/kt; subcutaneously, and ethylnornicotiue in dosage of 7.5 mg/kg subcutaneously, caused sigttificant delay of the test mire compared to the controls (15.9 * 2.3 and 14.2 i 1.2 vz 7A s: 0.7 sec, respectively). rornicotine, alh•lnomicotine, acetvl- nornicotine, bensoylnornicotine, and carbantinoylnorairo• tine, injected subcutaneously or intraperitoneally, were with- out effect. Pre-treatment of mice with reserpine tended to shorten the delay time on the hot-plate; such treatment slightly decreased the analgetie action of nicotine, but this decrease was not significant. Keele (1962) studied the eommon chemical sense [pain] by applying chemicals to the exposed nen-e•endings in the base oi a blister induced in human skin (forearm) by canthariditi. In concentrations of 0.1-0.5 nng/ml, nicotine tartrate pro• duced pain; but, after initial stimulat:ott, nicotine prevented the action of subsequently applied acetylcholine. Apparently, the substance of Keele's report xres derived from D. A'.rm- strong's "Chentical Excitants of Cutaneous Pain" (L'niverxit.• of London [Ph.D.] Thesis, 1957), and I(eck and Armstroaf; (1964) have published further details. The numerical scale used for subjective estimates of pain was: 0= no pain; 1 a slight pain; 2 o moderate pain; 3= severe pain; 4=.er,v Fevere pain; and the subjects.vere left to work out the values for themselves. Nicotine tartrate in concentrations of 0.1--0.5 mg/ml produced pain of 1-2.5 units in 4 subjects, and this action was not due to the tartrate ion. The higher concentra- tion of nicotine tartrate, left on for I min, depressed the pain response to acet,rlrholine tested 10 min later; when left on for 10 min, the pain response to acetylcholine was abolished. 111. TE\fPERATURE (40) I\o new data.) 112. EQInLIBRII'11 (40) 1•King llle ability of tllice to halanre for 2 min on a hnri- zontall,r rotating rod as a criterion, \lattila,Ahtre an-l Vartia- inen (1.965) found that s.c. injection of 2.5 mg/kg nicotinw or 7.5 tng/kg ethylnomirotittc caused several of the anitnals to fall. 113. 6An1'alA'TH nuiLtT1t.; (-!0~ (No new data.) 114. Nystagaeus (41) The study by Dc Klc}'n and \'enteexh (10::) on the acthm of nicotine on the ve.,tibulnr ay~*tem of mbhilr, prec-iouay Produced btr The Council for 0002934 Tobucco fwsoaxh-USA, Inc.

SPECIAL SENSES reported (91b), has been published in another placc (De r ~.. ., a n... . ... .t , no.. , . .b.., . According to Melanoniki (1963), nystagmus sometimes occurred in certain individuals after heavy cigarette-smoking; end S. L. Shapiro (1964) made complete abstention of snok• ing an inflexible rule in treating petsons with diazipesa ac- companied by objective vestibular abnormalities, In Shapiro's clinical experience, dizaintsa, as well as the postural nystag- mus accompanying it, disappeared in a very sbort time follow- ing cessation of smoking, before any other thernpy was in- stituted. This writer attributed these disturbances to the vasoconstrictor action of niootine on the smaller blood-vessels of the internal ear. Teceling and Kruse (1961) stated that SI'alter (Dent. Z. f:a. (ierirhtl. Med. l,3:232, 1954) had repnrted,erperiments with 3 persons who smoked a cigarette while fasting in the morning, and who exhibited a nystagmus of 3-5 min duration, which took a vertical direction in 2 and ¢ diagonal one up- wards to the right in the third. The former authors them- selves reported tests on 70 students, aged 20-25 }earc, who elso smoked a cigarette in the morning (before, rising) and while fasting. Forty-one of the 70 were affected by a nystag- mus, vertical nystagr»us (always in the upward direction) predominating. Latent period averaged 3.5 mi¢t from the beginning of smoking; duration averaged about 10 min. Smokers and non-smokers were about equally a6ected; women were affected more frequently than men. Vegetative symptoms occurred in all but 8 subjecta; pulse-rate increased in all. 115. HItiTOPLTHotAOY oF TEE iNNEB EhH (41) In the guinea pigs subjected to acute or,cbronnc cigarette- smoke exposttre by aflnffei and Itfiani (1960), no noteworthy alterations in the histological picture of, the semicircular canals were found. In acutely exposed animals (smoke from 22-65 cigarettes over a short period of time), the endothelium of the intima of the cochlea became particularly evident, with distinct showing-up of the nuclei of these vascular sheathing cells, a finding which lt3afei and 1lfiani (1962) attributed to subistitnal edema linked !o the action of tobacco-smoke. The organ of Corti and the acoustic nerve 0 showed nothing frankly pathological, and the spiTal ganglion .. ~ .. C}.p:.:.:~.. .... .., b......r :.. b....~ .v...u::v... v~.,~.....::va.o v: the eustachian tube~in this group of 4 guinea pigs revealed a normal tubal epitheUum. In a secoiid group of 4 animals exiwsed for 2 months to the smoke of 8 cigarettes for a total of 4 hours daih•, the ganglion of Corti in some animals showed degenerative factors, with mild rarefaction of the ftanglial cell; and it was sometimes possible to mbRerve a slight increase of connective tissue present between the ganglial cells. Hemor- rhages were frequently observed, pArticularly at the levei of the stria vascularis in its lower portion. The presence of abundant exudate in the lumen of the eustachian tube, and mild initial involvement of the epith.eGum of the salpinges, were noted. In a third group of 4 animals exposed to the smoke of 7-12 eigarettec daily for 17 mnntha, vamnlar ufgna in the cochlea were less intense, and were most obr-ious in the peeineurgl zones. The limits of the stria vascularis appeared to be especially altered, revealing many blood cells ooeinR out from the vasal lacunae of these formations, due to diapede- sis. Intense degenerative signs were,pre.cent, inVolving the cells of the organ of Corti; Reissner's membrane was generally situated in such a position as to justify eventual hvperteusion of the endolwnphatic fluids. \othins pathological involved the acoustic nerve, while, in contrast, the spiral ganglion revealed a reduced number of cells separated from each other by obvious intercellular edema; in these, degenerative sixns, both protoplasmic and nuclear, were observed. With resliect to the eustachian tubes, there was tharked involvement of the tubal mucosae, poor secreting, with 9attened cells poor in cilia, and with submucrosaa reduced in thickness and poorly supplied with blood. Additional study of the encephalon showed degenerative factors of marked degree involving the cerebral matter, with cellular vacuoliaation affecting the nerve tissue, in the animals acut«7y exposed to cigarette- smoke; and this finding was ptrs ent to a much slighter degre: in the guinea pigs exposed for 2 or for ti months. 116. REVIEWS Schievelbein (1962) presented a re%iew of the newer litera- ture (mostly over the prehxding 20 yeuts, but not limited to this) covering smoking and its effects c1n sense organs. Produced by The Council for Tobacco Research-USA, Inc. 0002935

.' NERVOUS SYSTEM 117. INTECRATBD EfFFECfS ON THE NERVOUS SYST0t AS A WHOLE (42) This section is again devoted to a discussion of anidial experimentation on the effects of nicotine and tobacco or tobacco-smoke on the nh..rvous system as a whole. For an account of notably sce.rce recent reports of effects of tobacco and smoking on mental and physical efficiency in man, see Chapter 17, 1178 and 1182. The advantage of experiments with animals is that causes and effects, can often be kept in better focus; but, as against this, Van Proosdij (1960, p. 101) went on to say, it is precisely in the intellectual field that comparison between man and beast calls for great cireu m- spectdon. Also, bearing in mind how greatly human smoking differs from the smoking experiments performed with a'xii- mals, it must be conceded that the latter scarcely provide a satisfactory eaperimental model. 118. Spontaneoue Adiaity (42) In addition to a cephalic hematoma which developed almost immediately after application of 5 mg nicotNne to the chorio-allantoic membrane of 10-11-de',y.old chick embryos, Gatling (1964) noted a second response, to the drug, namely, central nervous system (CNS) depression; this was reven,ible, in that surviving embryos demonstrated gradual return of activity (motion of different parts of the body) after several hours. This CNS depn'ss"on was prevented by pretreatment of the embryos with phenothiasine derivatives, but not by tolasoline; and tolerance cculd be developed to the effect. Application of nicotine to older, "y embryos caused no or minimal )esions; bavtiever, CNS depression wae not inhibited in the older embryroa An imtroved spring-mouated cage for measuring the motor activity of mice has been described by lionta and co•workers (1960), who also published a table of results obtained with their apparatus following i.v. injection of 01g-0.43 mg/kg nicotine; the table shdvred graded increases in motor activity with increasing dosage. Following a(toaYc) oml dose of 10 mg/kg nicotine, the motor activity of Cl"`W female mice was markedly depressed; ataxia was slightly Increased (l3astian,1961). In dnsafte of 5 mR/kg i.m., nicotine tartrate was reported to have rommonly; reduced spontaneous activity in CFI-strain mice (\tafii and Sondn, 195g). Like nicotine, nornicotine, etltylnornicotine, and allylnornicotane increased spontaneous activity in ntice, but acetylnomicot$ne decreased spontaneous activity and carbaminoy)nomicotitte caused a slight decrease (Illattila and Vartiainen, 1902; Tlattila,1983a, b). In t1rese experiments, spontaneous motility was determined by use of a photoelectric motility-box, and the several compounds were administered by i.v., s.e. or 8.p. Injection (Mattila, Ahtee and Vartiaj4nen, 1905). On ~.v. administration, only 0.1 mg/kg nicotine and 0.4 mg/kg nornicotine significantly increased spontaneous motility over the controls; 0.5 mg/kg ethylnorndcotine, 1.5 mg/kg alh•lnor- nicotine, 25 mgikg atclyluorni.rotioc, and 4.5 mg/l:g bcnaoyl- nornieotine had no significant effect; while 1011 mg/kg carba- minoylnomicotine significantly depressed spontaneous motility. In contrast, on s.c. injection, 2.5 mg/kg nicotine, 7.5 mg/kg ethylnornicotine, and 15 mg/kg alwluornicotine significantly depressed spontaneous actit7ty, whereas 7.5 mg/kg nornicotine bad no effect. Following i.p. administra- taon, 30 mg/kg acetylnornicotine, 30 mg/kg benzoy)nornico- tine, and I0D mg/kg carbaminoylnornicotine were without effect on spontaneous motility. Bovet-Nitti and Bovet (1980) employed computerized activity-wheels to record running activity of Wistar Sh1 rats, aged 3 months when introduced to the apparat.os and 4-plus months at the start of pharmacologic tests. The exist- ence of a rhythm of voluntary activity was confirmed; locomotor activity, which during the day averaged about 150 m/hr, could attain at aight even 30 times this value. The effect of 0.2 and 1.0 mg/kg nicotine (calculated as the base), injected ac. at 10 a.m. or 10 p.m., on voluntary activity was studied; and performances over a 3-hr period follovring ad- ministmtion of nicotine were compared to those over the same hours on preceding days. The dose of 1.0 mg/kg signifi- cantly stimulated (P n 0.02) activity in the day tests, and showed a depressant (sedative) effect in the evening tests (P n 0.05). Effects of 0.02 mg/kg were in the seme opposite directions, stimulation in the day tests (P = 0.01) and depres- sion in the evening tests (P = 0.1). Steinhaus, Schultz and MLron (1952) had measured the spontaneous activity of white rats in freely-revolving drums in fresh a'v and in air filled at regular intervals with cigarette smoke during 12 hours of each day (43a); and, later, $tirewalt and Steinhaus (1900) suggested that the reduced spontaneous runnng activity observed in the animals subjected to eigarette.smoke-lnden air, or given s.c. injections of nicotine, might be related to an increased serum-potas4ium level, such, as they had observed following cigarette-smoking by human subjects (see below, 701). According to Frankenhoeuser and Lundervold (1949), blowing ciRarettR.smokc on to the nose of rabbits under urethane narcoeis cau-1-4 r*x inhibition of "spoutaneous motor activity" in 00- 701, of trials. in rnbbils, i.v. injecu,m d 3 oi 4 mg/kg nicotine did not potentiate the narcotic effect of small doses of Somniffte [diethyl• + alIy•lisoproln•Imalotn•lunmJ (l3aridty, Kohtcr and I3arbe, 1953). Neither nicotine nor norniootine affected sodium-pentobarbital sleeping-time of mice. Sleeping-time was significantly prolonaed only by allylnorniootine, aeetyl- nornicotine. benaoylnnrnirotine, and carbnminoylnornicotine (afattila, .lhtee and \'artiainen, 1905). Administered to mice 24 Produced bv The Council for 0002936 ;Tobaeco Rescaich-USA, Inc.

I NERVOUS 8Y8TEM just awakening from the barbiturate sleep csrbaminoylnor- _ were given by s.c. or i.p. injection in the doses specifled above in this section.) 119. Behdoio. Since the CNS is so delicately adjusted, and so susceptible to the effects of neurotropic dnup such as nicotine, we have suggested that it would be highly desirable to investigate the actions of such agents on behavior of the organism-as-a-whole within the range of min{snnl egeclire dosage (Silv ette et al., 1962; Imtson and Silvette, 1965b). In animals, as well as in man, we may surely believe that the optimal effects of neu- ropharmacological drugs lie within this range of "minunal" d- ~*age, not.cithin that of "maximal" or supm•ma.Yimal (todc) dosage. In behavioral studies, the animal species and its handling are of scarcely less importance than drug dosage (see, respectively, Chance (1957) and Dews (195g)(42a-b)j. Equally important would appear to be the use of a variety of experimental techniques in testing the effects of the same drug, in order to define adequately its chsracteristic psycho- phannacology (Robustelli, 1963). Also, in animals as in man, personality affects drug-response (Lancet 1: 805, 1984). The conditions of experiment must be carefully controlled, for repetition and experience can alter results. As more work is done, so the skill increases with which useful and appropriate behavior tests can be chosen and applied. Afost of these conditions appear to have been met in the studies by Bovet (1985) and Bovet-Nitti (1965) oql the action of nicotine on conditioned behavior in naive and pretanined rats (see below, 120 and 121). In her discussion following these two papers, $ovet-Nitti pointed out that , these experiments clearly demonstrated the importance of the nature and form of conditioning, the motivation, and the genetic characteristics of the ar.imal.sttr.in in the analysis of the effects of a ps.yebo. tropic drug. "Concernit.g the ability to transfer into humans the results obsermd, in animeis, the elements of judgernent are reprwente<: by the fact that the technique has proved to he useful in the screening of psychotxopic drugs. Also the active dosage levels in animals are relatively near those taken in by man (inhalatAon by smokers): ' In investigating the effect of nicotine on the behavior of experimental animals, Werle and Schievelbein (1988) wrotc, one can distinguish a stlmulaut component from a sedative one, corresponding to the effect of smoking. Them writers also s sted that some investigators feel that nicotine has a positive influence on the leanring of conditioned behavior, but that one must be careful in applyvrg these results to humans, since the dose is so drastically different. Marley and Key (1963) reiwrted that nicotine was without effect in the new-born kitten, but produced behavioral drowsiness in 1-2-week-old animals; and, by the 3rd week, the drug produced behavioral alerting. At 4-5 weeks, the response to nicotine was similar to that evoked in the adult animal, namely, behavioral alerting (sa below, 150, for corre-ponding cerebral electrical activityJ. In attempting to analyte the pecking behavior in pigeons induced by apomorphine, Deshpande and associates (1961) noted that nicotine (dosage not specified) ptvduced a behavior pattern akinn to that seen after apomorphine, but in lesser degree. In dosage of 0.2-0.4 mg/kg i.p., nicotine intensified apomorphine pecking, making it so compulsive that the birds pecked incessantly. Only in a dose of 4 mg/kg, which produced vomiting in all pigeons, did nicotine block apomorphine- inducvd pecking. Vaillant. (1964) ddescribed the behavior of t5 white male Carneaux pigeons (maintained at 80% of their nA-!ib wrrnhtR hv nartial food denrivstion for the duration bf the study), using apparatus for operant behavior similar to that designed by Dews (J. Pherm. l•:xp. Tber.)l8: 393,1956) • the schedule in effect in all experiments was mult F2 FR. Nicotine caused suppression of responding under mult PI 180 sec (key light illumination red, and the fvst peck on the key after, 180 aec had elapsed resulted in the immediate presentation of food) FR 30 /4y illumination blue, and every 30th peck resulted in immediate presentation of foodJ. A dose of 0.3 mg nicotine administered into the breast muscle produced equivocal change~, of behavior of a gTossdy observable kind, and the suppression of responding lasted about one•half hour. There was no suggestion of antagonism of the nicotine supprerWon by either atropine or methcl- atropine. In an important pioneer study, van Laer and Jan-ik (1963) have investigated smoking behavior in rhesus monkeys. Sua animals were given access to metal tubes through which they could suck air passing over hevted tobacco, or over an empry heated chamber; they were tested in 4tfay cycles, receiving tobacco on the first 2 days and heated air on the second 2 days. All animals developed a strong preference for tobacco: the average sucking-rate over the 2 tobacco-days increased, whereas it decreased considerably over the 2 non-tobacco- days. An additional study compared the amount of sucking on a tube holding a Gght.ed cigarette with one holding an unlighted one; here again, the 4 monkeys tested showed a preference for the lighted cigarettes. The results, taken together, indicated that something exists in tobacco-smoke, or in the volatile products of tobacco, wh:ch can act as a positive incentive for monkeys, so that they will spend some time during the day smoking, in preference to the other possible e.ctivities available. In an additional experiment, 4 monkeys were given access to cigarettes containing 2U mg morphine sulfate, which led to a marked increase in smoking for I animal, but not for the others, who generally had a lower smoking rate. Jarvik (1987) has amplified this pre. )iminary report, and summarized his results: A small group of 6 monkeys aould be induced to smoke cigarettea, but the amount of nicot inP in standard cigarette-tobacco did not seem to have any in8uence. -'t msrked preference was shown for a very fragrsnt pipe-tobacco over cigarette-tobacco, although here too individual differencea were large. When smoking was forced by means of a stronger incentive (water for thirsty monkeys)R the preference for tobacco over air persisted, and the amcurnt of tobacco vapor inhaled was greatly increased This worker concluded that it was possible to elicit and maiti- tain smoking behavior in monkeys with no other incentive and on a free-choice basis, but that such behavior is relatively weak compared to other ingestive behavior (such as drinking) which satisfies an obvious physiological need. Relative to this sobject ar+r the studies by Dcruau and Inoki (1967) on nicvi- tine self-admirdstration In rhesuq monkeys, in which it was was found that, under certain circumstances--whicb in no way rr.serMbled the natural habitat of the animals, however-- monkeys iv ql relf-administcr the drug intravenously. But the fact that nnonkeys will self-administer nicotine at all suggedted to these later workers that this alkaloid ma,v be one of the sub- stances in tobacco-smoke which is responsible for the use of tobacco by men (see belanr,1131). >\fam' of the aepccts of the "smoking behavior" of humans may be derireii ;•-om thr account, below, 1129, of "1ypes of Smoker.:"; a• %% eI, as 11y_1. Smoking Mocior. Produced bv Thn Councii for .Tobacco ptcscarch-USA, Inc. 0002937

26 TOBACCO-EXPERIMENTAL ATD CLINICAL --TUDIFS. $UPPLE.IIE\T I 120. Learning (43) ,.ouu/!W`• un.n a- .."...u...u of nicotine tartrate to rats trained to discriminate betaeen colors according to the method described by Cain and Ex- tremet (C. tettd. soc. Biol. 148: 115, 1954). Doses of 0.5 and 0.75 mg/kg depressed performance on the first day, but not on the second; increasing the dose to 1.5, and then to 2 mg/kg, depressed performance for the first 4 days, with return to normal on the fifth day of injection.ll'ith a dose of 2.5 mg/kg, a relative tolerance was reached Ly the fifth week. Following a rest of 15 days, 2 or 2.5 mg/kg nicotine tartrate dropped performance to zero, but recovery to 8VC- and 100';0 occurred in the next 2;dags. The authors conrluded that the rat adapts rapidly to the initial fxrturbations produced by nicotine (compare I. Gcllcr et al., 1980, Lcln.c, 121). Using a 4-uuit, 8-cul Lashley IlI alley mase, Robustelli (1963) studied the effect on learning of 40 male white Wistar rats given 0.2 mgfkg nicotine s.c., compared to 40 control animals receiving the same volunme of distilled water; each rat was maintained in an isolated state for 15 min before being placed in the labyrinth. In the first study invo/ving half the rats, the ry'totivating influence .vss food, the animals being maintained att weights of 85% of the values recorded on the second day after a 24-hour fast. In a preliminary training period of 9 dgys, a straight corridor was used leading to the "goal box". This was followed by an 8-day training period in the labyrinth. in which, in one eeperiment, nicotine treatr ments were confined to this period, and, in a second experi- ment, injections of the drug were begun 7 days previously. The difference between the mean of the errors committed in tests 2-8 by animals in the nicotine-injected group, and the mean of t%e errors made in the same period by animals in the control group, was not statistically dgniflcant. However, the learning curve showed that, in the serond phase of the learning period, the animals of the e.eperimental group showed an average performance which was progrecsively supermr to that of the ratP of the control group. '1b evaluate the level of performance achieved by the two groups at the end of the training period, the number of animals which had traversed the labyrinth without an error on the last day was determined; and, in this comparison, there was a statistically significant difference in favor of the experimental group. In a second study on 19 nicotine-treated rats and 20 control animals, the motivating influence was thirst, in nhich the weight of the rats was spontaneously stabilized at about 90% of nor•nral value. Again, a preliminary training period of 9 days was ttsed, followed by an 8-day training period in the labyrinth. In two experiments performed, nicotine injection are.c begun 3 days before beginning of the tests in the labyrinth. In this study, the mean of the errors made in tests 2-8 by the nicotine- injected animals was significantly less than the mean of the errors made by the control rats during the same period. The results of the two studies indicated to Robustelli that nirotbre in the dose used facilitated the learning of tsts in the lsbyr)nsb. The limited facilitation obtained in the study in which motivation consisted of the search for food was attrib- uted to two factors: first, since nicotine decre,a.ces the appetite, it is possible that there was a diminution in the motivation in the animals treated nith this substance; second, it ia neressary to take into consideration, as had been demonstrated by Petrinovitch and Bolles (J. Comp. Physiol. Psychol. 47: 480, 1954), that different types of motivation produce different "patterns" of learning. Thus, it is Iwssible that a drug, n•bile not having a direct effect on motivation, may nevertheless act differently on the specific "patterns' of learning deter- mined by different motivations. In contrast to the effect of nirotinv in riw'rrAalnn Rmwfi}P 1hi. auhq/nnrP h»rl no mnro than a"conirol-drug" effect on selectn-ely-induced drinking in rats (Stein and t3eifter, 1961, 1962), which might have some bearing on Robustelli's finding that thirst a•ar a ntiorc potent motivator than food. Robustelli s results have Iyecn summarized and discus.a4 by liovet-Xitti (1965). 'l'hc con• tributions of I3ovet, (1965) and Ilovet-Nitti (1965) on the action of nicotine on conditioned behavior in naive 'and pre• trained rats, a+rd that of Domino (1965a) on the effects of nicotine on both established conditioned evoidance behavior in rats and on acquisition of conditioned tivoidance behavior, all of which were presented before the Btotkholm Svmlw.aum on Tobacco Alkaloids and Related Compounds, are more appropriately described in the tollou•ing section. The possibility mtist be considered that unfavorable effects on performance may not be due to any specific interference with the learning prucess or pattern kself, but may be ex- plained on the basis of nmt•specific effects upon the general health of the animal; that is to say, probAbly any ribstainrc administered in to:dc dosage would be apt to have a delete- rious influence on perfortnance or learning (1'. S. Laescm and Silvette, 1905b). As we have indicated, however, liovet \itti considered that the active dosage lerel., in animals studieiE in their IaborBtory were relatively near those, reached on ;nho.la- tion by smokers; and Domino (1995a, p. 163) stated that, in general, his own doses were lower than those employed by the llovets. At the eame time, it mu-.t be remembered that the effect of nicotine on leaming in the rdt is somethind imt a priari akin to t.bPr effect of smoking on "exlmrimentol" learning, or learning processes, in man (42). 121. Conditfoned Ra•,flexes and CmWiHorjed Beharior (44) I. Geller, DeMarco and Seifter (1960) 16ained hungry rAts to time precisely, by rewarding with food those lever-pre.~Ing responses spaced 20-22 see apart. No significant effect occpr- red until the third day following a test dose of 0.1-0.25 mg/kg nicotine bitartrate, at which time there appeared a flattening of the interresponse•;time distributions, an increase in the average response.rate, and a marked decreese in the total number of reinforcements (food reu•ards),obtained. At this time, the animals were observed to be somewhat agitated in the experimental chtvntber, but not in the honte cage:; but no such agitation had been observed on the day of the drug injection. One wonders how such an effoct as thv above, noted 3 days after tF single small dose of nicotine, can hc reconciled with the fact that, by this time, the drug hed presumabl,v long since been entirely metabolized. It scems imposcible not to conclude that these animals were eoudi- t5oned to some other part of the eamrimental pra•elum, rather than that the effect was due to the nicotine per sc: witness the "agitation" correlated with the •'experimental chatnber", but not with the "drug injection" /tvnrywne Merc.ier and Des.migote (19130), above, 120J. Tobin (1962) stated that he and co-workers had shown that nicotine inhibited the conditioned avoidance response of trained rats, which he took to be a sign of nicotine addie• tion or habituation. In Bovet's laboratory, however, anerc the effect of drugs on the acquisition of a simple conditioned avoidance reaction in naive rats was studied, using light as the conditioned stitnulus and elcctrie shock as the uncrntdi- tioned stimulus, s.c. injection of 0.2 mg/kg nicotine producwl a clear-cut facilitation of avoidance conditioning (liovet, Iiignami and Robustelli,1963; bilttiami and Robustelii,19ti3). 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NERVOUS SYSTEM These mtter results were said to confirm the findings of Lucromzsa)a tlaor, cnea uy Iwvet et at., ltJOd). .3cquisition of avoidance conditioniug reaction with Gght as the conditioned stimulus and electric shock as the uncon- ditioned stimulus was further studied by Bovet (1965) using a Warner-type shuttlebox and male albino R'istar-+derivcd- strain rats and the split-litter technique to reduce variability. Kirotine tartmte was injected s.c. 15 min before the trial for the fust 3 days of the esperiment in 3 groups of 8 rats each at dosage-levels of 0.05, 0.20, and 1.0 mg/kg; while 2 group;; of 8 control animals each received an equivalent amount of distilled water. Performance of the rats receiving 0.20 and 1.0 mg/kg was significantly better than that of the controls; however, interruption of treatment from the 4th to iBth day was followed by a rapid lowering of the performance level. Statistical analysis of 8 ecperiments comprising 96 kontrole} and 96 rats receiving 0.20 mg/kg nicotine tartrate revealed a significant difference between the two groups, the prcaportiom of conditioned responses in the course of the first 3 days being 33% for the controls and 42% for the treated animals. t/om, pariron of the different eqleriments indicated that, for 5 of these performances of nicotine-injected rats, performances were significantly higher than the controls; in 2, the dilferent* was not significant; and in 1, a lower performance by the nicotine-treated animals bordered the limit of significance, Ordered in relation to level of performance in the control group, the positive facilitating effcct of nicotine on learning appeared more clearly demonstrated in those experinients in which a relatively low level of performance was noticed in the controls. In the course of conducting these conditional trials, a particular t3qre of response-called intertrial or non-motivated movement-was observed, consisting of spon- taneous activity of the animal during the time t?etween successive conditioning trials; this did not correspond either to the unconditioned escape-reaction or to the conditioned avoidance-reaction. Under the experimental conditions em- ployed, the animals which tried to go from one part to another of the box during the intervals between the conditioning cycles received an electric shock on the grid, and returned to the original side. Normally present during the initial period of conditioning, the frequency of the ir.tertrial reactitons was higher in the nieotine-treated group; for the 2 groups of 96 animals previously mentioned, the relative frequency of the intertrial responses was 10% in the total group and 16% in the nicotine•treated group (p < 0.01). The increase in fre- quency of these intertrial reactions corresponded to the central stimulating effects of nicotine. An investigation on the genetics of behavior, conducted in Bovet's laboratory by llignami (1964, unpublished results cited by Bovet, 1965), permitted establishment of two strains of rats char.t•terized by very slow or by very fast acquisition of avoidance condi- tiotting. I3igtutmi, Bobustelli, Janku and Ilovet (1905) studied avoidance conditioning in rats selected on the basis of slatrneF.a of their ability to acquire conditioned avoidance; the animals were placed in aWarner-tyl)e shuttlebox and experidnental conditions consisted of a conditioned light stimulus prcn•eding the unconditioned stimulus consisting of the passage of elec- trical current through the grid. Administration of 0.2 ittg/kg nicotine s.c. signifit•antly improved the performance of such rats; but, as Werle and $rhierrlbein (1906) warned labove, 1191, one must. be careful in applring such results to htimans, since the dose of nicotine is so drastically different. The relatintnhil-.c observed betn•een the facilitating effect of nieo- tine and the level of perfotmanre in the control ttmu)r of 96 rats led )iovet (1965) to study the action of nicotine in a 27 series of animals belonging to Dignami's low -avoidanct, strain. i-uuuai+ euuumg it very lua• lecel 01 conuittontng at the end of the 5 daily sessions of 50 trials each were eubtuitteil to much longer daily sessions consisting of 250 cond6tioning trials hmting 125 min. After a certain number of sexion's, some of the animaL exhibited a behavior characterized by (a) the slowness with which they Mscquired an avoidance-behat•ior during each daily session, and (b) the almost complete abxnce of retention, which assured a relative constancy of the Eearning-curve during the successive daily trials. These chnr- acteristics pet•Rnitted direct comparison between the perfornl- ance during treatment with that of the preceding and folldw- tng days; and such comparison showed an aspect of nicot•lae action which rlemonstrated a very marked reduction of the time necessary for the reacquisition of the conditioned behavior in the "low avoidance rats." The effect of 0.2mg/kg nicotine was found to be comparable, in this nexpeet, with 0.5 mg/kg aui,phetamine. Q.l mg/kg Delysid"', or 5 uq/kg benactysine and Ditran®. Observations were also reported on the action of nicotine on motor and inhibitory conditioned res)wnses prei7ously acquired, by means of esperituelits performed on rats trained for a double instrutnental escape conditioning with single or associated stimuli. In the course of two series, the animals successively learned two t31x.r• of conditioned reactions; the first, motor conditioning consi;ted of an avoidance-response to a conditioned atimuhis represented by a light-:~gnal (light CtnS); the second, inhibi- to,-y conditioning consisted of a double stimulus, both stwitd and light (sound and light aS). Follou•ing the doulile stimultn., the animal was not to move to the opposite com- partment to avoid being punished. The data indicated that doses of nicotine (0.2-1.5 tng/kg) active on the formatron of conditioning, did not modify motor and Inhibitory rnnditioit- ing previously obtained. With higher (3-5 mg/kg),dtses, motor conditioning was more or less cloarly depressed; pnhitii. tory conditioning was only n•eakly depressed at a dose of, 3 mg/kg; but these findings with the higher dosrs were tittrib- uted to nonspecific toxicity. The results obtained with nicotine in the Warner cage, just described, led l3ovet-\itti (1965) to extend the tests to different or more complex forn.ts of conditioning. The Yerkes-Thompson-I3ovet-Nitti ap)larn- tats, consisting of a large box, on one side of which arc 2 to 5 doors, only one of which is marked by a particular paltterht, and may be opened by the animal, permitted study of the escape-reaction induced by an electric shock and then of the action of nicotine on visual discrimination. Patterns of itictra;- ing complexity were presented to rats previously trained to distinguish between white und black colored doors, the i+e.ults being calculated on the basis of the number of errors (Osralm sttempts through the blocked domx), the nuniber of errorle=cc trials, and the time between the entry and exit from the elee- trified part of the cage. Control and nicotine-treated animalv were ,rtbmitted to learning I rial w,drr ,.vnparablr conditiaie (10 trials a day for 5 to 10 consecutive daya); atnl liartud results from experitnents still in prot;recs at the time of n, porting were said to indicate that nicotine can facilitate certain forms of visual dircrimitwtiun. In xnme triah wing a 2-door cage and diccriminating beta-er•n two sintilar pattern-, pretreatment with nicotine sigttifitmntly accelerated leatving of 2 of the 4 couplea of patterov utilized; in the 5-door cage, far1tation u•aF observed only for I of the 3 couples of pattenn5 used. Studies were aW reported on the action of nicotine in Sidman-Gatti lever-prering avoidattre; this method tva• desrribed as involvittg regietration in a Skinner box of the operant mslwntes (lever-pmsing) of a rat, whirh permits the Produced by The Counc;I for 0002939 Tobacco Reseai-ch-IJSA, Inc,

28 TOBACCO-EXPERIAIENTAL AND CLINICAL STUDIES. BUPPLEJIIENT I animal to avoid a nociceptive stunulun (electric shock) follow. ino nnnn fho mmt;#;..nwt Y..t.o ovi....,r.w TL.. .,..rl.... .+..,..1... sised 't.hat, during trials in the shu¢tlebox, any intertrial response is punished, while,, in the Sidman-Gatti type of avoidance-conditioning, each intertrial response leads to a 20- sec postponement of the begunning of the next cycle. Data, graphically shown, indicated that 0.2 mg/kg nicotine caused a reduction in reaction-time, and an increase in the number of operant and conditioned reisponses, and, at the same time, produced a reduction in the number of unconditioned and intertrial responses. These wene considered to be chatactetistic effects of nicotine, related to those of amphetamine, and diametrically opposed to those of chlorpromasine and thymo- leptic drugs (C. L. Gatti, 1964, to be published, and here cited by Bovet-Nitti, 1905). A study by Gatti and Bueei (1964) of effecta of nicotine on the learuing of a conditioned behavior by Wistar rats involved two techniques: one, the pole-cGmbing response to the sound of a butuer to avoid re- ceipt of an electric shock; and two, lever-pressiug at a liight sigual to avoid receipt of an electric shock (discriminative avoidance). In the first technique, 5 animals received 0.2 mg/ kg nicotine s.c. on each of 5 days, and 5 controls received sa- line; in the second, 5 rats received the same dose of nicotine for 10 days, while 5 controls received saline. With both tech- niques, the tests were made 15 min after the daily injection. In the pole-climbing-response tests, the nicotine•treated ani mals showed faster learning ol the "avoidance behavior"; in fact, by the 5th day, the percentages for the conditioned re- sponses were 87 for the treated rats ardd 60 for the contnds, and the not-conditioned resr.,roses, respectively, 13 and 40. Better results were obtained in the discriminated-avoidance study: findings here are graphically shown in the original ar- ticle; and, in the treated rats, it is possible to perceive a con- tinuous constant improvement of performance under the ac- tion of nicotine. The percentage of conditioned responses increased day by day in parallel lines, with a decrease of notV conditioned responses, while the percentage of spontaneous responses was not significantly modified. The °reaction.tin:r' also progressively decreased, while the ratio between the re- celved stimuli (conditioned + not-conditioned) and the re- sponses obtained (conditioned + not-conditioned) was in- creased. The authors concluded tBat these findings showed the facilitating effect of nicotine on the learning of a condi- tioned behavior in the tat, and they consideted it important to note that• the percentage of spontaneous responses was not modified, thus demonstrating that the effect of the drug is specific, and not due to a generic, aspecific hyperexcitability or hypermotility. In addition, tests carried out after 2 to 7 days were said to have shown the persistence of a good per- formence and the definitive character of the lmrning. In her excellent discussion following her ov~n paper and that of Bovet, Bovet.ll'itta (1905, pp. 142-143) said, in part, that the reported results suggest that nicotine administration can impmvn the perfonnence of the naive animal during :ondi- tioning and learning, and that the drug exerts a r.tamulant action on some form of previously-acquired hehavior. With respect to mechanism of action, the effect of nicotine on conditioned behavior may be related to two more general problems of plt,vsio- and psychophamtacologv: the fust con- cerns the role of eholinergic transmission in the CA'S of mam- mals; the second deals with the physiopharmacology of wakefulness and, in particular, the conception concendug the existence and various functions of a double makefulness system. According to 8oulairac's (.Annales Endorrinol. 6: 731, 1958) concept of a double wakefulness system-in which the first system, sensitive to amphetamine, has under its control .. ~,: ....... . eN....wu.vue activity, consciousness, and elementary forms of conditioning; and in which the second, cholinergic in nature, controls the more specialized and elaborate forms of activity-, the effects of nicotine on behavior, according to Bovet-Nitti, may be attributed to a facilitation of synaptic transmission at the level of the second higher system of wakefulness. In the next following paper in the Symposium, Domino (1965a) reported the effects of nicotine on (A) established conditioned avoidance behavior, and (B) acquisition of con- ditioned avoidance behavior, in male albino Holtzman rats. Initial observations under A were directed to effects of nieo- tine on conditioned pole-jumping, in which each of 30 animals was given a total of 60 training triala, 5 per da,v for 12 dey s. The conditioned stimulus was a fhvolt buat.er presented for a 15-see interval, followed by a 30.sec unconditioned stimulus consisting of ad electric shock to the grid floor. After training, animrls manifesting 95-100'io avoidance-behavior were se- lected randomly into groups of 6 for s.c. administration of nicotine in saline solution in increasing dose.c of 0.25, 0.5, 1.0, and 2.0 mg/kg; each animal was tested 5, 15, 30, 60, and 120 min after injectaon. Prior to the injection, and at each of these tame-hrtervals, every rat was given 5 trials, and its avoidance- and escape-behavior determined; all animals were injected with each dose once, and then discarded from subsequent nicotine experiments. Doses of 0.25 mg/kg had no significant effect on estab6hed conditioned avoidance behavior, whereas dom of 0.5,1.0; and 2.0 mg/kg produced depression of avoid- ance. Escape-avoidance, however, wtis not as markedly de- pressed, indicating a more selective depre,.ion of avoidance- behavior. Doaa~,of I and 2 mg/kg produced definite convulsive movements within a minute after injection; these gradually rrbsided. It was said to be unknown if the depressant effects of nicotine are secondary to poststimulatory depression, or it they are a mdiufestation of an action of nicotine unrelated to convulsions; but Domino noted that animals which had the severest convulsions phowed the greatest depression of con- ditioned avoidance behavior, suggeFting that the two actions might be relat(d. In these ex1teriments, the greatest per-cent depresQion of conditioned avoidance behavior occurred 5 min after nicotine injection, and, in general, the largest dom-a producrd the longest. 4uration of effect. Administration of 2 mg/kg s.c. 2 hours after the initial dose produced lex` depres- sion of avoidance than the first dov, suggesting mild tolerance development. With shorter intervals between nicotine injec- tions, much more tolerance Itachyphylaais) was observed. The author next studied antagonism to the effects of nicotine on established conditioned avoidance behavior by various gangliohic blocking agents; 5 mg/kg mecamylamine, 5 mg,'kg trimethidinium, or equal volumes of 0.917, saline were itqectnl s.c. 1 hour prior to similar adimnistwtion of 0.5, 1.0, or 2.0 mg/kg nicotine. Sinee quaternary compounds (e.g., trimethi- dinium) may be postulated to be relativell• ineffertive in penetrating the blood-brain barrier, in comparison with tertiary ganglionic blockers (e.g., mecam,vlamine), an indiea- tiou was rought as to whether the effects of nicotine on established behavior are peripheral, central, or both. The equipment used here differed front that in the first study, in that the pole in the center of the box aa.- connected with a mierosaitch, wluch the n-eight of the animal on the pole was suficient to activate, thus terminating the burter and shock applied to the grid floor. The data showed that rniramyla. mine effrctivcly antagoniacd depn:~ion of conditioned avoid- aore hehn.ior by nicotine at all three dose•IeveLa. Trimethi- 0002940 Produced by The Council for Tobacco Research-USA, Inc.

IJ NERVOUS SYSTEM 29 : : I : : , t : dinium also effectively antagonized 0.5 and 1.0 mg/kg doses .r ..c..~.C.... 6,0 .,+... ....,..1, 1.,.-,. ../r...s:... .1..... ......+......t....:..~ in antagonimng 2.0 mg/kg nicotine. These results led Domino to conclude that depression of conditioned avoidance bebavuor by minimal doses of nfcotine has primarily peripheral compo- nents, while large doses of nicotine act, not only peripheralEy, but also through central mechanisms. [ASecAmylamine pro- tected +tgainst the tremors and convulsive movements induced by larger doses of aieotine, while trimethidinittm was less effective in this respect. Binx, in Domino'b first series of e.periments, those auimsls w hicb had the severest nicotine oonvuiaions also showed the greatest depression of condi- tioned avoidance behavior, this differential antagonism of mecamylamine and trimethidi.nium against nicotine convel- srons may well be of firsGorder importance in interpredbg thin worker's results] In this series (A), the effects of small doses of nicotine on established conditioned avoidance bw havior in the monkey were also studied. Four young adett male and female 111aonca mulafla monkeys tcere trained to avoid an electric shock to their tail by depressing a telegraph key, using flickering light as the conditioned stimulus. AU animais were given saline or 40 µg/kg nicotade i.v. (this dose of nicotine was twice that found effective in causing EEG activation in the brainstem-transected monkey (D. E. Knapp and Domino, 1962)), and latency of avoidsnce behavior aas determined by an automatic stop-watch. No significsnt effect of nicotine was found, however, avoidance, latency being slightly prolonged in some animals, and slightly reduced ?o others. In the second series of studies directed at effects of nicotine on acquisition of conditioned avoidance behavior (B). initial experiments were made to determit<e the action of nicotine in rate with assisted training in pole-jumping, and the tesC-situatinn was similar to that used In the ganglronit- bbcking st4dy described above. Four groups of 12 naive mta were used, one group receiving saline, and the others, lA, 40, and 80 pg/kg nicotine base s.c., respectively. Training aas begun I min after injection, each animal being given ~0 training trials before the per-cent avoidance behavior w6s determined; and this requence was repeated approximately 24 hours later. On the third and fourth days, the animals arre subjected to 20 extinction ttials; no injections wePe made; the animals were simply placed in the conditioned avoidance situation, and run with the unconditioned stimulus off. The effects of 20 pg/kg nicotine and of saline were similar; 40 pg/kg facilitated slightly avoidancn learning (p < 0.05), and the rats in this group were also more resistant to extinc- tion during the first 10 trials, but, subsequently, extinguished similarly to the saline controls; go pg/kg signi8cantl,y depr~ed avoidance learoing and facilitated e~~tinction (p < 0.05). Domino was uncertain whether the improvement in aryuidtion observed with low doses of nicotinte repirsented an increase in learning, or whether some other non-controlled variables were operative. A study was next undertaken of the elUwk of nicotine on avoidance leanung in a completely isa inted situation; in this, the naive animals wete in the pole, jump apparatus within a sound-reducing enclosure, a one-way uindoa permitting observation of the animad. After 5 ntidt acelimatisation in the box, the injections were made, and the Uial, begun I min later; the injections consisted of either taliue or 20, 40, 80, 160, and 320 pg/kg nicotine. The result's indicatedl that large (160 and 320 µg/kg) doses of nicotine dccrea.wi avoidance learning; but some evidence--thougb oot near)y as conclusive as the foregcing-was,obt,ained that Mµe/kg nicot ine might facilitate avoidance leattiing. Domino ronddrred that these results must indirate that these quanti• ties are within a dose-range,which is critical for nicotine to ..... . .. .a.. ~. . ..:.. ..t .. »..r . .. . . .. . . a ...~ ..t,....ta m... to emphasize the potential importance of such a quant5tation of the biphasic action of nicotine on the CN6. In a third study of pole•jumping, a double-blind design was used; the interior of the box in this experiment was circular rather than square, and the box was , placed in a sound-dampened enclosure with a one-way cvindow- S.c. injections were of saline or of 40 or 80 µg/kg nicotine, but no significant differ- ences in the learning curves were obtained. Lastly, effects of 5 daily s.c. injections of 40 or 80 µg/lig nicotine base on acquisition of conditioned avoidance belhavior in rats were determined, using teehniques similar to those in the second and third experiments in this (B) series, and a group of saline. injected controls were studied for comparison. Data were presented and analyzed in several ways, leading to the con- clugion that small doses of hieotane improved acquisition of conditioned pole-jumping, but whether these effects were just barely significant or were non-signi6cant depended upon t,he statistical test used. In an over-all ionclusion, Domino stated that the behavioral actions of small doses of nicotine tu+e subtle, but consistent with the hypothesis that nicotine stimulates the brainstem activating system to produce behavioral arousai (see also below, 150). Domino (1985b) went on to compare certain pharmaonlogical actions of (-)-nicotiae, its optical isomer, and related oompounds. To determine relative effects on conditioned avoidance behavior, conditioned pole-jumping was eatabllished in trained rats given s.c. injections of (+)- or (-)-nicotine in dosage of 0.25, 0.5, 1.0, and 2.0 mg/kg. Increasing doses of both isomens progres- sive;ly depressed the conditioned avoidance behavior, (-)-nicotine being the much more effective of the two. Calculations gave a potency ratio of 7.1 for 50% blocked avoidance; but, since the sample of (+)-nicotine used in them studies tras only 86.4% pure (the rest being (-)-nico- tSne contaminant), a potencrv ratio of $.01 of (+)-/(-)- ntcotine would therefore indicate that the (+)-isomer was inert. Additional pharrnacological studies of the comparative acti'ons of the immers on other body-systems or functions reitifomed the conclusion that (+)-nicotine is pharmacologi. cally very weak, but not inert, (-)-Nicotiite, nicotine methio- dide (in which the pyrrolidine nitrogen is m.^ihylated), isonicotine meth,odide ~in which the p+,,rldine nitrogen is methylated), and nicotine bismethiodid' e (in which both nitrmttens are methylated), and the ganglionic stimuiant, dimethylphenylpiperazinium iodide (D\ifil'), all in dosages of 2 mg/kg s.c., were cortlared for effeets on conditioned pole-jumping in rats. All eucept nicotine were relatively ineiliective in blocking this conditioned avoidance behavior. The author noted that all bitt nicotine are 100% ionizable quaternary nitrogen compounds, to which the CNS is poorly accessible. The discussion following the paper by Domino (1985a, pp. 163• 188) is interesting, i¢tformed, and far-mnging, and the discussants also eommeqted upon the papers by the Bovets. (Domino, Incidentally, congratulated the Bovete on a superb piece of work, and added that, as usual, they were several years ahead of the thinking of many investi- gatorg.) Such a meaty discussion can hardly be summarized, and anyone interested in the subject of nicotine and experi- mental animal behavior should, of oourse, read It at first- hand. 13ut one question asked by A. K. Annitoge of Dr. Dwninu and Prof. Aovet seems to us to be even more far. sightpd thnn far-mnginR. In,their (ilovet's; Bovet•Titti's; Dontitni.l cxpcrimenta describing behavior which were re. Produced hv The Ninicil for o 0 o z s 41 Tobacco Rasea,ch-tjSA, lnc.

30 TOBACCU-EXPIat1AlEKTAL Arl) CLINICAL z,'TUI)IEa. SUPPLlt2L%tf•:\T I corded automatically, Armitage wondered if they were able to Kn) a ueuier tne alitma/. wunrv au)' uap ner ur /e.+ .uc~xi. Domino replied that he did not think t~e rat looks halbpy after nicotine; of cotitse, he ssirl, he renll)• could not tell, but his impression was, they looked a little sick. Bovet stated that all their rats-nirotine-treated and eontrola alikr-secmed to be very happ)•, if they, could encalM from the normal box when they saw the experiment in the room, and they asked to go to trial in the disc•rimination-box, even if they had the, electric shuck. Sinee human smoking is above all a pleasurable behavior-pattern, and often said to be a"tel><<ion•redueer", these inteqpret,'ations of the ••feel- inq.•" of the nicotine-treated rat are intri ~p;uing. \icotine depressed the lever-rcswn.ce rnte in ratF trained to depress a lever sMitch for a fund reu•e!•1 delivered af'ter every 10th reslwnee; or on a multiple rwhedulc consirling of 2 components: (a) fixed interval, and (b) differential reinforcement of low tsles of nstiondinR 'illareal, Schuster and Domino, 1965). Atropine did not block the nicotine depression 122. INTEiiRATED rFFE1.Ta, Pltlaf.lRIL1" SEN>;Om' (45) 123. Sensory TAreshold (45) E\o new data.) 124. Viareref Pain (45) iDistinguished from cutaneous liain, above, 110.) 1'erhalu closer to ".isceral" than to "cutaneous" pain is the observation by Cofiman aml Javett (19fi3), who noted that all 6 subjects :eceiving 4 me, nicotine bitartrate i.v. in 100 ml saline over a 10-12-min period esperiencet! aching in the arm into which the nicotine was infused. 1;):5. tNTE6RATr.D EFFRC7s, PRIxtARILl' NoTOn (45) 126. Nu+otine Colwufeeons If the dose of nicotine is sufftcient, tremor (see below, 180) is usually followed by oonvulsions, Orcutt, \lichaekwn and 1'rytbPrch (1963) observed and described ei8bt progressive stages of cerebral nicotinic effect in rats injected with 0.5 mg/kg nicotine in the dorsal tail• vein: 1. Prostration-an extreme astheltia, including the inability to ata.nd; collapse in an awkward position; disin• c6nation to any acthity; without paral_vsis (exhibited by 100,70 of 78 animals tested); 2. Fihe Trrmm-a rapid (10 or more per sec) shaking or vibret6on of restricted extent, particularly noticeable in the extremities (exhibited by 100r'e); 3. Clonus-rapid (about 31xrt sec) alternate contrac• tions and relimtiolw of groups of muscles, causing an exeur- sion of the feet of I em or more (exxhibited by 100%); 4. Cfonic Conotdsiona-Involuntary, generalieed patoxysms of alternate contractions of opposite );rouiu of musrles over the entire body (exhibited by 100 0); 5. OptaUwfonus--backward arching of the head and hind•limbs from tonic spasm of the muscles of the back (eNhibited by 83 %); 6. Tonic F7exion-- tonic spasm of the fleuror mtucles of the fore•limbs, acenm- pany-ing opisthotonus (exhibited by 47%); 7. Tonic lixtcWnn -tonic spasm of the eitensor rnusdcK+ of the fore- and hind- limbs, iMereostals, and diaphragut, accompanying opistho- tonus (exhibited by 42 c,-,); 8. DeatA-respiratory paralysis fronm prolonged tonic e.pasm of the respiratory muscles (ex- hibited by 18 0). 12"s. Con.•ulaf-re Dose of I\ieotline (49). Fatal doses of nicotine generally cause colrvuhiom: in all laboratory animal<, uut u1e cwnuiai%e uobr ~i.ii/ ui 1tl'WtN/P 10 grjmuu~% uu4rJ- sitood to be that dose which inducccconvulsions from •a•hirh the animal recovers virtually unharmed. lionta and co•n•orkels (1960) reported that low (up tu 0.6 mg/I:g) doss of nicotine i.v. indured only tremorA in ruice, whereas, with higher doarN, the tremors were ralndly follou•ed by clonic convulsions or even by seisures of the tonic-extensor type. The i.v. t:D.r in this strcrie+ war re- riorted to be 0.4 mg/kg (Hans and lilavchn, 1955); the C:1),,, 0.43 mg (Kerley et al., 1961); and 0.45 rng/klt was said to be a surely CD (I:roncberg, 1955). On i.p. injection. CD,o averaged 12.8 mg/lat; (range, 12.0-14.0) (Fujiwam, Yamawaki and Shimamoto, 19G1). A dose of 7.5 mF/kt; tyir•otine tarlrnto i m. Prndlnrril etmd- mnvulcin•rc in CVI- strahl mire (>IaNn and Smein, 19M). lntracerebral injec- tion of nicotine by the methqd of Halcy and :11cConnick (Brit. J. Pharmacol. 12: 12, 1957) caumd immediate a/ai siometimes violent convulsions kl mhr (Gok6ale and Culati, 1962); the CDeo was calculated to be 1.0 f 0.52 µg/kF. With an effective dose, convuLdurts bcc•urmd as soon as the dnl>: was admini=tered; these were followed by hyfa~nentilation and a period of hyperexcitabilily lasting for about 4-6 ntitn; none of the animals showed apuoea. ht the rat on i.p. injcc.tion; the CDa, the CDw, and tEle CD., , were found to br, m:.lKctivel,r, 1.0, 1.15 * 0.15, and 1.9 mg/kg (Cmkhalc iund Gulati, 1962). On i.m. ilqcc- tion, the ('D,e was reported to bc 20 mg/kg (l. Yalnamoto, 1963). Do+ax of I and 2 mK/kµ nicotine s.c. produced definite convulsive movements within a minute after injlrtion, r•hieh gisdualh• subsidcd (Domitlo, A65a). It may be noted saain here that rats which had the z<cvereet convulsions aho shoN•ed the greatest dblur,-Aon of, conditioned avoidance behavior, sugmsting that the two action+ might be related (tzee above, 121). Administration of 0.10-(N.15 mg/kg nicotine in daily s.c. injections for 2 weekr in 50 male ratc was said to have produced severe symptum, of acutc• intoxication, with touic• clonlc convulsions which s1>.read to all of the mttscks- and which produced tmnsitory res,piratory paralysis; most of the animals survived (t'era.nee, d965). Intracerebral injection of 2 mg/kg nicotine (pre<timabh• as the hydrogen tartrate( in 12 rats was immediately follcdu•ed by intense motor excita- bility and convulsions, the animals recovering within a feu• minutes (Carmo and DiaR Da SIva,1966). In the rabbit, the convttlfsh•e do.a on i.v. injection ha; been given as0.45 and 1.0 mg/kg nicdt;ne (Denisenko and Pratuse- vich, 1962, 1964) and 1 nig/kg nicotine acid tartrate (J. J. Reuse, 1960). With respect to the oomi>taradive comvlsant action of the optical isomers of nicotine, Domino (196lib) obtained a potency ratio of 6.2 for (-h)•oli~bthlc/(- )-nicotitte in the production of tonic extensor rrisures in 50% af test wtP, when the materials were Riven i.v. When the purity of the isomers used was taken into rfmxideratiun, a ratio of ti.01 would indicate that the (+)•iwumer u•a• inert (see abore, 121), so that the ratio actually found for the convulxant action showed that (-F)•nicotitde is phartuaeologicall}• very w eak, but not inert. On i.p. ilqection in white mice, nicotine, nornieotiue, ethy-Inomicotine, and allylnorttirothle all provoked convul• sion.a; convulsions caused by aeet-vlnornirotine and benzo)•1• nornicotine were milder, while earbaminoylnornicotine lvas apparently not convulsive in an%' • dose (\lattila and Varti• ainen, 1(I02; \lattila, 1963a, b; ilattila, Ahtce aud 1'arti• ainen, 1965). Tested on hydrated ratx, i.p. injection of 2 mK/ / e Produced by The Council for 0002942 ' Tobacco Research-USA, Inc.

KER1'oUa t;Y: Tk:.Nl 31 ekus v~uginga.uyuw.u.wuu~ay v;u.uuuwu.u:• simLr; but the other deriv&tives did not produce convulsions in the doses employed (Aldttila, Ahtee and Vartiainen, 108.i'). Like nicotine, the p}•rrolidiue V-0xide and pyridine N-oxide of nimtine caused tremor and clonico.tonic spasms in mice and clonic tremors in rabbits (Arutyunyan and \IaskovFkli, 1961). 128. Site of Convulaionb (45). Lendle and Ruplxrt (1942) (4.10a ) had suggested that apnoea or respiratory arrests might be the cause of nicatine couvulsidns; but Gokhale and Gulati (1962), noting thi lack of production of nicotine apnoea in mice, and the fact that, in the rat, nicotine con- vuhsions always preceded the period of apnoea but never followed it, pointed out tbat this propoEed mechanism there- forr can not be the cause. 129. Local.zation of Co'nvuleione (46). With few excep- tiotu, earlier workers who had investigated the antagonistic action of drugs against nicotine•induted convulsions had used as their criterion the weakening or enppr+ession of visible seizure,, although this is a crude and often misleading indica• taon of site or localization x<nd mechanisln of action (Silvette et al., 1962; Larson 'and Silvette, 1905b). A more refined technique for the investigation of the site and character of nicotine convulsions is oBtted by the electroencephalograph (EEG'r, and recent use of this instrument has yielded interest~ ing, if not conclusive results (described below, 150. Brain Potenlials). ln brief, nicotine produces a typical IiEG pic• ture of "grand tnaP' convulsions. In 3 experiments reported by liumagai, Sakai and Qtsuka (1962), 18-60 pg nicotine, iajected apparently into the midbrain reticuhtr formation, faiacd to produce seizure discharges in cats, as determined by EEG recording. Most recent workers, however, have preferred to ir.ject nicotine (and other drugs) intravenously, in )mrt in order to avoid loral destruction of brain tissue (see Silvette et al. 1962, p. 142). lo an extension of the work by iong,o and llovet (1952) and von Berger and Longo (1953) (ah+ehsfy described (46b- 4ia)j, Longo, von Berger, and )lovet (1954) attempted to elucidate the specific tylie mtd locus of action of several drugs on the convulsant action of nicotine. EEG studies were perfotmed on 80 rabbits and on some cats and dogs. In the norntal and "eru+ep6ol0 iaofls" ("isolated-brain") rabbit, nirotine caused some EEG changes charizcteristic of a grand ms) seizure; tt.eae changesfould be divided into three periods: During the first phase of 20-BO sec duration, the spindles and normal components disappeared, at8d were replaced by an uninterrupted series of vaves of 5- 6 eyclos/se¢ at 100-1S0 p\'; the second phase, lasting 70-120 see, was characterized by the convulsive seizure, in which spikes of 0.3-0.7 mV apfftred, particularly in the parietal lead; the third phase was long and variable in duration, u•itli convulsive spikes, together with slow waves of high voltajse (1.6 c/rec, 0.5-7 m1'). The site of the andinicotinic effects of caramiphen (Panparnit), ethopropazine, (Parsidol), cAlorprotnaaine (Lar- gactil), and trihea-)•phenid)-l (Artane) was considered to be at the level of the reticular substance, and probably involved a cholinergic mechanism, such as blocking acetylcholine ac• tivity centrally. Ftoris, \torocutti and Ayala (1962, 1964) measured Elsl: activity in rabbits (curarieed; tracheotomized under local anesthetic; under artificial re_spiration) with silver electrodes in contact with the dura (cortex), and deep eltv. trotlea in the hipixrcampus and int.prlaminar nuclei of the . ... ..... ...... . .....:... at the intereollicularulevel~through the prepontine area; in others, the cortex was isolated by a spatula after a subpial section. l.v. injection of 1-2 mg/kg nicotine in intact anintaL, induced an immediate de,ynchrotoiration of the rfwntaneous electrical activity of all of the explored anesu:, the cltanfte lalso referred to as itnmediate activation in all areas of regis. trationJ being similar to a phgsaological alerting response. Larger (2-6 mg/kg) do..es caused immediate alch•ting or activation, followed by bio.electric crisis, typical of a gen eralized convuL,ion of the whole animal. Identical effects were produced by the same doses in auuna4 with blainKtem sectioned at the intercollicular level. While there was a convuLaive response in the intact cortex hl atimalK with isYrlated curtex, there Na.+ none hi the isolated nrvar (Fluri,, Aiorocutti and Ayala, 1962). In 2 of the bminstem-xectiuned rabbits, the convulsive activity appeared first in the hippo• campus or in the thalamus, epMading aflerwanis to the cortex; but when the seizure activity became general, the discharges in the cortex and those of the hilqpocampus were not necessarily contemporaneous: the hippocantliu.+ dir• charges were continuous, while the cortical ones were ireriodi• cally interrupted by lalises into normal activity (Floris, \lorocutti and :lyala, 106A). In 4 animals, a large, rcctaugu• lar (8-10 mm) slab of cerebral coTtex w•as deafferenfed, and also isolated from the neighboring intact cortex by subpial section, the vascularizaCron of the slab remaiuiup intact ("rmrau isole" or "isolated cortex" preparation); two eler• trodes were placed on the slab, and two on symmetrical atras of the intact contralateral hemisphere. Lv. injection of 1-2 mg/kg nicotine induced cortical dwynchrourization in the intact hentisphere, but no changes in the activity of the isolated cortex; i,jection of 2-t} mg/kg produced eon- vulsive activity only in the intact cortex. In comparison, the activity induced by Metrasnl (pentyleuetetra*ol) ap• peered sytnmetrically in both the normal and the isolated cortex. These observations seemed to the authors to indicate that the nicotine-induced convulsive activih• originated in the hipporampus, and involved primarily hiplwcampal• thalamic circuits, while the cortical seizures were due to secondary sl.re.ad along affeirat pathways (Floris, Morocutti and Ayala, 1962, 1964). Dcnir.enko and Pratuowich (1063, 1964) reported that nicotine in do.¢es of 0.45 and 1.0 mg/kg caused convulwous in 12 intact rabbits, while, in decerebrate animaln (brain divided at the level of the inferior colliculi), nicotipte con- vulsions were observed in only I of 5 experinments. The authors compared arecoliue com•ulsions to nieot ine eonvulCions under various expcrimental conditions, and concluded that spatially distinct cholinergic atriuctures were wtsponsible for the development of rom•ulsioti.a after administretion of these drug.c EEG recordingR were said to have verified the h.•pothesis. 11io.electric potentials were recorded by \'ardapetyan (1903) from the cerebral cortex, the hypothalamic region, the upper cervical s.•mpatletic ganglion, and the central (cranial) end of the vattus nen•e of mnture, unanesthctiaed rabbit.a (meehanically immobilized) following i.v. injection of 0.8 mg/kg nicotine. The appearance of nicotine npasitt (convulsions) was preceded by inktrase in the bio.electric activity of the sympathetic nervous system, as indicated by increase in the amplitude and frequency of the bio•elec• trir Iwtentials. On this barkgrouud', the hypothalamic region and cerebral cortex were gradually involved, leading to a sudden seizure of generalit:ed epileptic spasms, during which Produced by The Council for 0002943 Tok^^0 t?~~~~~~f, t'C 1 Irc

32 TOBACCO-EXPERIMENTAL AND CLINICAL STUDIFS. SUPPLEMENT I all the investigated parts of the nervous system generated strong high-voltAge discharges. Cessation of the spasms was connected with the development of depression of the bio-electric activity of the ganglia of the sympathetic nervous system, followed by reduction of the bio-electric activity of the hypothalamic region and cerebral cortex. On the general background of depression of the bioaelectric pulse, the con- vulsive seizure keased. Identical sequences were said to have been obtained following administration of bfetrazol, camphor, and picrotoxdn. Thus, the site of the convulsive act'¢on of nicotine bas been variously localized; and, in this paragreph, we shall sum- marise the conjectures of some writens taken from Tobacao, as well as of later ones. Silvestrini (1956) considered that the convulsive effect was due to a general state of excitation, probably including the cortical neurones ;69a). Vardapetyan (1963) apparently regarded an increase in the bio-electrical activity of the "pathetic nervous ;aystem as the primary event, after which the hypothalamic region and cerebral cortex were gradually involved. Longo; von Betger and Bovet (1954) implicated the reticular, substance, although experi- ments (perhaps ttnsatisfactory) have been described Isee Kumagai et al. (1962), aboveJ in which injection of nicotine into the midbrain reticular formation failed to produce EEG seizure discharges. Stumpf and co-workers [St.umpf (1959) (69a-b); Dunlop, Stumpf et ql., (1060)) have implicated the hippocampus; and Floris, Itlorocutti and Ayala (1962, 198A) posLulated the origin of the convulsive action of nico- tine to be in the hippocampus, from which it spreads Lo the cortex by afferent pathways, Stem and McColl (1958) be• lieved that nicotine produced its central cenvulsant action at the level of the diencephalon (49a). In any event, the upper levels of the CNS are probr,bly broadl, involved as sites of the convulsive action of nicotine, and it has been suggested that the biochemical structures excited, at least in the reticu- 1ar formation, involve a cholinetgic aoecbanism (Longo, von Berger and Bovet, 1954; Ilyuebenok and Ostrovskaya, 1962; Denisenko and Pratusevich,1963,196Q). In his paper "On the Relation of the Content of DOPA and Chtecholaininea in Several Rat Tissues to Nicatine-In- duced Convulsion", Nagai tl96$a) discussed the implication of catecholaminea on the various stages of nicotine convul- sions; but neither the Braxiria abstnact (Nagai, 1963a) nor the full report (Nagai, ll963b) details this discussion. 130. B,>Tcet of Other Drups on Nfcotfne Cmmala£ons (47) A large number of ne« and old drugs continues to display a greater or lesser inhibitory effect on nicotine cenvulsior•s in several speciea 1n general, these drugs may be classified as having (A) anesthetic (or otherwise CNS-depteasant), (B) atropine-like, (C) adrenolytic, (D) ganglioplegic, or O;) ant3-histaminic effects (47b). In ®ddition, a variet.y of otherwise classified, or unclassifiable, agents have also been tested as nicotine antagonists (1:'). 130•A. Anesthetic (or Otherwise CNS•Depressauat). Rapid i.v. injection of 0.1 ml/10 gm body-weight of 2% nicotine tartrate in mice resulted ienmediately in clonic seisures, followed 5-7 sec later by maximal tonic-extensor seizures of t.he limbs, and death (0. Chen and Bohner,1957). The tonic-extensor component slres found to be suppressed first before the clonic seisutm by minimal effective doses of morphine. isonipecaine (meperidine), methadone, mepro- bamate, barbital, and bensctysine; while the order of the anti-seisure effect was reversed with chlorpromasine (49a). These findings were extended and published in detail by Chen and I3ohner (1900). In addition to the drugs mentioned above, diphenylbydantoin and mephenesin more readily abolished the tonic-extensor component of the nicotine convulsion, whereas the initial clonic seizure wres more effec- tively abolished by promazine as well as by chlorpromazinc. According to O'Dell, Napoli and Alirsky (1903), the appro.i- mate Protective Dose.o of inepheneain i.p. against 1 mg/kg nicotine bitartrate (as the base) for mice was found to be >400 mg/kg. Orcutt. Aiichaelson and PryKberch (1903) studied the inhibition of nicotine-induced convulsions in the rat in the following manner: Each experimental anti-nicotine drug tested was made up as a suspension or solution in u.5 -;i. sodium carbox}rnethyl cellulose in such concentration that the entire dosage was contained in 5 ml, which was then administered perorally by stomacb-tube exactly 0.5, l, or 3 hours prior to tail-vein injection of 0.5 mg/kg nicotine. With one exception, the dose used was the highest at which no visible change in the animal's appearance or behavior occurred within 24 hours (_ "ADo") [butacaine sulfate was the exception, and was tested at about 77% of the ADo ; see below, l30•FJ. Phenobarbital (60 mg/kg) inhibited clonus and clonic convulsions in about two-thirds of the animals tested, and suppressed opisthotonus, tonic flexion, and tonic extension in virtually all. In rats, prior oral administration of 280 mg/kg phenacetyL urea (Phenurone; phenacemide) had no effect on clonus induced by i.v. injection of 0.5 mg/kg nicotine; clonic convul- sions, however, were suppressed in 9 of 10 animals tested, and opisthotonus and tonic 6exion and extension were suppressed in all (Orcutt, 1lichaeL.°on and Prytherch, 1963). Phenacety4 urea also showed a marked protective action against nicotine- induced clonic convulsions in mice (Otori, 1961b). Otori (1964a) reported that central depressants were, as a clm, the most efficient of the agents tested for protection against c',onic convulsions induced by nicotine in mice, but the greatest protective action against tonic convulsions was attained with tranquilizing drugs. Chlorpromasine, perphena- sine, and benactyzine showed the highest LDso/I:Dw retio-• for protective action against death, snd protective effects against death paralleled those against tonic convulsion (Otori, 1004b). In small doses, prior administration of beuactysine lowered the nicotine convulsive threshold in mice; high doe" raised the threshold as measured by continuous i.v. infusion of nicotine (Volicer et al., 1964). Jan extract of SrAiiondra cAfnensis had a similar effect; see below, 130-F.] Yen and Day (1965) also reported that chlorpromasine, as well as chlor- prothixenc and meprobamate, antagonized the convulrive action of nicotine in this species (for experimental details, see below, 192). The following tranquilizers (mg/kg by mouth) were relxrrted to be effertive in nudrolGng cntivul.ive sPizur'~ in 50% of mice receiving 1.75 mg/kg nicotine scid tartrate: hydroxysine, >100; Dixynasine (10-[2-methyl-3•(1-hydroxy- ethoxyethyl 4.piperaainyl)propyl phenothiasineJ), 50; pronta• sine, 75; chlorpromasine, >100 (Lewis et al., 1961). On i.p. injection in mice, 71.0 mg/kg mepasiine supprPcced ronvub sions in F;1 % of mice given a convulsive dose of nicotine i.v. 1 hour later (Kerley et al., 1961). Also in this epecie.c, the convulsive effect of nicotine was antagonized by an oral dose of 5 mt,/kg Prenolon (I~V•(Rox,vethyl)-a".(-y-3.cbloro-10- phenothiasinyl)propylJ-piperasine-3.4, 5- trimetho\Tbeneoic- acid-ester difumarate or diethancsulfonate; T-5) (Csstnyi, Borsy and Toldy, 1960). 2-Phenyl-4-nminobutyric acid Produced bv Thq ('nl tnci I for 0002944 Tobacco Reseaich-uSA, It1GR

NERVOUS SYSTE.1f 33 (l't::U9A)-consrdered to be skm to tranquWsers--ord not protect mice against the convulsive effects of nicotine (Khaunina,1964).1n rats, an i.p. injection of chlorpromazine HC130 min prior to 1.9 mg/kg nicotine i.p. (m CDw) exerted a protective action against nicotine convulsions, which was found to be dose-related, in that 14, 2.0, and 3.0 mg/kg of the former drug progressively decreased the incidence of convulsions (Gokhale and Gulati, 1962). In rabbits, 5 mg/kg chlotpromazine (Largactil) antagonized convulsions resulting from i.v. injection of 2 mg/kg nicotine bitarWste, but convul- sions due to strychnine or pentylenetetrazol were not antsgo- niseil (Longo, von Berger and Idovet, 1954). In. doses as high as 100 mg/kg i.p., phenyramidol J2-(8- hydrwyphenet3tylamino)pyridine; 1TW offered no protec- tion in mice against convulsions induced by nicotine (0'Dell, 1960; 0'Dell et al., 1960), nor did the 4-methylamidopyridine derivative (0'Dell et al., 1960). (These compounds are stated to have both analgesic and muscle•relaxant (interneuronal• blocking) actions.) For the anticonvulsive effects of reserpine and tetrabena- sine (which also have general tranquilising aetion), see below, 130•C. For the effect on nicotine convulsions of certain carbamates, see Homblev (1962), below, 130•E. 130•1B. Atropine-Like (Including Anti-Parkinson) Dg' s. Kroneberg (1955) studied in mice the protective Zt of belladonna alkaloids given s.c. ;A min before i.v. e administration of a surely.convulsive dose of nicotine; the alkaloids used were atropine, and the d- and 1-forms of hyoscy- amiae and hyoscine (scopolatnine). No appreciable difference was found between 1• and d.elkaloids as central inhibitors of nicotine-induced convulsions. JOn the other hand, the i•alka- loids were found to be many times more active than the d-isoixsers with respect to antagonizing the peripheral effects of nicotine (Kroneberg, 19fi4){307b).J Atropine and hyoscine suppapssed the toniaextensor component before the clonic seisu9'es of nicotSne-induced convulsions in mice (G. Chen and Bohner, 1960). However, atropine and scopolamine did show a marked pmtective action against clonic convulsicnv induced by nicotine in this species (Otori, 1964b). In rabbits, i.v. injection -of 5 mg/kg atropine did not modify clonic convulsive movements of the bind-limbs of tsbbits given i.v. injection of 1 mg/kg nicotine acid tartrate (J. J. Reuse, 1960). Benalctysine has stropine-like effecta; see above,130•A. H. Haas and Klavehn (1955) tested the effect of a number cf anti-parkinson compounds on nicotine convulsions in white mice; the agents were given s.c. 15 roin before i.v. injection of 0.4 mg/kg nicotine (a dose which had caused severe convul• sions in 46 of 50 animals). The following iiguree (n,mg/kg) represent the threshold protective dose of the several com- pounds which prevented convulsions in 50% of the nicotine• injected mice: atropine, >30.0; cstamiphen, 8.8; trihexy- pheniilyl, 26.4; Athopropasin, >20.0; oxycyclohexylphenyl acetate dietbylaminoethyl ester, 5.9; Akiaeton 13•piperidino• 1-phepryl•1•bicyeloheptenylpropanol-(1); biperidenJ, 4.6; 1- bicyefoheptyl-l-phenylpiperidinopropanol, 26.2; 1-bicyclo• osyheptyl•1-phenyl 3 piperidinopropanol, 24.3; 1-bieyclo- 6cptepyl•1-phenyl-3-piperidinehpropene, 5.3. It is appsrent from these figures that Akineton (biperiden) proved the most effective compound in protecting against nicotine convulsions. The yuthors also sun.rnarised, in table form, information from the literature bearing on the inhibition of nicoth e con- vulsions in rabbits by various substances, including some of those given above (also see Longo, von Berger and Bovet, 1954, below). In i.p. dosage of 32.8 mg/kg, chlorphenoxamine tila was enecuvh m suppressing convuMons w ouya 01 mtce given a convulsive dose of nicotine i.v. 1 hour leu-r, and the 0-diethyl arhalogue of chlorphenc:smine icalled Keitbon) had an EDeo of , 24.5 mg/kg (Kerley et al., 1961). The ED&o for other anti-parkinson compounds, determined in the same manner, were reported to be: orphenadrine BCI, 14.8; car- amiphen, 23.0; ethapropasine HCI, 28.0; procyclidine HCI, 34.5; cycrimine HCI, 46.5; triher.)pbenidyl HCI, 58.0; atro. pine sulfate, >1p.0; bentotropine methanesulfonate, >32.0. S.c. injection of brpbenadrine 15 ruin before i.v. injection of 3.75 mg/kg nicotine bitartrate in mice failed to prevent nicotine convulsions, although death was prevented (Harms et al., 1902). Procyclidine antagonized the tremor as well as the convuhione induced by nicotine in mice (Yen and Day, 1905). Diethazine HC) was among the drugs which ahowe5 the highest LDw/ED,o ratios for protective action against nicotine tonic convulsions and death (Otori, 19frfb). The anti-parkinson agent, 9-IN•methyl•3-piperidyp-methyll. thioxanthene HCl (Tremariq, given i.p. to mice 30 min prior to i.v. injection of 1.2 mg/kg nicotine, had no effect on nico• tine convulsions in doses up to 48 mg/kg, but did show a protective effect dn survival (Caviezel et al., 1963). Increasing the dose to 64, 80, and 128 mg/kg Tremaril progressively antagonized nicotine convulsions, while protection against death became prdgtessively less. Depending upmn the time elapsing between oral adminis- tration of 220 mg/kg cwamiphen and i.v. injection of 0.5 mg/kg nicotine to rats, the forrner drug inhibited clonic convulsions in from 50% to 100°,'a of the animals tested; opist)•otonus, tonic 6exion, and tonic extension were sup• pressed in all animals (Orcutt, Michaelson and Pre•therch, 1963). L, expetimenta on rabbits, 3 mg/kg caramiphen (Pan. pamit), 5 mg/kg etbopropasine (Parsidol), or 3 mg/kg triheayphenidyl (Artane) antagonized convulsions resulting from i.v. injection of 2mg/i:g nicotine bitartrate (Ioongo, von Berger and Bovet, 1954). Convulsions due to strychnine or pentylenetetra:ol were not antagonized. In tests with two new central chounulyGce, DPUisenko and Pratuserich (1983, 1964) tepcrted that 10 mg/kg Afetamisil did not affect nien• tine convulsionb in rabbits, altheugh much amaller dasea prevented the development of arecoline convulsions. On the other hand, small (3-5 mg/kg) ilotws of Methyldifacil markedly antagonized the excitatory action of nicotine, while these doses almost completely failed to prevent the onset of arecoline eonvvlsions. For mcphenesin (which has been used as an anti-parkinEon drug), see above, 130-A. 130-C. Adrenolytics. According to Otori (1904a), admno• lytic drugs showed more potency against nicotine-induced clonic convulsions in mice than against death. Phentolamine metLnncsulfatc and tolszoanc IICI had a marked protective action again.-t clonic comvl•iviu, while reserpine protected against• both clonic and tonic convulsions (Otnd, 1984b). On the other band, reserpine was said to be ineffective in antago. nising the convulvant action of nicotine in mice (G. Chen and Bohner, 1980), and even facilitated nicotine conwlsions in rats (Gokhale and Gulati, 1902); while, in rabbits, nsserpine altered the character of nicotine conwleions (J. J. Reuse, 1960).'Chere may be a epecie4 difference in re,ryxutse; but the matter of dosage, and of inten•al between tftvpine and nicotine administration, may he equally important. In rat.a, teseq>ine injected i.p. 2 hours prior to the i.p. injection of 1.0 mg/kg nicotine (CDte) facilitated nicotine Produced by The Couicil for 0002945 Tobacco Reseafch-USA, Inc.

34 'f'UI3ACCt>-EXPEIi15fENTAL A" U C1.ISICAL STCI)Ih~. vCl'NLE11E\T I convulsions; and this action was related to the dose ot re,;er. hide, 0.25, 0.5, and 1.0 mg/kg progressively increasing the cotivulsion-rate (Goklwle and Gulati, 1962). In unanes- thrtise•I rabbits prepared for recording of movemew of a hndd-foot (indicating convuleions), i.v. injection of I mg/kg nicotine acid tartrale re:,ulted in a series of clonic movementa of the limb (J. J. Reuxe, 1960). The same animals were in- jected with 0.5-1.5 mftYkg reserpine Lv. or Cp., followed in 24 houns by a second dose of 0.&5.0 mg/kg, and the reactions to nicotine were re-studied 24 hours later. Two types of re- sults were obtained: with moderate doses of reserpine, motor responses remained unchanged; but after strong doses, the responsa to nicotine, habitually exclusi+•ely clonic, became clohico-tonic. This modification of nicotinc convul-inua w•a.- eonsidered to be due to a central action of reserpine. According to fltjiu•ara, Yamawaki and Shimamoto (1964), the CDao for nicotine bitartrate when injected i.p. into dd- atroin mice of either sm was 12.8 mg/kg (range, 12.0-1d.0). A number of mono amine releaFers were studied for their efkxt on the convul.iant action of nicotine. Prior i.p. in- jection or 0.1 mg/kg ,rserphne ulcrea.sed the ('Dm without tnodifying the convulsive pattern; 1.0 and 3.0 mg/kg de- creased the CDao. Prior injection of 1A mg/kg tetrabenazine did not significanth• affect the CDoo ; 5 and 50 mg/I:g de- creased the CDoo. 13t1-D. Ganglioplegics. Minimal doses of hesamethonium suppressed nicotine-inducen convulsions in mice, thc lonicr extQnsor component being affected before the clonic seizures (G; Chen and 13ohner, 1980). Hexamethonium bitartrate showed a marked protective action against such clonic con• vutlsions (Otori, 1964b). Hexamethonium chloride, a, well a% chlorisondamine, antagonized not only the tremor induced by nicotine in mice, but also its convulsive action (Yeu and Day, 1905). )`.p. injection of 1 mg/kg mecamylamine abolished the convulsions in hydrated rats induced by i.p. injection of 2 mg,/kg nicotine (liattila, Ahtee and Vartiainen, 19Gi). S.c. injection of 5 mg/kg mecamrlamine protected against the tremors ald convulsive movements induced in mice by s.c. injection of 1.0 or 2.0 mg/kg nicotine, but 5 mg/kg trimethi- dintum was less effective in this respect (Domino, 19l>,5a). )These d,>Qes of nicotine pr duced definite con.-ulsive move• ments within I tnin after injection, which graduall,v suh.aded without anj prior treat:nent.] Kieotine-induced convukion.. ht mice were also inhibited by certain aminobicvclo12.2.1 J- heptanes (cong-nets of ineeamylamine) and bicyclo13.2.1 J• asaoctanes (bridged congenen: of pempid'1ne) (Edge et al., 1960). ' 130-E. Antihistaminfes. \Iinimal doses of diphenhydra• mine and promethazhm more readily abolished thc tonic• extensor component than the initial clonic seizures of nicottne-induced tonvulsions in mice (G. Chen avid liohner, 1960). Alto in this slecies, diphenhydramine showed marked protect.ive action against such r)onic convulsions (Otori, 1964b). With respect to do.cage, 16.5 mg/kg d'qlhenhy'dramine HCI w•as reported to be the Lp. Ehao against i.v. injection of a convulsive dose mf nicotine given 1 hour later (Kerley et al., 1961). S.c. injection of diphMthydramine or IiS 0W (2f• bHvllxnshydryl ether of dimetM•laminoetha)ol N(71 15 min before i v. hljection of 3.75 mg/kg nicotine bitart rate in micr failed to prevent comvWons from the latter drug, al• though death w-a.a antagonized (Harms et al., 1962). t.tu-r. .Aasixudireuue carbon dioxide lowered the thre+hold dose of s:c.-injirted nicotine tarlmte, causing cona•uhiotu (Lananickel et al., 1955). The animals were sa:d to have e%hibited initialh• clouie convul iot>s s}•nchronou.s with inspiration, which neither carbon dioxide nbr nicotine alone produced. Injection of 4 mg/kg 7.069 R. 1'. (:1'.methylpiperidyl•2- etM•1-3-indule HCI) produced a 50% reduction ir: nicotine convub:iotu in rabbits (Julou, Courvoisier et al., 1'B57). Chaikovska}•a (1959) studied the effect of spasmolytin, tremsentyl, and liplmcyi, avid their quaternary derivative.~, iodumethylate, i4docthylate, aud chlorbenzyIate, and re• Iwrtcd that iodomethylate was the most effective, ald chlor- ixmzvlate nevt, in the treatment of convulsions chused in mice avid rabbit:- ; by nicotine; iodoethylatc w•a< 6neffective. Korablev (1962) reported that, in doKes of 100 mg/kg orally to mice, Antabuse (dinulfiram), tetrathioac, bisxthy1• aanthogen, sodium diethyWithiorarbamate, sodiun~ dimetM•1• dithiocarbanlate, thiourea, and urea, all had an effect on the cour.m of hrperkinesis reFulting from s.c. injection of 10 mt;/k4 nicotine, as well as that from strychnine avid atx~.roline. No data were given fmr these; but, in a table of data of effects of lheee compoun(b, on spa.4mH induced by corazol, i;t appeared that urea prevented their development, while the otpanawtlfur cumpounds intensified the spa:::noltenic action. lu ratn, prior oral administration of 220 mg/kg (lrocaine or 170 mg /kg butaraine had no effect on elonus or clottic convul. sion• induced by i.v. adrnini>tratiun of 0.5 mg/kit nicotine; the incidence of opi~thotona» a•a% 4i}thtly reduced, however, and the incidence of tonic fle\iou and tonic extension was ven• areath• reduced (Orcutt, .llichaelron and Prythereh, 1963). The alticonvul••ive effect of 60 mg/kg Dyclonine (p•butosvs-piperidy1 propiopheuone; 3-lulmriditno -1'-butoxy- propiophenone HC); P.2G7) w•ax verv much more tnarked, clonic convuhiutwr and upistholonur being Ruppre:ewd in one-half or more bf the animals tested, and tonic flexim, atd extension in all of them. (S-ce above, )30-A, for details ef test procedure.) , According to Nagai (1963a, b), who tested the effect of 1)(1P.1, tyrosine, dolusmine, isoniaaide (isonicotinie acid hydraside), hydrosclamiuc HCI, a-methy-ldopa, and pyriduxal phasphate on convulsions induced in mice by i.p. injection of 17 mg/kg l-nicotine, DOI'.t and ty'rasine markedly delayed, and dopamine (Nagai, 1963m. b) and decartw.x.vlase inlubitot. (Nagai, 19(i3a) quickened, tlm owe thue of each convulsion. When injected in mice 15 min prior to i.v. injection of I mg/kg nicotine bitartrate (aq the bare), the approximate Protective Doseao o! i.p.-injtrted 2-(8•hydro\c-Q.pheneth,vl• amino)-py'rimidinp was found to be 150 mg/kg (O'Dell,Napoli and Jlirzk.•, 19G3). Pretreatment cf alhino rats a•ith 200 mg/kg thiamine at 5,10.20. or 30 min prior to i.m. injection of 20 mg/kg nicotine (a dose which caused r•onvuhsiua+ in 80 of 83 animnla) Inarkedll' decreawd t)le incidence of convulsions (l. Yama- moto. 1963). Other sign-, such as tremor and apnea were also lessened, but paralCtic Si(•,Ila remained for a considerable time. Nicotine convulsiom% were a>`o inhibited by 212 lnK/kK thiamine-prop.•1-disulfide ac. (Pretreatment with 100-200 mg/kg thiamine had no effect on comvlRimte elicited by electro.ahork. pentylenetetrasol, strychnine, or picrotokil).) In tests with other vitamins, the strength and duration of nicoline convu)rions in mire a•aa snmetvhat influenced by pretrPatment with psrido.ine snd cyetxmobalamine (('hem- nitius, 1961). QaQ2U6 Produced bv The Counc;l for Tobacco rescaich•USA, Inc.

.NBItvlWb 8yt¢Tp-w Small dusex of a purified netrol-eq.her extract of the plant, Srkizandra r/rtnenafe (kontaining 40% of the active principle, schisatidrin, of ligna,n structure), lowered the convulsive th" hold for nicotine in mice, whereas larger doses antago- hiacd n,icotine eonvuldions (Volicer et al., 1963). 1Cht:n meas ured by continuous a.v. infusion of nicotine 30 min after pdminiKtration of Schizondra extract, the latter lowered the threshold in small doses and raised it in high ones (Volirer et al., 19l)4). [)fenactyzide had similar effects; see above, 130•B.J l:gul'nikov (1965) reported administering 300 mF/kg thvroidin to male rats once a day for 15 days. Before thyroidin treatmgnt, the reactiofi~ of thexe anirnak to 1.5 mg/kg nicotine s.c. had been relatively weak, but, after 9, da~•s of treatment, the nicotine action appeared sooner and he intensity of the ~tui wr iu,rra.•e.I; after IS days, tlie reactiun lMVVu,c wcu more intense. Two n•ehks after cessation of thyroidin feeding, the reactions to nicotine were said to be hot uniform and to vary n•jth individual animals. bttracerebral injection of 2 mg/kg nicotine Ipresumably as the hydrogen tanratej in 12 \1-istar rat.s was immediately followed by intetue motor excitability atod convulsions, the animals recovering within a few minutes; repeated injections of the same dose elicited the same symptoms (Camto and Dias Da Silva, 1966). Subsequent injectimn of the histamine liberator, 48/80 (49a),,produced effects less intense than those observed in untreated, animals; however, the same dose of nicotine injected intracerebrally in rats made tolerant to 48/80 by tel>efl.ted injections produced full-size effects. IGiven by itself, 48/80 prodUces moderate neurologic disturbances; but, although this compound is a potent liistamine iiberator, antihistaminics did not protect against the neurologtc symp. toma.J Ilenzy-dryl-l-piperotiyl pipera.zine-said to be a new coro nary dilator--in dosage of 25 and 50 mg/kg i.p., failed to inhibit convulsions elicited in mice by 1.25 mg/kg nicotine Lv. (Laubie, Le Douar2c and Schmitt. 1964). Lyrergic acid diethylamide (LSD-25) antagonized not only the tremor induced by nicotine (see hwlow; 192), but al,o its convulsive action (Yen and Day, 1905). 131. Bffeet of 1\ iootine on Conoyrlafons IndueRd by Other .1l eans (49) :lccorifing to Afiua and Domino (1953), i.v. injection of 0.1 mg/kg nicotine increae;kd seizure duration induced in cats by electroshock. There whs a latency in its effect, which the authon; suggested might be due to adischarge of epinephrine- like sub4tance4 from the adrenal medulla, since injections of epinephnne and norepinephrine markedly prolonged seizure duration. Following an oral dose of )0 mgN nicotine, the tlrre=hold for Lv. pentylenetettgzol convulsions in female mice ass slightly increased, and about a 20c block in extensur tonus occurred (liastian, 1961). Nicotine was reported to have had no effect on arecoline convulsions in rabbits (Denisenko and Pratusevich, 1903, 19fr1). 132. Nicrotdne CalatmiEo (5I ) 133. It'fcoline Paralysis (51) [\o new data.] 134. J/isretlaneoua (521 INo utaclaacifiable data.) 3a 135. Tt1E AITPO\O.31fIC Nfl:RVOL'S Sl'STEI11 t52) houzett and Rot.hlin (195.3) have teviewed the action of syuapNntropic substatuv,. on certuin cifereut and afferent structures of the autonomic oen-uus ryxtem, into Nhich the actions of nicotine were integrated. C. Heymans (1952) has reviewed the effect on autotromir ganglia of other pyridinc derivatives with pharmacological actions similar to aicotinr: nirotrrine; nomicotiue; anaba.,inc; ni.Yrtellinc; mj.•u=miuc; and othen. lo the Stockhulm sy-nqrosium on "Tobacco .11- knloid, and Related Compound:", 1'. Treudclenburp: (191U1 presented an analytical review of nicotine and the Iwrriphcrnl autotromic nervou:c system. 136. .1 utononiir CangliQ (53) lik:ckman, Gita:borg and co-workers (1963) rel4oncd a study of tran.rovssion iu sympathetic ganl;lia of ihe frog, the preparation consisting of the i-•adatcd sympathetic chain %cith indivadual celL• imlmled n•ith nucro-elertrodes. \icotinc caused a transient depolarizatiun of the cell, followed by a pcrsistent depression of synaptic trau:mis,ion; the dclM,lari- aation caused by nicotine was graded, and was not aecotn- panied by any obvioua increav in the frequency ot spontaneous release of quanta fronr the prenynaptic teriniuals. ln asub.uryuent study of the action of drlalarizing drugx on sytinp.athetic ganglion celh: of the frog, Ginsborg and Guerrrro (1964) reported that nicotine de)wlarized the ganglion rrIW and depressed synaptic tran_mission; in its continued prea- etue, hoW ever, the depolarization subsided, but the dep.rcxxion of sytitaptic transmission Ixtxirted. In cats under hexobarbital anasthe.ia, with the superior cervical ganglion ananged tor periusiou and isotonic controc- tion of the nictitating membrane ren•ing na an iutdex of ganglionic st7mulatiotl, injection of 5-10 µg nicotine tarttate into the ve.•rls bathing the ganglion caused stronger:Stimula- tion than 50 pg of acetylcholine (Fehir et al., 1961). Gan- glioniw• stimulation by aceh•Icholine was strougly drpmxvtl by 5 ug, and abolished by 10 µg of nicotine, but transference of pmgangliouic stimulation was not affected: blockade of pnytanglionie stimulation required about 100 t.g of nicotine. IIt may be noted lure that, in thPir studies on tratv;,mic.ion 1Arough the right supenor cemva-) ganglion of spinal cat<, 11oFoch, Yaasonen and Trendelenburt; (1982) found that the prepatntio»s responded well to the usual (10 pg) dose of nicotine injected into the arterial blood supply to tlie gan- Rl'wn.) With a dw of 50 ;y( of acetylcholitM injected along with fticotine, sy'nergism resulted up to a nicotine dose of about 8 µg; preganglionic stLnulation alro exhibited n)nerghcm with nicotine, madmal at a nicotine dose of glcg, and taiiing off at higher nicotine dosage. (For an interpretation of the+e re:ulla, see below, 139.) Pelikau and ('arten (1962) also studied the differential effect of nicotine on ganglionir trans, mis`ion and reRpomse to aretylchnline, and found that nicotine impaired gan,tlionic transmission ttithout altering respotu:ive- atss of the cat superior cervical ganglion to idtjected acety9choline. 1n 47 pentobarbitaliaed cats, responses of the nirtit,ating membrane to alternate ipsilaternl lrrr. ganglionic nerve stimulation and inlra-artetial ercetylcha line injection, before and during the time•coutse of ac. timi of nicotine or hearemethoniutn injected intta-artetially to the ganglion, were recorded kymographicalh•. tCith I50- 250 µF of hesamethonium, the time-course of impaired gang;lionic transmission to stimuli of 1.25 and 20/sec paralleled thc time-coursse of imiaired reepm>.-* to 100-200 µg of acetyl- choline. In contrast, after initial ganglionic stimulatuon by Produced by The Council for o 0 0 z 9c; Tobacco ResearclrUSA, Inc. vilift

36 TOBACCt)-EXPERIMENTAL AND CLINICAL STUDIFS. SUPPLEMENT I 20-200 µg of nicotine, impaired ganglionic transmission to both stimulation frequencies persisted after rmpottses to acetylcboline had returned to control levels. After nicotine, but not after bexarnethonium, half-time for rebovery of ganglionic taansatiesion was longer than half-time for re- covery of responses to acetylcholine (means, respeetively,14.1 and 9.4 min; p< 0.001). These results were said to be consist. ent with PeGkan's hypothesis [see Pelikan and Garcia (1959)(bBa); Pelikan and Garcia (191i0); Pelikan (1960); and below, 139) that prolonged impairment of ganglionic trans- mission with nicotine, but not with hexamethonium, is caused by diminished release of neurotransmitter (acetylcboline) from presynaptic nerve terminals, rather than by occupation of postsynaptic ganglionic receptors for neurotransmitter. In studies on cats reported by Gebber and VoBc (1986), tetrsmethylammonium (TMA)-induced block of transmission in the superior cervical gan!fion was found to be chataa- terized by a sequential pattern of (1) blockade 'associated with ganglionic depolarir.ation (early block); (2) recovery froni block during the failing phase of depolarization; and (3) the reappearance of ganglionic blockade associated with gaa- glionic hyperpolariaation (late block). Injection of 5-20 pg nicotine into the common carotid artery produced a blockage of transmission persisting for 6-12 min. Owing to technical difficulties, it was difficult to determine whether the later stages of nicotine-induced block were associated with gan• glionic hyperpoletisation; however, it was found that the ganglionic spike monitored during the later staxes of nicotine• induc:ed block was followed by an accentuated negative after-potential. Cnlike TAIA, nicotine did not exhibit a period of recovery between the two phases of block. Repetitive preganglionic stimulation and ouabain (150 µg i.a) ;eBectively antagonized the later stagrs of nicotine-induced blockade ol transmission and had no effect on the blockade associated with depolarization. R:. C. Elliott (1965) studied the ac:ion of some centrally- active drugs on transmission by the isolated superior cervical sympathetic ganglion of the rabbit, of trains of irnimises at rates between 1.4 and 61 lrer sec. The drugs were found to be divisible into 3 groups: (A) those thn ganglion-blocking action of which was intensified by repetitive stimulation;, (B) those the ganglion-blockirtg action of which was relatively unaf- feeted by repetitive stim•alation; and (C) those the ganglion- blocking action of which tended to be reversed by repetitive stimulation. Nicotine fell into group C. Barlow and Hamilton (1985) have presented a quantitative comparison of the activities of the (-)- and (+)-isomers of nicotine at a variety of sitw in the periphcral nervous system. For details of each preparation, the reader is referred to the authors' original descriptions, and also to 978, below, for their discussion and conclusions. On cat sul>prior cervical ganglion (block), a ratio of 13.7 * 1.2 was obtained (bastd on 3 results). This finding is e%>ressed in equipotent molar ratio, In which, if the number is greater than 1, it indicates that the (+)-isomer is less active than the (-)-isomer. I n contrast to the effect on the normal ganglion,, tachyphy- laxis to nicotine did not occur on the chronically denervated cervical ganglion of the cat (Reinert, 19b7-SB). 187. .Bffert of DrupF on the Canglionfc Action of Nicoline (56) The antagonism of other drugs to the ganglionic action of nicotine has been studied for two reasons: as a means of elucidating the mechanism of action of nicotine, and as a phatmacological tool for investigating possible ganglionie actions of the other drugs or tor stuoyrng certaw pnrprr uva of ganglion cells and/or gatuglionic ttansmission (SBb). In general, presentation of these two aspects of nicotine--othe;r- dtug antagonism may be more or lew logically separated here into 137-A: the effects of nicotine following the administration of some other drug, which would appear to be primarily ditected at some further elucidation of nicotine aetion; and into 137-B: the effects of other drttgs on the nicotinized gakt- glion, which would seem to be primarily directed to h-wming something new concerning the other drvg and/or the ganglion, rather than of nicotine itself. M does not imply that such a division according to order in time of drug administration (that is, betsreen 137-A and 13t-B) is neressarily valid from any other point of view than that of reviewers whose pti- mary intere-t liec in the mechanism of action of nicotine; a neurophysiologist, for example, whose interest might Be in the mechanism of ganglionic trensmission, might well be better served by some other arrangernent of these data; bot, one assumes, this neurophysiotogist will already have ap. preciat,ed that these quite few ddts involving nicotine one woy or another are but isolated bits and pieces of a grand design, and be will not expect to find his complete picture in any specialized monograpb on any dne drug. 137-A. Nicotine After Other Drugs. In cats, retrograde injection of physostigmine (eserine) into the lingual artery (the external carotid artery being occluded during the injec- tion) did not potentiate the action of nicotine on the superior cervical ganglion (D. A. Brown, 1962). Mth transmission,in the superior cervical ganglion assessed by recording the responses of the nictitating membrane in eats under pherpo- barbi.al-sodium anesthesia, close arterial injection of phyao- stigmine reduced or eliminated , the response to close arterial injection of nicotine (D. F. J. Alason, 1962a). This inhibition ares not modified in the presence of atropine. Close arterral injections of neosiigmine to both normal and pnegangliotti- cally-denervated ganglia produced first a potcettiation of the response to nicotine, then, with larger doses, a contraction of the nictitating mtunbrane, and, concurrently, a depression of the response to nicotine. Pilocarpine, histamine, and eerotonin (trhydroxytr.•pta- tmne) IMtentiated the rwcponBe of the perfused superior cerl ieai ganglion of the st to' nicotine (U. 7lendclenburg, 1956a). [See also Trendelenburg (1955a, b; 19g1a), below, M-B; and Trendelenburg (1961a), below, 370.) E. Haas and Goldblatt (1960) determined the equipotent doses of five ga+tglionic blocking, agents by administering them at varying rates to the dog until an amount was found which, when infused in 10 min, antagonized the ganglion-stimulating action of nicotine. The test of equipotency was that the pressor effect (about 145 mm Hg) of a single (0.045 mg/kg) dose of nicotine was abolished, while an increase of the chal- lenging dose of nicotine to 0.1 mg/kg resulted in a slight pressar effect, indicating a state of pronounced comimtitive inhibitton of ganglia, rather than complete blockade. l;x- pressed in mg/kA/mitt, these doses were: tetraeth}iamtnoniitm chloride, C.b0; hexamethoniunt bromide, 0.07; pentolinium tartrate, 0.15; chlotisondamine'chloride, 0.04; and tnecamyla- mine HCI, 0.10. The previous addition to the organ-bath of hexamethonium pteventcd the tesporu;e to nicotine of isolated evlerior-cervical-ganglion preparations from the kitten (D. F. J. Mttson, 1962b). >;ojima and co-workers (1965) studied the combination effect of competitive ganglion- blocking agents on the nicotine-Induced ganglion blockade in the eat superior cervical ganglion using anhnals under pento- Produced h The Council for tTobacco Pesearch-USA, lm

NERVOUS SYSTEM 37 I barbital anesthesia, aad recording contractions of the nictitat- iag membrane elrcitsd by supmmaiamal eterctncal strhiwauon of the preganglionic nerve every b min. LTrhder such stitnula- tion; the potency of ganglionic blockade was calculated as follows: the decrease of contraction height was expreseed as a per-cent of control; each v'alue of per-cent block was summed up tagether until completa n~overy of th~ contrection wss observed, and this summed-up value was pxpressed as total blockang value (TBV). NAith intra-arterial injection (made through the cervical end of the lingual artery into the blood supply of the ganglion) of 150 µg nicotine and I.v. injection of 4 mg/kg tetraethylammoriium (TEA), the TBV of ihe com- bined dose was larger than the addition of each TBV of nicotine and TEA. Similar results were obtained with i.s.150 pg iii~cotine and i.v. 1.5 tag/kg hexamethonium. F°ollosring administration of hexamethonium, but not after stropine, nicotine failed to induce postgarrglionic firing from the superior cervical ganglion (Takeshige et a4, 1983; t;ee below, 136). Fadhel and Seager (1906) tested ganglionic function in dogs by three criteria, namely, blood-pressure response to left sympathetic chain stimulation, bilateral hd arotid occlusion, and #.v. injection of 26-SO pg/kg nicotine gtiHcylate. Nicotine gave the most marked responses, and these responscv were blocl:ed by Lv. injections of hexamethoniutiy in doses of 5-10 mg/kg or by continuous i.v. infusion of trimethaphan cam- phorsulfonate in the total dose of 1-2 mg/kg over 30 min. lorio and Afclsaao (1966) reported a sttrdy designed to chatgcterise and compare the effects of nicotine, pilorarpine, and histamine on the superior cervical ganglion of the pithed cat by recording isometric contractions of the nia;titating membrane resulting from injections of these drugs into the iromroior, carotid artery. Hexametbonium, atropine, and pyrilamine were found to be specific surmountable an(agonists of nicotine, piloaatpine, and histamine, respectively, leading to the conclusion that these three agonists act at different receptor sites on or in ganglion ceps. Chloml hydrate administered to cats by )j. A. Brown (1962) iIn the manner described above bad a variable effect on the actiop of nicotine on the superior cervical ganglionq in over half the animals tested, no change in sensitivity was produced. In the spinal cat, pretreatment aith 10-30 µg morphine or 10-100 jug methadone failed to reduce thF: response of the non-atropiniaed superior cervical ganglion to lingual-artery injecEion of nicotine (A. Jones, 1963). Cocaine In small amounts also failed to reduce the nicotine response. Prbcainamide reduced or abol;sbed the ptessor response to nicotine in cate, and this antagonism was shown by Paton and Thompson (1984) to be due to block of ganglionic trans- mission. I. Yamamoto (1961, 1963) demonstrated an antagonistic action of thiamine to nicotine at nerve ganglia, and concluded, from concentmtion-action-curve studies, that the antagonistic actioir of thiamine to nicotine p! nerve ganglia appeared to be a so-called competitive one (Yarrramoto, 1961). Tnis com- petitive block was said to br more typical than that of hex- amethonium and tetraethylanunonium. An.ong related compounds to thiamine, thiazole derivatives containing an amino group at "2" and/or phenyl group at other than "2" elicited an augmentatlon in antagonistic activity to nicotine. 1'amamoto (1963) later published findings on nicotine-thia- mine antagonism on isolatcd guinea-pig small intestine, which were taken to support the assumption that the anti-niootine aetion of thiamine involves competition at the receptor site. Considering the wide-range of varistion of the antaFgoniatic ratios of thiamine against nicotine, depending upon the kind of excised organ used (thiamine dose/nicotine dose: guinea-pig smal/1ntk5tnle, GUU; excisea rNOpn-e8r, ll'lU; 4UlW Wllu-aWiciC preparation, 10), Xamamoto concluded that the aati-nicotind action of thiamine io not attributable to a mere chemical antagonistic facior. In cats with the cervical syrnpathetic ganglion perfused (with and without section of the preganglionic fiber), doses of veratrvm alkploidt which were just effective in producing contraction of the nictitating membrane increased the re- sponse to submaruno) doses of nicotine as well as that to acetylcholine and potsssium chloride (Konsett and Rotblin, 1955). The effect on the response to acetylcholine could be explained by an inhibiting effect of the veratrum alkaloids on cholinesterase; bowevRr, this would not explain the potentiatg ing effect noticed with nicotine and sotassium chloride. Ouabain (150 µ$ intra-ar¢erlally) effectively antagonized the later stages of mcotihe-induced blockade of transmission in the cat superior cervical ganglion, but ld<d no effect on the blockade associated with depolarization (Gebber and Volle, 1906) [see above,1361. 137-B. Effect of Otlper lbrugs on Prevfrouely Nicotinized Ganglia. In studies by I',fbos. Itfdssilt and Ssab6 (1960) on the perfused superior cervical ganglion of cats, low (ganglion- atimulating) dosw of nicotine antagonirRd the stimulating effect of acetylc'i<oline without altering the effect of pregao- glionic stimulation; the latter aas blocked only by higher doses of nicotine. The difference was attributed by the authors to the kinetics of binding of free and innervated acetylcholine reteptors. The stimulating action of pilocarpine and histamine on the superior cervical ganglion of cats was inhibited by paralyzing doses of nicotine (U.. Tb'endelenbutg, 1955a); and nicotine also abolished the potentiating effect of pilocarpine and histamine on preganglionic impulses (Trendelenburg, 1955b); but the mechanism of this "sensitization" was said to remain obscure ('Itcndelenbprg, 1961a). ]Se.e also Trendelenburg (1956a), above, 137-A; and Trendelenbttrg (1961a), below, 370.1 AIcN-A-343 14.(m-chMrophenylearbamoylo)q)-2-butynyi- trimetbylammonium chloride] and AHR-602 IN.bensy6S. Nyrrolidyl acetate methylbromide] share the ganglion- stimulating effects of pilocarpine and muscarine, and these muscarine•lihe agents hare been used as experimental tools for studying ganglionic transmission. As measured by oonttae- tioa of the nictitating membrane and postganglionic action- potentials, respectively, intra-arterial injection of l1fcN-A-343 exerted a stimulating action on the superior cervical or the inferior mesenterric ganglion of cats (Aiurayama and Unna, 1962). The action was not blocked by large dases of nicotine, but preganglionic stimulation after nicotine reduced the contraction of the nictitating membrane obtained with rlclv-,1-343. In >in extension of these experiments, Afure,vama aud LTnna (1963) reported that large (0.1 or 0.2 mg) doses of nicotine injected intra-arterially to the superior cervical ganglion of anesthetized cats produced a transient contraction of the nictitating membrane, followed by complete block of responses to prel;anglionic nerve stimulation; and mtbsequent in]ection to the ganglion of 5, 10, or 20 vg of DfcN-A-34S still caused a significant contraction of the membrane, al- though ita onset was delayedt however, subsequent stimula- t3on of the pregangltonic nerve caused a distinct relaxation of the tone of the nictitating membrane, this effect occurring only during contraction of the membrane by MeN.A-343. Iotra-arterial injection of 20-200 pg dibenamne between or Produced bv The Cdiincil for Tobacco Rcscarcii-USA, Inc, 0002949

I 38 TOBACCU-EXPkltIrlE.h'TAL A:gl) CLl\ICAL tiTt!1)IES. SIiPPLE1t1MT I after the administration of nicotine and 11c\-A-3t3 did not alter this paradoxical respuu.c of the membrane to pregan• glionic stimulation. I'ilocarpiue injected to the ganglion in doses of 20 or 50 pg produced contraction of the membrane, and potentiated the contrartiotts following aubmaximal stimulation of the pret,mnglionir nerve. Nicotine t100 µg) completely abolirhed the action of pilocarltine. I'retreatment with 100 or 200 pg nicotine completely abolished contraction of the membrane produced by injection of 4 mg pota.ti-qum chloride injected to the gan)tlion. When mu>;carine, lIc\.A- 343, and AHR-602 were iojected into the central end of the lingual artery of spinal catG imtnediatelc after an intra-arterial injection of 0.2-0.6 mg nicotine to the non-atropinized su- imrior cen•icrsl ganglion, these at,•ente: failed to stimulate the gangGon (judbmd by nirtitatin'g.rncmbrane contraction); full recovery of the stintulanl action was observed 15-20 min later (A. Jones, 1963). Fr.rther studies showed that the block to thesee muscarinic compounds was confined to the depolaris- ing, and not to the uon.deliolariaing, phase of ganglionic block by nicotine. lntra-arterial injections of small amounts of synthetic d,l-muscarine, \Ic\..t-843, and aHR-602, alone, into the blood supply of nohnal and denervated superior cervical ganglia of cats causnd stimulation (A. Jones and Trendelenburg, 1983). During the second (late) phase of nicotine blork, the ganglia regained their resiwrtsirenera to muscarine•)ike agents, while nicotine-like drup remained ineffective (A. Jones and Trendelenburg, 1903; ('. Trendrlen- burg arrd Jones, If)fw). Trendelenburg and Jones (1963) cotr cludec! that this s)•mpathetic ganglion ]uu muscarinic, in addition to the well-known nirotinic, acetyleholine retirhlors- a good entample, it may be noled, of the use of drug antago- nisms as pharrnacoloFical tools with which to e%plorc cell function rather than drug action. In experiments on cats, the stimulation of the superior cervical ganglion by angioteusin and bradykinin «as pre- vented during nicotine-depolarization block, while, during the competitive block, the actimhs of thcw peptides were not reduced, but were in fact enhanced (G. P. Lewi: and Reit, 1986). 138. Gangfiontc .lu'ion-Potentidla {58) From their study of action-potentials in an isolated prcymrn- tdon, Douglas and Ritehie (1958) eoncluded that the accrisvey ceroical gvnglion of the rabbit appeared to be cholinergic. Thus, n•be, the region was pointed with neutral solutions of nicotine hydropen tartxate or, hexamethonium bromide, the Iwst{Sanglionic•'6ber req>otuce to preganglionic•fiber stimula- tion disappeared. Transmission in the fibere which passed through the atxessory cervicsl ganglion, -both the pteRan- glionic and postganglionic fibers, was nott abolished, provided the nicotine was applied only a short time, and then an..hed cdt. Pa,a•oe (1956) has described a method for investigating the effects of drugs on the polarization of the superior cervical ganglia of rats and rabbits in vitro. Nicotine depolar- ised the gang)ion, and the depolariaation was confined to the ganglion cells. After removal by washing, a positive overewing occurred, so that the ganglion became temporarily positive to the postganglionic trunk. D. F. J. Mason (1982b), tusfng isolated auiterior-cen-ical-gangaon prepamtiorts from rnta and cats, also found that nicotine produced depolarization, which usually reached a masimum in 4-8 min, and subsided only slowly on washing. On the isolated superior cervical ganglion of the rat, nicotine in concentrations of 2 µglml and greater produced delwlarizatiou, which reached a maximum in 2-4 min; on washing, the effect subsided stot*ly over a period of 8 or more min, and was sometimes followed by a small hygerpolarization, which sluwly subsided. In exl>rrimentF reported by Vollc (1962) on the superior cervical l,ronglia of intact cats anesthetized with urethanr, the re.4pnrur of the post-ganglionic thyroid nerve to preganRliunir stinmlatiun hy repetitive rubnia%imal electric shocka Ha• characterized by a bimodal artion-lrotential (S. aud t•',.), while the action-lwtent4at of the I0stgangli(nic eNternul- carotid nerve was char•6ctcrirzd usually by a single mnde tS,.). Nicotine lpr•e;umab;ly 0.5 ty; ky rlu~v arterial hnja•tum; see Takeauge et sl. (1963), inrmediateh• folloeiryrj potenti- ated all three mode.., but the greatest effect was observed un S.. In further experiments reported by Takeshil,m, 1'apano, lleGroat and Volle (1QG3) un ca6 under lhal•urethaac ancethesia, in which drup were admini.tered by close intm- arterial injection and pustgangliunic actiorolrotentials i+en• recorded, 0.5 µF nicotine caused a bur-l of aeytu•hronuu• firing, which was inunec#iate in omrt, aud Iuen.-i.cled for 2-s sec. The firing evoked by nicotine was markedly enharnrrl following repetitive preghngliunic atimulation at a rate of 3n c}•cleslset• for periods of 15-30 sv. 1'nder bhn•kade of tireu- glionic lratutmission by Q.3-1.5 mg he%amcthonium, nicotine failed to induce pactgangiionic firing; but I µg atropine (whirh completely blocked the re.slwnn~• to methurholine) had no effect on the response to nic•otine. During repetitive pregan• gliunie stimulation w ith supromacimal volleys at a mte nt 0.5 eycle/u'c, utjection of 2-6 pg nicotine caused deprr.,,iun of the amplitude of pos,lFanglinnic spikes; occasionally, the depre.~iou was followed by a prolonged (5-20 min) Iierioal ai enhanced tmrtsanirsion, and durinR this period, I µg atnipine reduced postgaoglitmic action•Irotential:; to control valui~-. See aLar Gebber and Volle (ifNHi), alwve, 136. The mode of ganglioriic block produced by several drugs was im-er:tittatcd by Shai,d (1965) by mca.•uring their effects on potentisl.z recorded esler+ralh• from the isolated superior cervical ganglion c1 the mt. Xirotine delndarized the ganglion, and, with 10-min ey>o`tqre., this depolarization was directly prolwrtiotml to the degrre of gaaKliunir block. The cBert of nicotina on the gangliopric actnm-potential was ssid to IH• qualitatively the sarne as that dtw•tihed by R. M. l:crh, 1(19G6)(59a)) on the isolated suimri-)r cervical R~ glion uf the rabbit and by Paton and Perr~ ((1953)(59a)~ cm the intact ganglion of the cat, and .~-a,+ de,tt•ribed by Shand as followr: "With a low concentrotnm (I X 10`0), the negative after-potential (N wave) was ralured in size and the posi- tive after-potential (P wave) was aut~mented with little or no reduction in the height of the +pike potential.lt ith greater concentrations, the spike petemia) was decreased and the \ wave was abolished. The 1' wave was filst shortened in dumtiott, returning morr rapidly to isopotential than with low dorts, before beittg gradrialh• abolished by high drug concen- tratiotu. (•i x l0"°)." Skok (1959) im•estigated the effect of nicotine on the 1oitive after`Iwtential and on port-actieation depressiun in the stellate ganglia of cats. FulluuinK hontosy7utptic, hetcrn. synalttic, or antidromal stitaulatfrnt, depression was meaqunvl by the diminution of the test artiun-Ixttential sul*rimlxwed on the conditioning Iwsjtive after-Iwternial. Lotc cotn•entra• tiona of nicotine increased the paaitir,-e after-potentialN and depres4on; higher (25G-1,000 µg/ml) corn.entrationa nm. trarted and diminished the liesitive after-pntential-a up to iomhlete dizuppearancc, and the depression was also dintin• i:hed. A slight inctease in the Iwr.itive afterqxttential %vn> occasionally observed, without any le:uening of delorc. iun. Produced bv The Council for Tobacco Research•USA, Ine, 0002950

NERVOUS r't•r'TF.M .. . .....~.. ....a .t. .. . .t .t-• .... ..., iine,tinal tract removed, injectiotd into the celiac or inferior nncvmterrc artery of 2-50 pg nicotine clo:je to the inferior mesenteric gangliou inrrea-vd the frequency of the ganglionic actiun•Itotcntiels (G)•errrmk and Herr. 1959; Herr auti Gvermek, 1960). Nicotine w•as found to be less potent than dimcthylphenylpiperaainium (1)~1PP), but more Iwtent than aeet)•Icholine (Herr and G,vennek, 1960). 139. Aleclwnisnr of the Gangfconic ,Icteon of Nicotine (59) The finding of Paton and Pcrry 1(1951e„ b)(58b-59a); see alw I'atoh and Perry (1953)(59a)j that nicotine blocks tran.;- mi.wiun at sympathetic gauglia through depolarization of the ganglion celL+ has been confirmed by later rvorkers (A. Lund- ber);, 1952; Pavoe, 1956; I). F. J: Afason, 1962b; Blackman et al., 1963; :A. Jones, 1963; Jones and Trendelenburg, 1963; \fattila, 1963a, b: Ginsborg and Gyierrera, 1904; Shand, 1965; Gebber and Volle, 1966). From his study of the effect of nicotinih on the positive after-poterotial and on post -activation depres,don in sgnpnthetic ganglia (see above, 138), Skok (1959) concluded that hyperpolarirstion of ganglion neurnnes was ahways attended by depression; however, depression might sometimes exist atithout any percept'ble positive after- potential. [See Gebber and Volle (1966), above, 136.1 1'eiikan and co•workers 1YeRkan and Garcia (1960); Pelikan and Carten (1962)) have again suggested that nicotine may preventt ganglionic transmission by impairing acetylcholine release (see Pelikan and Garcia (1059)(56a; 59b)J. Nicotine in concentration of 10 µq/uil also prevented release of acetyl- chuline from segments of guinea-pig ileum incubated in vitro; and I'eGkan (1960) summed up the mechanism of ganglionic blockade produced by nicotine as follows: "tiicotine may impair ganglionic uafismWou by preventing the release of acetylcholine from presynaptic nerve fibers, rather than by blockitt;X the tuetyleholine receptors on gsnglionic cell bodies." With resitect to receptor , the experimental results of the effect of nicotine and acetylcholine on the superior cervicr.1 r,anglio» (see abo••e, 136) were interpreted by Feh6r and co•.corkers (1961) according to the concept that ganglion cell., nave both "free" and "innervated" receptors. With increasing concentration, nicotine was said to bind first with the free, and then with the innervated receptors; its stimulat- ing effect was considered to be exerted on the free receptors, and its blocking effect on the innervated receptors. According to Pelikan and Carten (1963), intraganglionic selectivity of the action of nicotine (that is tosa,v, selectivity to stimitlate, rather than to block, ganglia), is inherent in the p>•rrolidine moiety of the nicotine molecule. Selectivity was evaluated by these workers from geometric mean molar doRes, equipot;ent in stimulating the superior cervical ganglion, blocking ganglionic transmission, oh causing shortening of the nictitating membrane in barbitalised cats; drugs were given by intra-arterial injection to ganglia or membranes, and each agent was studied in 3-5 cats, e'ach animal receiving one agent only. Strongly basic pwrolidine and weakly basic p)•ridine selectively stimulated, not blocked, ganglia, the former being the more potent, in this respeet. A'oruicotine, nicotine, and 2-pheu)•Ipyrrolide were alsro selective in stimu- lating ganglin; bensylamine and, phenethyhuttine prefer- em!islly blocked, not stimulated, ganglia. Pyridine, pyrrolidine, nicotiue, and nornicotine were more selective than 2•Iihe'nylpyrrolide, benaylanrine, and pheneth,rlamine for uanglionic, as compared to membrane effects; pyddine, 39 .....I t., ...nl:ri:..n rnnfnr,wl , thi. M1M•IiYHt• Ri wnl) Ra potency, on the nicotine moletule. The meth' vd group on the p)•ridine nitrogen atum of nieoQiue iucrea..etl lwteney, relative to nornicotine, but made nicotine hm selective than nornico- tine in stimulating, as olqwst4,i to blocking, ganglia. Studies of the pharmacological action of nieotiim mcthiodides had led Shimamoto and co•workers (1958) to conclude that, "though the rationic head of pyrimidine ring of nicotine molecule played an important rolc for the development of the action of nicotine, the anionic head of pa•ridiue ring of the molecule aho affected the same binding." Barlow and Hamilton (1962a) prepared a number of uwmerv. and humologues of nicotine+ some of which they tested for their ability to stimulate the superior cervical ganglioa of the cat (wing contrscture of the nictitating membrane as an indicator), and all ol which were tested for their ability to block transmission in this ganglion~ but for details of these estperirotents, and the authors' dihcuh- siou of structure-activity rela'tiunehil^, the reader mum be referred to the original article, or to the extensive review and analysis by Barlow (1965) of work which has been done in attempts to correlate chemical structure and biological ac• tivity of nicotine and related compounds at ganglia and at the neuromuscular junction. [See also liarlow and Hamilton (1965), below, 978.) In his investigation of the pharmacology of nicotine, nornicotine, ethylnornicotine, allyhtornicotine, acetybrornico• tine, carbamhtoyhwraicotine, and benzqvlnornicotine, \lattila (1963a, b) found that the actions of most of the derivatives on the superior cen-ical gangliou of the -at were of the same nature, causing depolarization (with the poccible exception of beuaoylnontirotine). Two other ashects of the "selectivity" of nicotine and/or ganglion cel.ls may be considered here. Using a preparation consisting of the excised rat superior cervical ganglion. with its postganglionic internal-rxrotid nerve and preganglionic cervical sympathetic trunk, Qttilliam and Shand (1964) compared th, effect of nicotine on ganglionic transmission and on the pre- ah•1 pactganglionic nerves, and found that nicotine blocked lratumission in ^otrcetdrations which did not affect nervous conduction; thus, nicotine was considered to be highly selective in action. pSat Shand (1965), above, 138.) appelgrer, Hans.co.t and ScSmiterl6w (1963) mstle intra-carotid injections of (-)-nicotine•methyl-'4C in cats, aud, 5 n.in later, removed the supetior cervical ganglion and the nndm-e ganglion of the vagus, which were then studied using auto-adiogrephic and micro-autotadiographic tech- niques. The micro•autaradiograms shon•ed that almost all of the radioactivity was located in the ganglion celLs whereas satellite cells and the connective tissue contained ver}• little radioaetivity; however, some of the ganglinn cells contained much more radioactivity than others. Possible explanations con.Qidered by the authors n•ere, that nicotine has a specific affinity for those cells which contaitted a high amount o1 acetykholinestetase, and that sympathetic ganglion cells are funct ionally heterogeneous. Compared to the superlor cen ical ganglion, much less radioactivity accumulated in the nodose ganglion, a phenomenon which might be explained by their different structure and physiological mode of action. 140. Spfna! Gangtio (60) Curtis and Eccles (1968a) described methods by which drugs can be applied electrohhoretiealh• to the s/xrial group of intemeurnnes, called Renshaw celh, located within the vent ml horn of lumbar segments of the spinal cord of the cat. Produced by T;ze Council for 0002951 Tobacco rescarch-USr1, Inc.

-!0 ;•OBACCO-E.CPEIt1MENTAL AX1) CLINICAL STCTllIES. SUPPLEMENT I .\Il'UlUll' tlU atlpUPU Wl'IYJLICU NIC 1LLlC V/ WMI1µ/(St V/ u.Ml. cells. (See P. St.erld (1961), below, 148, on serotonin and Ren- shaw cells.) Neither edrophonium nor neostigmine prolonged the response of a cell to nicotine; dihydro-fi-erytbroidine blocked t;he stimulant action of nicotine (Curtis and Eccles, 1958b). Fserine had no significant effect on the action of nicotine (J. C. Eccles. R. M. Eccles and Fatt, 1955). [See also Eccles. Ecclea and Fatt (1950)(81b).J Further studies supplied evidence that each Renshaw cell has both nicotinic and muscarinqc receptors (Curtis and Ryall, 1964). In this connec- tion, it may be rrlentioned that nicotine, electropboretically passed as the cation from a saturated solution of nicotine HCI (pH 5.5) in harrel electrodes, did not depress synaptic excitation of neurrlnes of the lateral geniculatF nucleus of cats under peiriobarbital anesthesia (Curtis and Davis, 1962). 141. Autonomic Verees and Nerve-Fibers (61) Armett and Rit,chie (1960, 1961) studied the action of nicotine on conduction in the mammalian non•myelulated nen•e-fibers, using the sucrore-gap apparatus described by Stimpfii f Experientia la 508, 1954) to measure the action- and resting-potentials of the desheathed vagus nerve of the rabbit. Perfusion of the nerve with 0.6 mJ1 nicotine hydrogen tartrate caused a derrease in the spike of about 15% (Armett and Ritchie, 1961), and perfusion with 1.7 null reduced the spike by about 35%, which was the reduction brought about by a similar molar concentration of acetylcholine chloride (.lrmett and Ritchie, 1960) The ability of acetylcholine to affect conduction in mammalian C fibers [arrnett and Ritchie (t901)) is shared by the acetylchoiine-like drugs whirh excite ganglion cells and depolarize motor end-plates (such as nico- tane, carbachol, and tetrttmethylammonium); and it thus seems that parts of the nerve membrane of C fibers are capable of pharmacological reactions reminiscent of those occurring at the motor end-plate and autonomic ganglion cells (Douglas atd Ritchie, 1962). In dogs under tliorphine and chloralose anesthesia, Jewett (1904) re•.orded activity of single efferent fibers in the cervical vagus nerve. Retrugrade injection into the superior thyroid artery of either 50-200 pg potassium cyanide or (infrequently) i0-20 pg nicotine caused 1 or 2 deep rapid breaths, and the following effects (none lasting longer than the respiratory stimulation) on t.he 21 Type I single fibers tested: heart-rate slowing and increased activity in l l fibers; a slight slowing of the heart and no change in 3 fibere; no change in either heart- rate or activity in 7 fibers. Using dogs under chloralose and urethane anesthesia and axtificial ventilation (opett-chest), Sleight and 11 iddicombe (1964, 1965) recorded action-potentials from.single-fiber and sma0 multifiber preparations of the cardiac and cervical vagus nerves, the fibers coming from epicardial and m,roesrdial receptors in the wall of the left ventricle. None of 7 prepara- tions was stimulated by intrapericardisl injections of 5(FIW pg nicotine (Sleight and Widdieombe, 1984). Cooling of the nght cervioal vago•sympathetic nerve to 7-8° C a•hile recording cephalad to the point of cooling ares found to block conduction of action-potential.p from intrapericsrdial injections of 60 µg nicotine (Sleight and 1l'iddicombe,198S). After a receptor had been Initially idenEified by Its fiber response to palpation of the ventrides, its response to nicotine injected into the peti- cardial aae was recorded, from which it was apparent that the receptors could be arbitrarily divided into two groups, a nicotine.sensitive group (peak frequency of discharge over 10 impulses/sec when' stimulated by nicotine) and a nicotine. insensitive group (0-10 impulses/sec). (For further chamc- _ ,. the complete article (Sleight and •Widdiconibe, 1965). The cardiovascular depressor reflex from the epicardium of the left ventricle in the dog has been described by Sleight (19&t).I 142. Cptoehemistsy of A vlorromic A'emes and Conplia Von Euler and Lishajko (1961a) ir•olated traw-miner granules containing norepinephnne from homat!enatea or pressed juice of adrenergic nerves, and reported that, in rc- sublrrnsions of t}IeQe granules, certain agents could effect release of noreoinephrine or blockade of release. In this re- spect, nicotine appesrcd to be without aclion. AW, nicotine in concentrations of 3-300 pte/mI had no effect on norepineph- rine release from transmitter grsnules 4.olated by high•g centrifugation of prpss-juice Aom bovine sldenie nerves (von Euler and Lishajko, 1981b). Holland, and Yamov (1965) described localiiation of catecbolamines in the inferior mesen- teric ganglion of rats and rabbits by means of a fluorescent histochemical procedure. Fonnaldehyde-c•ondensed mono- anuues were also localized in ciseeral organs. CateEholat.unes, characterized by a green fluorescence, were seen as beads, fibers, or larger fluorescent structures. In the intestinal ple..•uses, as well as in the taenia coli and heart, there was an abundance of fluorescent fibers, many of schich ran along the muscle fibers. In view of the abundance of catecholamine- eontaining structures in the intestinal and cardiac tissues studied, these n•orkers suggested that the syhipathominrctic action of nicotine on the heart and intestine is produced by the release of norepinephrine. \lirzoyan (1961) considered that some reactive groups of protein bodies undoubtedly played a responsible role in the primary chemical reactions of pharmacologic agents with the receptors of effector organs, and be therefore Im•estigated the interaction of gangliolyGcs with the tissue sulphydryl groups in structural homogenates-the superior cervical ganglion and the ganglia of the vagal intestinal fibers in cats. Doses of nicotine which augmented the sensitivity of ganglionic ceilx to electrical stimulation of presynaptic fibers and facilitated tranFmission in intraneurosal synapses, caused an increase iu tisuc :hiol-groups content. ln large sfoses, the blocking effert of nicotine on ganglionic transmission fully spread over the sulphydryl.groups content lauthor's EnglishJ. This capacity of gangliolyties to decrease the thiol-groups content anJ tissue sulphydryl groups appeared to the author to have a definite significance in the chemical significance lai/•] of receptors towards ganglion-blocking agents. Vysotskaya (1903) studied the effect of gang:ion-blockrog agent.-:, including nicotine, on the metabolism as well as trans- miaion in tlre superior cervical ganglion of urethane-anesthe- tieed cats. The blocking action of nicotine (as detetmined by response of the nictitating membrane to appropriate elec- tricsl sthnulat.ion) was aclvml/enie.d by a reduction ia the adenosine ttiphosphate (.aTP) and phosrhocreatiae content of the ganalion, and the concentration of inorganic phosphorus correspondingly increaaed. To explain the shifts in the ATi' content, a study was made of the effect on the processes of glycolytic and oxidative dTP resyntheits, as well as on the ATP activity in the ganglion, which revealed that the de- prexainn of resynthesis (Rlycolysis and oxidation) played a significant role in reduciion of the ATP content of the ganglion. However, stimulation of the mechanism of ATP breakdown (an incrPase of ATPa_v activity) was aiso said to be importanL. Nicotine decreased the potassium ion and fiH- group content of the superior cervical ganglion. I Produced bv The Coumcil for 0 00295 2) Tobacco itesc%a;ch-USA, Inc.

i t "A NERVOUS SYSTEAt 143. Hfatopathologd of Atslortom{e Nernea and Ganglia (63) [No new datal 144. Azon-ReJlea: PilomoEor Resporese (64) J. H. Burn and Rand (1980b) reported that they had con- 6rmed the observation of Brltcke jIQin. rlt'sebr.1/: 7, 19351 that, when a large amount of nicotine was injected into the skin at the base of a tuft of hair of the cad's tail, the effect of stimulation of the lumbar sympathetic chain was blocked, so that, while the adjacent tufts, respectively, near to and farther from, the base of the tail, were erected during stimulation, the tuft at the site of injection remained unafi'ect.ed. [Examination of Brcicke's original paper has revealed that this worker had actually used only acetylcholine, not nicotine.) J. H. Burn and his colleagues (1959) found that nicotine, acetylcholine, or epinephrine injected intradermally into the tail of the cat caused a localized pilomotor response, and this response was much reduced if the animal had first been given reaerpine. Treatment with reserpine also greatly reduced the norepinepbrine which could be extracted from the skin of the cat's ta9, and reduoed the number of cbroimaffrn cells present in the arrector-pili,museles, and the number of granules in the cells. These studies suggested that pilo.erection caused by nicotine is due to the release of a norepinephrine.like sub• stance, probably from chromat6n cells present in the skin of the cat's tag (Burn, Leach and Rand, 1959). HeOmann (1963) found that a 500 µg/ml concentration of niootine caused contraction of isolated pilomotor muscles of the skin of cate tails at bath temperatures of 30 and 20°C, but not at 10°C. In the absence of contraction from nicotine at 10`C, contractions due to electrical stimulation were de• pressed. If the temperature of the bath was then reised, a nieotine cwntaaction was obtained when the temperature reached 1b°C. In this type of preparation, nicotine in concen- tration below 100 pg/ml produced no contractitms, but 100 pg/mi nicotine did cause contraction (Hellroann,1984). How- ever, exposure to 10 µF/mI nicotine, although not causing oontraction, prevented the subsequen! appearance ef ecn- tractions by any concentratiorn of nicothne; similarly, after a contraction had been elicited by 100 µg/mi n:cotine, no sub• sequent contraction could be produced 'uy this or by any other dose of the drug. Nicotine failed to produce a contraction in prepamtior.4 previously treated with 100 pR/rni guanethidine for 30 tnin or with 250 µg/ml hexametltonii:m for 20 min. Contractions elicited by nicotine were not sustained, the attainment of maximal height being followed by relaxation. Electrical stimulation immediately foAowfag a nicotine eon• traction produceu contractions which were greatly poten- tiated; on further electrical stimulation, this potentiation was followed by a depression reversible on vigorouv washing. 145. Azon-ItqAear Stoeating (65) M. Wada and his Japanese colleagues have extensively investigated nicotine-induced local sweating under a variety of conditions in several animal species and in human skin. After reviewing their own and others' findings on species posressing ecerine and/or apocrine glands, they concluded that the teceptots responsible for the sweating axonae8ex are associated with cholinergic sweat nerve-fibers, trreapective of the type of sweat glands innervated (Wada et a1.,1962). A dual sudorific effect of nicotine was u.icovered by 1Cada, fiakamum, Hatanaka and Aoki (1965) by applying a band ligature around a tue-pad of a cat with sufficient teneion to Prevent an axon-ref)ex-provoking agent, which had been iu- 91 jecte.d intra- and subcutaneously to the distal side of the bancY, trom drGusmg to the proxunal side. ln this preparation, after acute section of the sciatic nerve, I pg/ml nicotine in most cases caused sweating by a direct action alotie (limited to the injected side), while 10 pg/mi nicotine in most cases elicited axon-reflex sweating responses (spreading to the uniajected side of the band). With progressively longer time following sciatic-nerve section, the nicotine concentration necersary to produce an axon-re9ex sweat response became progtessively greater, until even 10 mg/ml was ineffective. At this point, sweat response to direct action of nicotine per. sisteai, and was rather acceatuated. Further studies showed that degeinerative section of the sensory nerves suppl}ing the toe-pads did not aBect the axon-reflex sweat rrKponse, while degeqreration of the postganglionic sympathetic fibers abol- ished it, leaving unimpaired the sweat reslwnse to the direct action of nicotine. Atropine injected along with nicotine also abolished the axon-re8ex sweat response. In several dogs which showed a marked sweat response to intradermal injection of epinephrine or acetylcholine, intra- dermal injection of 10-1,000 pg/mi nicotine (which was su($- eient to cause typical axon-reflex sweating in man) in the frontal aspects of the thorax and abdomen, or the ventral surface of the thigh, did not result in a.eon-re8ex sweating (Aoki and Wads, 1951; Aoki, 1955). Higher (1-10 mR;'ml) concentrations produced, at most, a very delayed appe,uance of a small number of sweat spots on the injection wheal, usually 5-10 min after the injection. Most of the animals did not show any spontaneous sweating on the general hairy slun, , even when excitement and panting were considerable. In several dogs, however, a slight sweating wres obsened to occur spontaneously in restricted areas, e.g., around the umbijicus, in the median part of tb:.• hypogastric region, or in the pubic region; and in these animals, injection of nicotine into these restricted areas caused sweating of axon-reflex nature, although to a slight degree. The sa-eat, glands in the hairy skin of the dog were said to be of apocrine type. When tested on 2 femalu horses (B7uss caballus L.), nico- tine in concentrations of I µg-10 mg/ml had no sudorific effec4 and no evidence of the occurrence of axon sweating was found (Aoki, Kimura and it ada, 1959). The horse saeatV glands were also said to be of the apocrine type. The species of primates, A'yctieehur roncang, is said to have ap-rine-type sweat-glands in the hairy skin, which ave sur- rounded by nerve-fibers containing specific cholincalerase. By use of the single•banu technique described above (1t'ada et al (1955)), Aoki (1962) demonstrated that intradetmal injection of 10-1,000 µg/m1 nicotine elicited local Ra-eat'rng, axon-refleex in origin. Simultaneous administration of 100 pg/ml pmcaine with this concentration of nicotine suppressed the axon• reffex, and 1 mg/ml cocaine completely i»hibited it. In con• centre'ttone of 100 µg/rnl, both at.ropine and hexamethonium completely blocked the axon-reflex sweating produced by 100 µg/mi nicotine. In another species of priruvte. Pcraduticut poffo, the sweat glands of the hairy skin were also of apocrine type, but have no cholinceterase•positive nerres around t.hem. In this species, using the same technique and nicotine in l0- 1,000 pg/ml concentration, there was no evidence of axon• reffex eweating; there ares slight and inconstant sweating, localized to the injection wheal, but in none of the tests was there 6 sareat response across the band. Studies by Nada and co-workers on the inhibition of nico. tine-induoed axon-reffex sweating In human Fkin by other drugs revealed that hexamethonium, dccanncthoniutn, and d-tubocurarine (in order of decreasing potency) blocked tbe Produced by The Council tor Tobacco Rescarch-USA, Ir!~. 000295;3

42 TUBACCt)-I::XPERLUE\TAL A\ll CLINICAL S+TUIHEN'. >aUPP1.F'AfENT I blocked, totalfy or almost totally, the reslwnse to 10 µg/ml sticutine and to 1001at;/ml acetyIcholine (liuribayAshi, 1959) ; and hordenine sulfate also blocked the aaron-refle% sweat responses of human skin to nicotine and acetylcholine (G. Tashiro, 1960). The blocking effect of these antagoni:uic drugs was said to be due to competitiou at the site of the ason-reflex receptor (Wada et al., 1957; I:uribayashi, 1950; Tashiro, 1960). Using the one. and hvn.ruuber-band technique, Asakura (1964) found that thr axon-reflex of the human skin resulting from intradennal iujection of nicotine and sodium chloride was inhibited by reserpine (Serpasila); however, this inhibitory effect was shown to be entirely attribui.ablc to its vehicle per ae, and not to its reserpine content. The vehicle- polyethylene glycol SOII-aas found to inhibit the axon-reflex by acting on the receptoas involved, without affecting the pathways carrying the aeon-refle% impulses or the e6ector endings of the sweat nerve or the sweat glands themselves. ln a study of the effects of sodium and lithium upon the receptor for the sweating axonaeflea in human skpn, nicotine was employed in concentmtion of 10 µg/ml, and the NaCl in its solution was reduced to aa, 2j, t¢, 'y, 1;, '(, and 15 of 0.9% NaCI; the subtracted NaCI was replaced by sucrose in 43 and by glucose in 35 e.yreriments (11'ada et al., 1962). Compared to the area of sweat response to nirotio,e in 0.9% NaC1 solution, the area was reduced to 89, 79, 67, 97, 28, 19, and 9%, respectively, with the progressively-diluted NnCI solutions; also, with sodium-deficient solutions of nicotine, the latent period for the appearance of the sa•eat response was usually more or less markedly prolonged. Raising the concentration of NaC1 to 150 1~ of the control increased the size of the response area by about 15 %.1 n similar exryz'eriments performed using LiCI instead of NaCI, the size of the response with isotonic (0.6500 LiCI averaged 59% of that with isotonic NaCI. Lowering the conccntrotaon of LiCI by adding glucose as before, resulted in the size of the response being reduced proportionate to the decrease in LiCI; delay in the response was again also noted. The resjwru;e with isotonic LiCI was delayed in comparison with that to ir.otonic NaCl. JleLaughlin and Sonnet>RChein (1960) found that previous injeM3on of 0.05 mg diethyl•9-otitropherrl phosphate (1>Bra- oxon) into an ams of human skin produced unequivocal inhibit'ron of the sweat response to 2-20 µg/mI nicotine in 16 of 19 experiments; in 3 trials, paraoxon had either no effect on the nicotine reslwnse, or produced a slight facilitation. When nicotine was injected at higher (l00 or 500 pg/ml) cancentra- taons into parao..ron-trested areas, the sugaestion of a facili- tatory effect was seen in each of 5 trials. 146. .4amt-lteffax.' 1'asom.'otor Axon-Refletea (66) Giniel and ICottega I = KotteEtoda) (1954) sought to eluci. date the role of aabn-re8exes, in the stimulating effect of nicotine and acetylcho6l>e on heart and blooda•cssels, and concluded from their e.h,eriment.s on normally innen'ated and chronically denervated otians of the rabbit that the stimulating actioruz of nirolioe end nretylcholine in iheFe preparations were not mediated through a%on.reflexes in the corresponding Iwstganglionic sympathetic fibers. 147. Central .dutononttr 8gatem (66) Certain experiments and assumptions by Tauberger (1984) led this worker to beliere that nicotine ntanifested a primar.' central stinwlation Jof autonomic cemerx), and that the "eentral" efleats of nicotine were subject to marked tachy- nrlt'IAVIF (/nr on neenunt of the,w ex,erimeotts, see helnw. 3.39). There have been no systematic attempts at invesligat. img such "centrai" effect.c, however; and, as ise ourreh'e3 had written in drawing attention to the probable importance of the central autonomic effects of nicotine and smoking, re- search in this area has been fmgmentary, if not haphacard, a'nc4 the olnen'ations below still reflect this situation (66a). In the course of her study on the concentration of sympathin in different Imrta of the central nen'ous e,yateln, Vogt (1951) administered nicotine bitartrate s.c. lo 9 cats in divided dosr~ totaning 12-17 mg/ka; the animel+ were sacrificed 4.25-6.5 hoteta after the Cirst injection, and mean b>ryiothalamic nor- epiu,ephrine content was found to be reduced to two-thirdr: of that of control animals. (In the doses used, nicotine eausal si'ckma in 2 cats aud cuna ulsiuur in the ulher 2 auimals.) ; In his book, Lh'nys, .lledfcfnee and Man, li. liunt (1962) disCus.sed pharnutcological and non-pharmacological reaecol>.i for smoking and, in connection u'ith the former, he cited studies with reserpine and DOPA suggesting that the nor- epinephrine present in the hypothalamus and elsewhere in the brain determines one's mood: when norcqrinephrine is absent, we becoRne depressed, so that it is possible that a state of normal cheerfulness delxnds on a normal release of norepinephrine from the store in the hypothalamus, and it may be, on the bther hand, that excessive release of norepi- nephrine leads to anxiety and tension. Since nicotine has been shown to releaae,norepinephrine from stores in the heart and blood-vessels and elsewhere, Nurn considend ot reasonable to suppose that thm drug may alxro release norepinephrine from stores in the hypothalamus and in the midbraid; and the effect of smoking may then be that the norcpinepha•ine released in this way elevates the mood and produces some degree of euphoria or feeling of w'e!I-being. Such an action too would explain the addiction to smoking of those w-ho become de- prrsced without it Grossman (Science )9m• 301, 1960) reported that eating or drinking could be elicited by the direct appl?ration of cr}•s- talline substances into the lateral hypothalamus of the rat. adrenergic substances (norepinephrine and epinephrine) selectively induced eating, and cholinergic substances (carba- chol, acetylrholb,e capped 1kv physostigrnine) selectively in- duced drinking. L. StPLt and Seifter (1961, 1962) cot.firnted and extended the cholinergic findings. Minute (1-5 µR) quantities of crystalline compounds were introduced into the brains of 28 rata thnwgh permanently indN'ellitqt cannulae. lluscarine had an effect equal to carbachol, producing strong drinking within 5-10 min, which persisted with variable in- tensity for almorat an hour. Nicotine had the same small "non-specific" effect as sodium chloride-about twice as much as no-drug conttoLw but only 20 % of the effect of carbachol or muscarine. The effects of carbachol and mus- carhte were largely blocked by prior application of atropine, suRgrcting that rhnlinergicall}•-induced drinking i,e a mus- carinir, not uieotiuic, action. A. Katz (1965) noted that prerious investigators had shown that mur•rarinic agents induc'e a temlwrary rage state in previously normal cats /when injected into the posterior hypothalamic ar'caJ. In his own experiments, this worker found that eleettically-induced rage failed to elicit the full. fiedged rage behavior caused by the drugs, bnt induced his.cing, escafm, ahd biting reactions. Testing of drugs on the electrically-atimufated cats showed that the hissing threshold was raised by anticholinergics and lowered by pro-choliner. plics, including nicotine; the eacape thre:chold was raised by auti-muscadnicp, lowered by pro-muscarinics, and was un- Produccd bv The Council for 0002954 Tobacco Research-USA, lnc.

TI;1tYnUS S)'gT1;Af 13 chanl:eal by nicotine; the biting threshold tras wsflected by any of the drugs employed. According to I. Yamamoto (1961), antidiuresis produced by cigarette-smoking was siynificantly inhibited by thiamine in ma.n, although this substence had no influence on the anti- diuresis induced by Iwsterior-pituit'ary extract. This .uggf~zted that nicotine antagonism by thiamine is connected with the In•twt6lamico-posterior-pituitary , system; and this w•orker further noted that no other nicotinolytic substances shonrd the same type of antagonism. ln this connection, it in interest- ing that a target organ for nicotine sertns to be the pituitary; 5 min alter injection of "C•labelled nicotine in mice, the mdio• acticity in the pituitary e•ds higher than in the brain, and remained so until 1 hour (Hansisron and Scbmiterlbw, 1962). 148. CERERILaL CORTEX (67) Itt may be recalled that, Qn his study of conditioned pole• jumping in rats (see above, 121), Domino (1965b) found that only nicotine of a number of related compounds n•a<eflective in blocking the conditioned avoidance behavior, and this was related to the fact that all the compounds but nicotine were 100 % ionizable quaternary nitrogen compounds, to which the central nervous system (C*iS) is Iworly accessible. Experi- ments with the physiological disposition and fate of "C- labelled nicotine in mice and mts fed Hmusson and Sehmiter- Iotv (1962) to postulate that nicotine has, indeed, some affinity for brain time ]a•hich its metubolite, cotinine, aptean/ not to have-but see Bowman et al. (1964), following). When a solution ol this radioactive nicotine was applied directly into the trachea of mice, autoradiogr.lms made 2 tnin later showed that nicotine had entered the CXS rapidly, and a 15-tnin auUoradiopnm revealed that the brain then contained con- siderably less activity t.han at 2 min (SchmiterCow and Hans• son, 1965). In autoradiogrants of, brain t;ections froni cats sacrificed 5 min after injection of "C-nicotine, the hitihe:ct level of tadioactivity was observed in the grey matter, compared to the white matter (Appela!en, Hansron and Schmiterl8w, 1962). The nuclei of the diencephalon and the medulla oblongata also had a tendency to concentrate radio- activity. inthebrainof cats sacrificed 15 min after i.v. injec- tion of nicotine, there was stili a distinct difference between white and grey matter, and there was a fairly high concentra- tion of radioaMivity, in the hipiwcampus, particularly in cr.r- tain areas, presumably corresponding to the cell layers. It appears that radioactive cotinine does concentrate, to some extent, in the cerebellum of mice (E. R. Aowman, Hansson, Turnbull, AtcKennis and Schmiterl8w, 1964). The difference in distribution betaeea nicotine and cotinine haa been related to the marked pharmacological activity of nicotine in contrast to the lesser activity of the nicotine metabolite, cotinine.l'he.ee demonstmtionathattdcotlnereadily penetrates the blood-brain barrier, and possesses an affinity for nervous tiscue not shared by at least one of its tnetabolites, must be considered to be of the greatest importance in atty consideration of nicotine as a neterotropfc drug, since it is "for its effects on the brain that nicotine ia used" (H. Burn, 1962). 3s to the ntechartism by which nicotine or tobacco-smoking produces its CNS effects, our previous suggestion that It is possible to conceive that nicotine•induced brain serotonin release may have "beneficial" efi'ects on CNS function (69a) has perhaps received some equally hypothetical support fmm later experiments Rclaevelbein, Werle and Jacoby (1961), studying dogs, concluded that nicotine (iberates serotottin from the cerebral hemispherea, and cotttryuentl,v also exerts a pot;sible in6uence on the function of the cerebrum; and Echiea•elbein and tt'erle (1962a) have discw:.ed the effect of . IIIlYI11111.' U// SYlvluul/1 Ixu;gib)e psychic e8^cts ol nicotine obtained through smoking. 13ut even the exact effect of nicotine on brtsin•serotonin con- tent i., still uncertain, or at least anlxnrv to be dependent on eximrimentsl rondition* which do not obtain in human smok- iuk (-,tr bclow, 151). We ntay alab refer the reader Nerr to certain studies by H. Haas (1960) on the tnodifiral,ion of nicotine to!ticit;• in mice by tirrotonin (see belou•, 991), ap- parcntly relating the sonien•Iwt protertiee effect to the z,rro- touin content of the C\a (brain). Certain extmrimentr re- Iwrteli by 1'. Steru (1904) appesr to be relernot. This irorker found that intravenous injection of 0.25 mt;/kF nicotine in mice transformed the static tremor produced by trentorun• tu an intentinn tremnr; nnJl. nn the aramptiml that nirnline relcascs serotonitt in the ('N5, he Ial.-u+lated that serolunin is the transition substance of the Renshaw cells a•hich inhibits the motor anterior horn cells. 1Chen the do)utntine and r•cro- tonin cotttents in the CNS were lowered bw• n_vrpine; nico• tine no longer caused intention tremor; when only the du)m- mine, but not the serotonin in the C\,t, a•as reduced by a-mettni•r++•tyrosine, nicotine produtMd an iMentiott tntinor from the static tremor. Dichlorisoprolp•hwtepinephrine, which inhibits some central dopamine effects, did not inhibit the nicotine effect; and the serotonin inhibitor, lynergir acid diethylantide (ISU), did not annul the nicotine eflect on static tremor. (LSD alone can produce inteutimt tremor following tremorine.) Whether the ens)-matic ,\••metF.ylation of serotonin and nornicotine described by .3eelrod (19t12s1, b) is of relevance here is questionable; see above. 56. H. Bum (19(12) has suareded another possible nteclt- anism-nicotine-induced norepinephrisc releate from stonis present in the hypothalanms and elsewhere in the brain (aY'e above, 1d7)-•by whic'.1 tobacco-sntokina may produce an cfiect on the higher centers. For experimental evidence tend- ing to 6upport this )wssibility, see below, 131. In the councc of iontophoretic studies of single neurane• itt the cerebral corte. of cats, rabbits and rhesus mdnkeys (usually anesthetiatii with diallylburbituric acid phv urc- thane), I:rtljevif and Phillis (1963a, b) found that nicotine excited only about a quarter of the celLs tested, and then usually after a very long delay; thi. %ras considcred to be an unspecific excitant Fffect of nicotine on cortical neuroneF. \icotine tended to prcaluce some mild deptexaion of excit- ability of many ceUs, whet her cholinotrptive or not (as tasted by releasing of t.•glutamate or acetylrholine); there was no specific antagonism to acetylcholine itself (Kmjeviti and Phillis, 1963b). According to Curtis and Ryall (1984), nicotine is only a a•eak excitant to liet.z cells Ilat•ge pyratnidal cells in the Holandic• area (motor area of the cerebral cortex, comprising the prr-central and post-central eonvolutions), esixcially in the cortex of the pre-central *mus]. httheirstudyof the phamtaculupy or neurola~in tlrc pyri- form cortex, Leg.Ge, RandiF and Straughan (191W) used cats, mostly under Dial anesthesia, and drugs were administered by the microelretrophoretio method, and edracellular spike Iwtentials from single units were reconhYl. \icotine hydrogen tartrate in 0.2 )1 concentration was tested on 24 unita in 5 preparatiotts; in 9 units, after a long latency of 20-40 nec, therewasa sntall incrcasein the firinq•rate of the satne negative spike which was fired by a.-glutamate or by aretyl-IB-methyl• choline, and the cells continued to fire for a time after the adminiatration of nicotine a•as stopped before regaining a norntnl level of exeitability. 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44 TOBACCO-EXPERIbfENTAL AND CLIN6CAL STUDIES. BUPPLEMENT I Turpaev and colleagues (1984) reported having detected a . _ _r .t., .h.!:_. ..... _ .~r ..:C.. ~. ~.: .. ..... rabbit (auricle; inbestine; uterine born; skeletal muscle; cerebntm) sensitive to acetylcholine and possessing neceptota of the muscarinic and rieotinic type; this protein was not found in tissuea and organa not sensitive to acetydcho)ine. Experiments with an extract fr+om rabbit cerebrum demon- stt•ated that high (100 Ng/ml) concentrations of nicotine eliminated or suppressed the effect of acetylcholinc. Certain effects of nicotine and some nicotine•like com- pounds injected into the cerebral ventricles of the cat have been described by Armitage, Milton and Alorrisoro (191i5, 19136). Nicotine was injected in both anesthetized and con- scious eats through an indwelling Collieon cannula implanted into either the right or left lateral ventricle, in doses ranging from I to 100 pg. In the conscious, unrestrained animal, regu• larly observed effects were: twitching o,f the eam, vomiting and retching, salivation, licking and crouching. With higher doses (25-100 pg), there also occurred panting, inability to stand, and unresponsiveness to noise and touch. Fu0 re:overy usually occurred within 30 min. The most striking effect was con- sidered to be the continuous twitching of the erus, which closely resembled repeated activation of the pinna Aex, and was observed in both conscious and chloralose-ariesthetiaed animals. Experiments on anesthetized cats with antagonistic drugs (mecamylamine; hexamethoniµm; neostigmine) sug- gested to the authors that this action of nicotine involves a central cholinergic mechanism. Nicotine given i.v. in doses of the order of 40-100 pg/kg also caused twitching of the eatB in both anesthetized and conscioua eata. The nicotine-Bke compounds, dunetltylphenylpiperazWum iodide (DDfPP), nornicotine, anabasine, and metanicqtine, also caused ear- twitch when injected Lv. in the conscious cat, but these drugs were less effective than nicotine. Alarkiewicz (1985) has bri--fly reviewed mechanisms of ae• tion of the principal constituents of tobacco on the cerebral cortex, etc. 149. PeychunwWr Centers (Sens-im.otor Punctions) (67) We bavre remarked that experiments on the effeet of nico• tine on functions of the eensorirnotflr areas appear to be rarely performed or reported today (P. S. Larson and Sil- vette, 1965b). At any rate, no recent ones have come to out notice. 150. Broin Potentials (6g) Summarizing most briefly the published work up to that considered in the Stockholm Symposium on "Tobaceo Al. kaloids and Central Nervou System Functions" (Larson and Silvette, 1995b), it is evident that nicotine ildministration causes typical "grand maP" seizure patterns in the electro• encephalogram (EEG), provided sufficiently large doses are given. Doses of less than I mgl/:g appear to cause on)y pro- longed desynebtrmisation. With somewhat larger dases, there may be tonic and clonic contract5ons, as well as muscular fibrillation, and the appearance in EEG tracing of convulsive patterns (see above, 129). The desynchroniaing effect of nicotine ie very probably due to excitation of the reticular formation; the convulsive effect has been ascribed to a general state of excitation, probably including cortical neuronea. I.v. injection of 1 µg/kg nicotine caused a distinct prolonga- tion of electrically induced c•ortacal after-discharge of the neuronall,v-isolated cerebral corcex of the rat, and this effect was not blocked by i.v. injection of 1 mg/kg atropine, either before or after the nicotine (Nickaqder and Yma, 1962). The 9Hav.rnevhqw.r.v nf D7nP icnt9lat cerebral cortex in unenesthetised, iEamobiliaed doka also was significantly iacneaeed by doses of nicotine as low, as 1.0 Kd/kg i.v. (Nickander and Y'un, 1904). This effect in dogs was generally observable 2-10 min following nicotine ad- ministration, and was still evident more than 1 hour later; arid it was unaffected by the subxequent administration of at-opine. Nicotine was also studied subsequent to a;dntinis- tra>.ion of arecoline, methantheline; caffeine, and tremorine, as well as atropine; and, in ell cases, nicotine csused signifi- cant increases in the mean duration of after-dischar)ge over pre-nicotine levels. The effect on the durstion af after-dis- charge of the intact motor cortex was found to be similar to that observed in the isolated cortex of the dog. Aforocutti and aergio ((195tl) (g9a)j had recognized four phases in the electrocorticograms of rabbits given, i.v. i.ajec- tion of nicotine: an initial brief phase of synchronization; a discharge of "spikes"; the appearance of slow a.~ai'; es; and a final phase of cerebral sileace. Floiria, rforocutti and Ayaia (1982,1964) measured EEG activity in rabbits prepared with silver electrodes in contact with the dura, and deep electrodes in the hippocampus and interlaminar nuclei of the thalamus. Iti certain animals, the brainstem was sectioned at the inter- collicular level through the prepontine area; in oathers, the cortex was isolated by a spatula in 8-10 mm areas, without vass:ular damage, after a subpial 'seetion. I.v. injection of 1-2 mg/kg nicotine caused immedipte activation in all arras of registration; 2-6 mg/kg caused transitory dctivation, followed by bio-electric cr.sis, typikal of a generaliaeti con- vulsion of the whole animal. The sqme effecta cvkre observed in animals with isolated brain. %'hile there was a convulsive response in the intact eortex in animals with isodatted,oortex, there was none in the isolated areas. The origin of the con- vulsive action of tacotine was postulated to be in the, hippo• campus, spreading to the cortex by afferent padways. Stumpf 0959) (69a-b)) had reported that i.v. injection of 0.b-3 mg/kg nicotine into awake, non-narcotized rabbits, resulted in immediate EEG changes characteristic of an "aroural reaction", followed by a discharge pattern, Ldrnilar to that seen in convulsions. In a sulrsequent series of 'experi• ments, Dunlop, Stumpf and co•woikera (1960) anre.c4igated cortical, reticular, and hippocampal unit activity in adult male New Zealand rabbits (immobilized with d•tubocurarine) following i.v. injection of 3 mg/Icg nicotine bitartre~te. These doses induced hippocampnl seizure activity in 44 ~;c of d'xperi• ments, while 6 mg/kg nicotine bitartrate was 90% effective. Regularisation (i.e appearance of regular 4-g.8 waves/see) of the EEG activity recording from the micro•eJectrdde occurred in 05 0 of all experiments; reguiarir,atien of the EEC in all regions was accompanied by increased unit excitability, and, during seiaures, the unit activity was depressed. Of the three regions examined, the hippocampus had the lowest threahold for nieotine eRect.~. Modification of neocortical atnl ret&•ulnr unit acti%tity, without any concomitant EEG changes in thesc regiorrs, was occasionally recorded after nicotine injection. Further experiments indicat.ed that these changes in unit activity were due to alterations in, hippocampal excitation elicited by the drug. Electrical stimulation of the hip)io• eampus, which evoked regularization and seizure diFcluirgeF, similarly modified the reticular unit excitability; but no con- clusive results could be obtained regarding the hippocampal inBuence on neocortical units. In a later publication, IirOrke and Stumpf (1901) reported that, during the first phase of its action in rabbits, nicotine i.v. caused a hippocampus theta t Produced by The Council for 0002956 Tobacco Research-USH, Inc.

1vERVOUS SYSTEM rbgtnm, accompamed oy an rncreaseu unug-raw ui ~ue Ny- ramidal cells. Following this, a bippocampus seiaure PatRern developed, with inhibition of unit firing. The nicotine•induoed theta rhythm. but not the seisure pattern, could be abolished by septal destruction. Ochs and Hunt (1980) examined the effect of nicotine and y-aminobutyric acid (GABA) on cortical transmission; the possibility that these drugs act as synaptic blocking agents in the cortex was said to be indicated by their reversible block of propagation of spreading depression. This was shoen in the molecular-layer preparation, where spreading-depression r.+mRn,i-:-n alth;,ugl, su cortical layers below the first are cut through. Nicotine was placed either on the re- cording point while direct cortical responses were elicited, or into a well-type of recording electrode while recording direct cortical responses. Nieotine either had no effect on these responses, or augmented them almost 2-fold. An increase in direct cortical responses after nicotine application could come about by a selective block of inhibitory synapses acting on apical dendrites. GABA either eliminated the direct cortical responses, or reversed them; and this wes related to direct application or use of the well-electrode. GABA has been suggested as a speci@c blocking agent for excitatory synapses. Block of spreading depression would then come about by block of inhibitory synapses (by nicotine) or block of excitatory synapses (by GABA). Alternately, nicotine may have an ac- tion on the release of, or reaction to, transmitter substance postulated in the "contiguity" theory of spreading-depression transmission. GABA corad also have this effect, or more probably, it acts in s generalized fashion on apical dendrites to inwrfere with transmission of spreading depression and direct cortical responses. D. E. Knapp and Domino (1961) reported t6r.c midpontine- traosected cats and rabbits showed EEG arousal following small (10-20 µg/itg) doses of nicotine. (it is worthy of note that the dozes of nicotine used in these e:perimenta were, roughly, witliln the "smoking" range, whereas some other workers have employed toxic or convulsive doses of the drug.) These animals also displayed EEC arousal to epinepbrine and serotonin; but, dissociating this effect from adrenal.catechol- amine release, epinephrine-insensitive prepontine•transected cats and rabbits were found to retain their sendtivity to 1-nicotine. Furthermore, in the midpontine•transecterl dog, in which epinephrine and norepiuephrine do not cause EEG arousal, i-nicotine was effective in the same low doses, while the potent ganglionic stimulant, dimetbylphenylpiperaeinium (DAfPP), in dosage of 5-10 Ng/kg, did not cause EEG activa- tion. Completely deaferented dog brains were prepared by pn trigeminal transection, bilateral destruction of cranial nerves II and III, and the topicsl application of 2% Iidocaine to the olfactory mucosa. In such prrparations,1-nicotine still caused EEG arousal, indicating that the site of action of nicotine was within the C:~*S. Subsequently, them authorz (Knapp and Domino, 1982) elaborated more fuily, on their studies on the action of nicotine on the ascending reticular activating system. Synchronization of the EEG was produced by midpontine bralnstAm urtion in the rabbit, cat, and dog, and by high pontinc transection in the monkey. After trsnsection, i.v. injectioru3 of 10-20 µg/6g nicotine produced EEG activa- tion in all four species. Knapp and Domino (1983) further reported that EEG activation was observed after i.v. adminis- tmtion of this dose of nicotine in midpontine lintercollicular, midpontine, pretrigeminnl section) rabbits, eats, aod dogs, and high pontine monkeys and in prepontine lintercollicular, prepontine sectionj rabbits and catR, but not in the prepontine 45 . .. ~ . rrn uu8. %Dlraw uwawu..e s... u. w w.~ .. ~.. .... activation responses were given as rabbit, 2.47 f 0.56 mic; eat, 3.80 t 1.07 min; monkey, 4.83 :h ?,.30 min.) Theae latter results led Knapp and Domino (1963) to point out the need for caution in transferriag results of heuropbarmacological experimentation from one species to another. The mechanism and site of action on the EEG activation effect aere explored by these workers in the dog (Knapp and Domino, ID02). Within 20 sec after administration of 10 pg/kg nicotine, the spindling, slow-wave EEG pattern of the seu~orimotor cortex was replaced by 35-45 cycles/sec wavea of approximateiy 50 µV amplitude. By 4 min after injectAon~ spindle bursts began to reappear, and 6 min after injection, spindles again were a dominant feature of recording. That peripheral nerve-re- ceptors were not essentially involved was shown by obtaining the same results in the deatferented midpontime dog. Blocking cf nicotine-induced blood-pressure ebapgea by the ganglionic blockers, hexeznethonium or trimetbidinium bismethosulfate, did not prevent nieotine-induced EEG activation; however, the ganglionic blocking agent, mecamylamiae, blocked both the blood-pressure and EEG effects of nicotine. Further proof that nicotane-induced EEG activation was not mediated by release of peripheral stnres of catecholamines~ or mediated by changes in blood-flow, was obtained by ttte use of the ganglion- stimulating drug D11PP, which caused marked changes in blood pressure, but did not cause EEG Activation. Additional evidence of the specificity of r.icotine in causing EEG activa- tion was obtained with nicotine monometbiodide, which produced a blood-pressure rise without accompan}'ing EEG activation /nicotine methiodide is a 1009r ioni8able quaternary nitrogen compound which does not pass the blood-brain barrier (Domino, 1985b)). Studies in dogs pretreated .•ith reserpine in dosage causing depletiop of both brainstem catechofamines and serownin, suggested that nicotine did not owe its EEG-activating effect to releaEe of central stores of these agents. No tachypbylaxis was observed to nicotine- induced EEG activation when the intertal between injections was 25~% min. At a 10-min interval between injections, EEG activation was not repeated in any of the 8 tn9als. Adminis• tered vasopressin did not cause EEG activation, but did block nicotine-induced EEG activation for a ieriod of less than 30 min. In view of this, the authors drew attention to the possi• bility that release of vasopressin may be involved in the development of acute tolerance to certain central actions of nicotine. Nicotine did not produce El•;G activation in pre- pont:ne or postmanunillary dogs, but did alter electrical activity of the dog reticular slab, leading Knapp and Domino (1992) to propose a ponto-mesencephalic site of action for the EEG-actlvating effect of niootine. No e=idence was obtained denying the possibility that nicotine pioduced EEG activa- tion by a cholinergie mechanism; and thRse writers tentatively concluded that a likely mechanism by which nicotine produces EEG activation is by mimicking, or fxwibl.• releasing, arctyl- eholine in the CNS. However, intrn%-entriculael,v-ndministered bemicholinium bromide to dogs (immobilized by decametho- nium and under artificial respiration), which produced in 3-5 hours a high-voltage, slow-aave EEC pattern, was nott entagonizod by 40 pg/kg nicotine; i.v. arecolMe, pilocarpine, and physostigmine did desynchronize the EEG after bemi- cholinium, whereas epinephrine and d-amphetamine, as well as nicotine, did not (Dren and Domino,19t)5y. Having shotvn that nicotine induced transient EEG aetiva- tion in midpontinc brainstem-tranrecced anirrtais pCnspp and Domino (;M), aboveJ, K. Yamamoto and Domino (1964) considered it of interest to determine whether this EEG effect Prodttced h Tha Cotcnc;l for lobacco Resetarch-U5A, lnc.

46 'j'OBACCO-EXPERRt1ENTAL AND CLINICAL STUDII:S. SUPPLEMENT I correlated with behavioral arousal or the "activated" or pwauuxiwi Niuur ui aiert,. iu nrre/puug ww wni$ Cuium; indwelling electrodes implanted in the somatosensory cort:exm hippocampus, amygdala. posterior hypothalamus, and mid- brain reticular formation, 5-10 pg/kg nicotine given as an i.v. infusion over I min produced behavioral arousal and EEG activation for 2-8 min; such phenomena were not, obeerverl following equal volumes of wanned saline. ).•quipressor doses of epinephrine (2 Ng/kg), arginine-vasopressin (50 mPli/kg), or D,*%fPP (3-5 µg/kg), all produced much weaker EEG activation and behavioral arousal than did nicotine; in dosage of 2 mg/kg, trimrthidinium (a quatemary ganglionic blocker which does not easily penetrate the blood-brain barrier) blocked the blood-pressure and electrocardiographic effects of nicotine, but did not alter behavioral arou-W or EE(: activa- tion; on the other hand, 0.3-0.6 mg/kg mecamylamine (a ganglionic blocking apent which can penetrate the CNS) completely blocked the actions of nicotine on both EEG and behavior (Yamamoto and Domino, 1964; Mi'nino and Yamamoto, 1955) lcompare Domino (1965a), above, 121j. The arousal effects of nicotine appeared to these authors to be an action on the CNS, rather than peripheral afferent stimulation or release of various neurohumors. In another report of the effect of nicotine on the eleep of the cat, Domino and Yamamoto (1965) supplied additional information. The studies were carried out in the unanesthetized state, 2 weeks after operative implantation of the electrodes, with the cats placed in a warni, sound-proof, insulated box with a one-way window, the natural sleep-awalte cycle of the animals being recorded throughout the experiraental day. In cats iut natural slow-wave (deep) sleep, nicotine in doses of 5-10 pg/kg pro- duced three distinct EEC and behavioral phenomena. FSrat, a brief period (ee. 3 min) of neocortical desynchronisat.ion and hippocampal theta actdvity, accompanied by behavioral wake-up. Subsequeatly, the cats again lapsed into slow-wave sleep, frequently into deeper sleep than before nicwtine injec- tion. After about 15-25 min, an increased incidence of fast- wave sleep was observed. The nicotine metabolite, cotinine, had no effects in doses equal to those of nicotine, but in mas- sive (25 mg/kg) dovs produced a transient EEG and be- havioral arou.Ral effect lasting only 1-2 min. Nicotine and arttininP vasoprtssin increased the time that the animals spent in activated sleep within a 25•min period after drug injection, and Domino and Yamamoto suggested that the abilit3 of nicotine to produce activated sleep may bi' relat.,," to vasoprescin release. Villarreal and Domino (1964) pre• sented evidetace for two types of cbolinergic receptors invoh•ed in nicotine-induced EEG devynchronization. EEG desyn- ehronisation was induced in brainstem-transected (ntid- collicular, high-pontine) cats by i.v. injection of nicotine and arecoline, the peripheral autononmic effects of a•hueo had been antagoniaed by atropine methylnitrate and trimr•thidin• ium. As before, 0.5 mg/kg mecamylamine blocked the EEG Wects of 40 pg/kg nicutine; but even large (5.0 mg/k'g) doses failed to block the F:EG effects of 96 pg/kg arecoline. JLarge doses of inecarnybmine interfered with the natural slekp-cycle pf the cat (K. Yatnamoto and Domino, 1909).J Illoekade of the desynclunnizing efiects of nicotine tras aku acl:feved by • repeAted administration of increa4ing doses of nicotine itself (totalling at least 3 mg/kg nicotine in less than 1 hour); but such nicotiniaation failed to block the desynchronization produced by arecoline. In an over-all conclusion rerived frnmi his work on conditioned avoidance bchavior in rals (see above, 121), Domhm (198La) stated that the behavioral nctions of small dow of nicotine atr subtle, butt conKietent aith the hypothesis that nieotine stimulates the brainslem acmsilug nyntent prooucmg benav7ora/ arousa/. ltyucbenok and Ostrovskaya (1962) investigated the role of cholinoreactive systems of the mesencephalon in the tmch- anism of EEG nicotine activation, and the results are sum- mariaed as follows: "Acute and chronic experiments on cats and rabbits demonstrate that nicotine arbninistration,caurrs phasic changes in the bioelectric activity of the brain. ln isolated brain and in rostral sections, in which a pan of the eticular formation of mesencephalon remains unconnected to the overlying portions, the administration of nicotine pro- vokes EEG activation as in animals with av, intact brain. With the section pass'ing from the dorsal side in front of the superior colliculi of corpora quadrigemina, and from the ventral side-directly bchind thc corpora mnmmilariA (tnr- mesencephalic section), nicotine administration provides no change in the slow high-amplitude activity characteristic of the section. The data obtained suggest that,the activating effect produoed by nicotine on the EEG is connected with the excitation of reticular fonnation of inesencephrslon. JAlthoug;h nicotine, injected apparently into the mesencelihalon reticular formation, failed to produce EEC seizure discharges in cats (Kunuigai et al., 1962); see above, 129.) The impossibility ot preventing nicotine activation by preliminary aminasidie ad• minist:mtion shows that the excitation of adrenorelictive structures does not participate in the mechanism of this . activation. On the basis ot studying the interaction of nico- tine•provoked activation with substances blocking the central Al- and N-cholinoreactive systems (benactisin, bensatsin, metamisyl, atropine, tropacine, trasentin, e)dopromaaihe) it is suggested that the biochemical structures ixcited in the reticular formation are T-cholinoreactive." (Ugol'nikoy (1965) studied the effect of thyroldin on the choknoceptor excitability nf the CA'S as manilested in nicotine and arecoline convulsions (see above,130-F), and concluded that th)rroidiu increases the excitability of central T-.holinorarpton; and decreases the excitability of 11-cholfuoreceptors.J The effect of i.v.-injected oxylidine on the EE,('s wres studied by alashkovskiy and Rascldna (1903) in acute experiments on nommtrcotised cats under artificial respiration aad in cl,tonic experiments on rabbits with indwelling electrodes. Oxylidine was found to antagonise the EEG-artivating action of nico• tine. Voronov, Bohadnecky and Benesova (1964) rcompared the efrccts of atropine (a cholinolytic) and atroitriptyline (a thymoleptic) on the theta activity induced by 0.1 ntg/kg nicotine (injected over a 2-mht period) in rabbits. Atropine (0.5 mg/kg i.v.) did not significantly change the nicotine- induced theta activity, but amittiptyline (2.5 mg/kg i.v.) completely blocked theta activity in 9 animals and shorlener) its dumtion to I min in I rabbit. L'sittg techniques developed for recording cereoral electrieal activity in couscious, unreslt>ained small animalk and for the remote i.v. injeMion of drugs, the maturation of thc eleetm• corticogrem and behavior in the kitten and gipinra piq was studied by ,llarley and Key (1963). (Findings with nicotine were repoNed only for the kitten.) Nicotine tiva.< <vithaut effect in the new-born kitten, but produced behavioral drow•sineas in 1-2-a•eek-okl animnh. lty the 3rd week, nicotine produmi behavioral alcrting, although bur:as of rh,vthnmic activity alrtmared in the clectrorortirogmm. At 4-5 wcrk.s, the response to nicotine was similar to that evoked in the adult, nameh•, behavioral alerting with high-voltage faat activity in the electroeortico>;ram. 11. 13. lirown and 5hr,rne (1964) sstudied the effects of ryieo• tine on brain eleetrical activity in afwntanrouc:ly hrha+•ing ProdUced Nr Th4 Council for 0002958 Tobacco 11 %Oseai*ch-USA, Inc.

1 i i NERWOUS SYSTEM rat.! F.Ffi were recorded from electrodes implanted in the fornix, septum and hippocampal formation (ahryme and Brown, 1961); and the drug was administered either s.c. (Brown and6hryne, 1964) or i.m.,(Shr) ne and Brown, 1964) in amounts comparable to the quantity of nicotine absorbe,/ by man in smoking 2-3 cigarettes. The actual doses used were reported to be 30-70 pg/kg nicotini: bitartrate s.c. (Brown and Shn•ae, 1964) or 30-70 pg/kg nicotine i.m. (Shry-nc and Brown, 1964). During the first 30-00 min, the animals were either alert or indicated uneasiness; but, during the second hour, they were lethargic, droasy, or in 6ght sleep; and, at the end of 90 tni¢t, they had returned to normal levels (Shryne and Brown, 1964). EEC traeings showed an alert pattern, with increased b'ippocampal theta for approximately 60 mir., followed by a persisting drowsy pattern; concomitant be- havior activity was manifested as initial uneasines9, followed by drowsiness. EEC•folloa•ing of photic flicker was enhanced, but EEG and behavioral arousal thresholds showed no eon- +tistent changes (Brown and Shtyete, 1964). When comparrtd to thresholds obtained in the four spontaneous behavioral states of alert, awake, sleep, and activated-sleep behavior, evoked responses of hippocampal structurm showed chanirs in thresholds for both excitability and responsiveness. S.c injections of 30 and 37.5 µg/kg nicotine bitartrate caused thresholds to parallel or lower those obtained during alert or awake behavior; dosesof 50 and 70 pg/kgcaused thresholds to parallel or lowiu tlqae obtained during skep, except for responses between fornia, hippocampus, and hippocampal gyrus. The responses of these last were consistently altered in the direction of alert behavior, although both EEG ahd behavior pattems resembled those of drowsiness. The results of the tests were saa id to be, in the main, consistent for given doses of nicotine and for the different brain areas stimulattd (Shryne and Biown, 1964). The reader iaako referred to the EEG studies ou nicotine convulsions by I,ongo, von Berger and Bovet (1954), and to the description by Vartiapetyan '1963) of the bio-electdic activity of centkal and sympathetic nervous syslems during nicotine., camphor., metrazol-, and picrotoan-induced spasms, abore,129. There appear to be no reports of EEG changes in human subjects following nicotine adutinistration in "smokit>R" dos• age. Such investigations would be highly desirable, if only to corroborate the impression that the EEG changes observed during or following cigarette-stnoking do not appear to be due to nicotine per se; although thenz is, of course, the pos'si- bility that any "pure" nicotine effects may be "covered up" by EEG changes brought about by the physical act of sntok- ing or by other constituents of tobaccosmoke. Such an e.w periment aould help to clarify, for example, the study by Hauser and cawaiters (1958) of EEC changes in healthy young adults on cigarette-stnoking. In this study, an increase in the dominant a-frequency of 1-2 cycles/sec occurred in over 60% of the initial group, but 6 records were considered equivocal because of poorly-developed a.rhythm or marked Q-inhibitaoa. Excluding these 6, then 16% of smokers and 30% of non-smokers showed no change in the dominant a-frequency, while 89 io of smokers and 70 0 of non-smokers did show a change. In over one•thin] of the subjects, the a-frequency ghifted during the initial inhalation of cigarette. smoke. All shifts occurred during the first cigarette, and, once the shift occurred, it persisted withoutt fluctuation in relation to inhalation. Opening the eyes for prolonged petiods caused a tempomry return to the basal state. As long as the subjects remained in the easy-chair with their eyes closed, the shift 47 persisted (up to Sf miu). 'Moving elrout for a shortt period MuWU u.u.-e u,c.uu. smoked another ciRarette, the shift would occur again. Five subjects wLo had shifted 1 -2 cycles/sec simulated smoking a glass cigarette stuffed with cotton, and also 2 specially-pre• yiared nicotine•free cigarettes; 4 showed sonm increase in a•frequenc, with the glass-cigarette, and ail showed an in• emat;e with the low-nicotine cigarette similar to that noted with the regular cigarette. There Nae no evidence from hlood• pressure and palse•rate data that the effect was directly mtdi• ated by cardiovascular changes, nor did data from hyper- ventilation suggest that it was mediated by a lowering of [tilood carbon dioxide. In fact, the inciearr in dominant young adults, smokers and a-frequency occurring in healthy tion-smokeihs alike, in tbis series, demonstrated to the authors that emoking in ronic way affects the C\s. The rhift u4-uld seem to represent a psycho-physiologic response related more to the act of smoking than to physiologic or metabolic effects front substances present in the cigarette. Birkford (1960) analyzed EEG tracings obtained in the above study, and considered that the shift in a-rhythm was related to some concomitant action of smoking, perhapa a shift in attention, and not to the effect of any inhaled agents from the ciFarette itself. Concerning these conclusions, the Advisory Committee to the Surgeon General of the CSPHS stated that wide gaps of information exist in this area, and the Committee pointed out that it is not meaningful to attempt inferences rwncerning correlations of electrical events in the CNS and subjective effects of smoking from the t.7.e of evidence currently avail-, able (Smoking and Health, 1961, p. 70). Hon-ever, it would surely be eash to minimize the role of nicotine in smoking (see below, 1131). H. Burn (1962), for example, like Louis Lenin long before him (53a; 531b; 539a), stated that nicotine i,s used for its effect on the brain; and P. H. Knapp, Bliss and Wells (lf)65) regarded chronic sympathetic stim tlation ax one result of hr'avy smoking; that sympathetic-nervous arousal is mediated pharmacologically by nicotine; and that heavy smokers seek a state of chronic low-grade arousal. But Knapp's hypotheses may also be regarded as an fitference concemhtg correlations of electrical events in the animal C\S with an "objective" effect of smoking in mau, w hich may be as harardous in its way as that of Hauser and Bick- ford that the act of smoking is more important than the com- position of the smokc. 151. CgfArlcemistry It has been already noted t.hat, following administration of toxie doses,of nicotine to cat:, mean hypothalamic norepineph. rine conteht was reduced to two-thirds of that of control animals (Vogt, 1954). lirain content of epinephrine, nor• epinephrine, dopamine, and nOl'.t, of mta given 20 mg/kg t-nicotine i.p. was detet•mined by Nugai (1f163a, b) at three phases of convulsion (tremor; clonic convulsion; tonic eon- vulsion) and at the moment of drath. Xorepinephrhm in• cressed at the tremor phase, but• decreased at the other phases; dopamine increasad at the tonic-convulsion phase; end DOPA increased in all phases. ln acutely adrermlertomired rats, brain content of DOI'.a increased at the tonic-convuleion phase, but; the other cattrholamines remained unchanped. Following i.p. or i.v. injection of nirotine into nrice, rats, and guinea pip, the eatechnlamine and etrotonin content of the brain remained mratffected (Hatucon, Jlasuoka and Clark, 1963). With mspect to times and dosages, no effects were found on total caterholamine and gerotonin levels in the brain of mire sacrificed 30 or 60 min following i.v. injection of Produced hv Thc Counca for 002959 Tobacco haseaict~-USA, lnc. 0

f 48 TOI3ACC0-EXPERibfENTAL AND CLINICAL STUDIFS. SUPPLEMEI\T I 0.05 or 0.1 mg/kg nicotane, or in animals sacri6ced 30 or 60 min fnllnwino i n od.nWor.ws:- nf m m Am n, n c o., 4 mg/kg nicotine (Hatmn, Masuoks and Clark, 1964; Hansson, 1905). Similar negat'rve findings were obtahted tor rats or guinea pigs sacrificed 60 min following 0.1 mg/kg nicotine i.p., or for guinea pigs sacrificed 60 min following I mg/kg nicotine i.p. every hour for 4 houra. In male Holtzman rats given s.c. injections of 0.25, 0.5, or 1.5 mg/kg nicotine 4 times daily, no significant effects were observed on either brain epinephriae or norepinephrine levels when animals were sacrificed on the 3rd, 7th; or 14th day of the experiment (Westfall and Watta, 19ti4b). With nicotine doses of 0.5 and 1.0 mg/kg, brain-serotonin levels were significantly increased on day 7, but not on days 3, or 14. Highly significant in- creases in brain serotonin were said to have been produced by injrctiou of the known effective drngs, a•phenytieopropyl- hydtszine (pheniprazine; JB•518j and iproniaaid. Schievelbein and his colleagues have been particularly interested in the effect of nicotine on serotonin metaboiiam. Removal of one cerebral hemisphere from each of 3 dogs, before and 10-30 min after i.v. injection of 0.31-0.50 mg/kg nicotine, revealed 30-58% decreases in serotonin content of that hemisphere removed after nicotine injection (Schievel- bein, Werle and Jacoby, 1981). No decreases occurred in 2 control experiments, in which saGne, instead of nicotine, had been injected. These workers concluded that nicotine liberates serotonin, and consequently also exerts a possible influence on the function of the cerebrum; and the nicotine obtained through smoking msy be of importance in this fashion from the standpoint of possible psychic effects in human smokers (Schievelbein and Werle, 1962a). Using this same technique, Schievelbein and Werle (1982b) found that release of sero- tonin by 1obeGne in the dog preparation was virtually nil. These workers injected 1 mg lobeline suJfate i.v. in rats, which were then sacnficed after 20 mia, the brain removed, the cortex dissected out, and the brainstem homogenized and analyzed for serotonin. An average value of 0.39 pg/gm was - found, compared to 0.46 pg/gm in control rats, equivalent to a decrease of 17%. This finding that lobeline, like nicotine, liberates serotonin from the brvin, is of interest in connection with the use of lobeline in the cure of the smoking habit as a kind of "substitute therapy" (see below, 1145-Ci. No differences in brain content of serotonin, b-hydroxy-3- indoleacetic acid, tryptanune, and 3-indoleacetic acid were found by DeClercq and Truhaut (19G3a) between control animals and rats receiving 0.5 gm/liter cotinine daily in their drini:ing-water over a period of 18 months. The elfecta of nicotine and smoking on serotonin metabolism are dealt with in greater detail below, 712. The mean values of the various lipid ratios (lipid/tissue; lipid/nitrogen; cholesterol/lioid) in the lirains of newborn young of nicotine.fed rats did not differ significantly (rom those of offspring of control mothers (Jlosier and Armstrong, 1964); for further details of thio study, ser heloe•, 817, 152. ttiafopatAofogy (70) Application of 5 mg nicotine to the ehorio-allantoic mem- brane of 10-11-day-old chick-embryos was almost immedi- ately followed by development of a cephalic hematoma (Gatling, 1964). Application of nicotine to older, 13-day embryos caused no or minimal lesions. CNS lesions were said to have been found in rats chronically exposed to nicotine and methyl-alcohol vapore (1'etrova, 1963); for further details of this investigation, sec below, 1088. White mice exposed to either Ught or heavy concentrations r ;4~. ... ..... ^:... r. : ~ ,' " .....,r N...:..u.. ~N :w 7'v;, ~iu .%e showed nerve-cell degeneration in the brain, accompanied by multiplication of the macro- and microglia; for the fiusl 10 months, the changes were limited to the cortex; after 10 months' exposure, the changes occurred in the white matter of the brain and in the brain membranes (Komcaynski,1958). The encephalon of guinea pips acutely exposed to ciganMte- smoke was said to have showed degenerative factors of marked degree involving the cerebral matter, with cellular vacuolieation affecting the nerve tissue; and this find'uiF was present to a much slighter degree in animals chronicslly ex- posed to lesser amounts of cigarette-smoke over petiods of 2 or 17 months (\faftei and Afiani, 1962). (For an account of accompanying structural changes in the cochJna of thaw gninPs pigs, see above, 115.) 153. THALAMUS Curtis and Davis (1962) reported that nicotine, electro- pboretically passed as the cation from a saturated solution of nicotine HCI (pH 5.5) in barrel electrodt's, did not depres.c synaptic excitation of neurones of the lateral geniculate nucleus of cats under pentobarbital anesthesia. Using this same technique, Andersen and Curtis (1964) investigated the pharmacological properties of acetylcboline•sensitive neurones in the ventrobasal nucleus of the thalamus, and found nicotine to be an effective excitsnt of t.halamic neuronm although less potent in this respect than acetykholine. 154. MEDULLA OBLONGATA (70); PONS f3radley, Dhaa'an and VColstencroft (1904) relwrted that nicotine, iontophoretically administete.l, had similar effects to acetylcholine, both qualitatively and quantitatively, on activation of cbo6noceptive neuroses in the medulla and pon.a of the cat. Complete or partial block of the excitatory and inhibitory actions of acetylcholine was obtainable on ionto- phoretic application of nicotine. These workers noted that, in their response to nicotine, these newone8 in the medulla and pons appeared to differ from the cortical cells described by Krnjevi( and Phillis (1963a, b) (see above,148j. Injection of 5-10 pg nicotine into the diencephalon, meseu. eepitslun, or the medulla oblongata (bufbe rachidien) of cats, lightly anesthetized with chloralose and urethane, had no effect on blood preasure recorded in the carotid and pulmonary arteries, or on contractions of the nittitat'tng membrane, or on rr:=piratory movements (H. Schmitt and HEIAne Schmitt, 1903). However, as measured by the above parameters, in- hibition of central autonomic reactions evoked by electrical stimulation (via a tubular monopolar electrode implanted into the several regions) did result. l.v. injection of 25-50 pg nicotine also inhibited the effects of electrical stimuli applied to the same regions. The et1M'tA of nirotine on certain meduUary functions (vomiting; tx;viration; vasomotor reactions) have beeu rc viewed and summarised by the compilers ($ilvette et al., 1962; Larson and Silvcttc, 19gSb); for additional detailY and/or accnunts of published work subsequent to theFe reviefr-articies, consult the relevant sections in this Supple• tnent. 1E5. CEREBELLUM (70) From eqwriments performed by E. R. Bowman and oo• workets (ltr3•1), It appean that radioactive cotinine conceu• trates, to some extent, in the cerebellum of mice (see above, Produced M The Cou nc:l for 0002960 Tobacco Researc'&USA, Inc. Is

NERVOUS SYSTEM 148). For the bistopathologiral effect of chronic admioi.stra- tion of cotinine ~(~an metaboGte of nicotine) on the cerebellum, CPP TrIVl/allt. 1/n ~IPHYI NII/t 1flli/It;PI /1pW M.D....• l+%4 • J1cCance and Phillis (1964) employred 5•batrelled micro- pilMttes to release iontophoretically a number o( drng,? in the irnnied•iate vicinity of individual neurones in the cerebellar conex, and recorded the induced extracellulai action-po• lentials. Acetylcholuw-seitsitive cells were found in all areaa ol the cerebellum investigated; these cells were also excited by a range of chofinomimetic ageuts, including in deacending order of potency, carbamyIcholine, acetyl-S-methyicholine, (acetylrhoGtre), nicotine, and arecoline. Dihydro•O-erythroi- dine n-as found to block excitation by either acetylcho8ne or nicotine. Yan Proosclij (1960, p. 158) has remarked that we po:,~,;Pss practically no reliable facta about the effect of nicotine on the mrebcllum, the brainstem, and the spinal cord iti inau. 156. SPINAL CORD (71) See the remark by Van Proosdij immediately above; see Fukuya (1961), below, 159. 157. Chroucsie of the Spinal Cord (72) 158. Spinatf-Cord Pofentiafs (72) J.Vo new data.) 159. Spinal Re.Beres (72) Perfusion of the frog spinal cord with 1:1,0U0 acicotine had a stimulant action on reflex activity, which was lost after perfusion with 1:1,00p,000 y-aminobutyric acid (Fukuya, 1961). Accord,ing to Jancsb, Jancsb-G6bor and Tahdts (1961), ganaiionic blocking apents reduced or abolished the sensi- tivity of guinea-pig comeo-conjunctival pain•endings to the excitatorT action of nicotine, althougb the cornesl reflex wres easily elicited by touch. This effect could be particularly well ol"`erved on rats. Certain nicotinolytic (antipap•kir-soritsm) drugs in low concentration also desensitized the eye agaitLCt nicotine, but did not influence the corneal reflex. The sneezing ret?ex induced in guinea piRs and rats by appGcatior. of nico- tine :o the naeal mueosa, and its inhibition by certain gan- glion-bloel:ing oigettts and nirotinolytic dr.,gs, was ALvo investi- galed by these workers, and their restlts are described more fully below, $14. In mamrr.als (cats, dogs), nicotine depreasec or abolishes the patellar-tendon (knte-jerk) reflex (see Silvette et al., 1962; Larson and Silvette, 1985b; and below). The patellar- mflex inhibiting action of nicotine in cats was neutralized by phenearbamide (Wirth and Gossw•ald, 1A&5). According to E. Fischer, Silvette, Larson and Flaag (1t1R0), exidence is available to indicate that the selective depression of the patellar reflex by nicotine is due to nicotine stimulation of the midbrain reticular formation, which has an inhibitory influ- enrc upon the spinal knee•jerk centers. Fulloeing inttacerebroventricular (ICV) administretion of d tulwrurarine chloride in cats, enhancement of t.heknee-jerk hm been reporied. According to llhattacharva and Pradhan (1/ki8), injection of 20-50 W.t nicotine by this roule reduced the enhanced knee-jerk in the majority of experiments. When giren ICV or intracisternally, 0.5-2 mg atropine also de- pftv1 the knee jerk to some extent, but this drug usually did not modify the depressant effect of nicotine. In eats under chlmsh,re anesthesia, both crphenadrine and mecamylnmine 49 were found to block nicotine-induced knee-jerk inhibition (Ginzel, 1988). 4 .......{.~. ..r ... ..a~::,. :... ..,~~m,ao aiie rniet ui nicotine on the monosynaptic patellar reflex to its effect on polysynaptic (bomolateral flexor, contralateral extensor) re- flexes (see again Silvette et al., 1982; l.arson and Silvette, i965b; and below). Considering the variety of the exheri- mental techniques employed, most, although not all, rt~ults agree in demonstrating that nicotine dimiuishes or abolisheA the monosynaptic patellar reflex, while the polysynaptie flexor reflex remains relatively unaffected, or i.c even enham•ed by the drug. It may be, as suggested by Curtis, Eccles aud Ecclca (1957), that nicotine excites the slxrial group of idter- nwncia) neurones called the Renshaw cells Isee above, 1•f01, which exert an inhibitory action on motor ncuronea; Renritmcc cells are known to be cholinertricalh• articated from the utotor axon collaterals isee also J. C. lredes, R. M. L:ccleR rsnd Fatt (1956); Curtis and Eccles (195f3a, b) (S1b). See also P. Stem (1964), above,1-18.j In cats under light pentobarbital anesthesia, with all lumbar domal roots either cut or crushed, injection of 26 µg nicotine into the lower aorta (with all branches below the renal arteries lii;ated, aith the exception of the lumbar arteries) was ahnost invariably followed by transient depression of the testing monosynaptie reflex in L-7 and S•1 segments (Curtis, I;ccles and Ecclec:, 1957). In a further study of monosyroaptic reflexes in cats, nicotine, applied locally to the spinal cord in a concentration (S00 pg/ml) which completely eliminated responses of Renshaw• cells to a testing ventral-root volley, had no siRttificant effect on presynaptic inhibition (J. C. Eccles, Schmidt and Willis, 1963). According to Koll and SchOtz (1960), there is much evidence which proves the presence of choBnergically-activated ele- ments in the C_VS, the aetion of which is increased by the in9uence of anticholinesterase drugs. The rise of reflex dis- charges under the influence of these substances is interpreted as a consequence of increase ot acetylcholine at chofinergie synapses, and the action of aertylcholine at these s,rmarses has been called a"nicotinic" one. These worketa therefore felt it would be of interest to investigate whether nicotine itself possessed a similar effect on these cholinergic elements; for this purpose, experiments were Ix rformed on several reflex systems of low-sirinal cats. Reflex discharges were elicited by ck.t-trical stimulation of (a) the afferent a-fibers of the gas trocnemius nerve and of the nerve to the fle.rrr-hamstrina group; (b) the aNerent, higher-thre--hold A-fil.ers of the sttme nerves; (c) the afferent a-y-fibers of the sural and superficial peroneal nerves; (d) the afferent Iwst-y-fibets; and (e) the afferent C-fibers of the latter. Records were taken from the homolateral L-7 or S-1 ventral root. In another series of expetiments, the mechanicalh•-elici:ed knee-tendon jerk, and the homolateral flexor reflex (elicited by electrical st'unulatiun of the superficial peroneal nerve), were recorded myoxraph- ically. In dose-ranges of 0.04 to 4 mg/kg nicotine bitartrate i.c•., only depression of all inve, tittated reflex-discharges occurred; increases were never ohwn•ed. Uonosy-naptic rr- flex-discharges were much mon• diminished than polygrnaptir ones. Also, the polysyttaptic nociceptive reflex-diseharQex, elicited by stimulation of afferent post-y- atd C-fibers, were only depressed; and the knee-tendon jerk and homolateral flexor teflex were never augmented by the action of nicotine, but were "larly diminished. The authors concluded from these results that it does nnt seem lwssible to speak in gencral temts of a"nirotinic" action of acetylcholine in the CNS. They considered thatdiscrepancies beth•ecn the depression of Produced bv Tfle Gouqcq for Tobacco ResearcirUSA, Inc. 0002961 wo~.6~..~a+n~

I I 50 TOBACCO-EXPERIMENTAL AND CLINICAL STLTllIEG. SUPPLEMENT I monosynaptic re8eses by nicotine and acetylcholine, and ineir augmentauun u.• anucnow"terkne urup, ueeueu further investigation, as did the much weaker depressive action of nicotine on pohsvneptic reflexea. Experiments performed by Skoglund (1961) suggested to this worker other possible mechanisms ol the nicotine effect. The action of nicotine on different spnajNic systems in the spinal cord of decerebratc or spinal cats was tested by inject- ing solutions of nicotine tartrgte of different concentrations into the blood-str:am. Remarkably constant effects were found on the monosymiptic system, wherein inhibitory effects were observed at threshold doses of 0.5 mg/kg, corresponding to blood concentrations obtained in man by smoking I or 2 cigarettes (amuming absorptiun of nicotine on smoking to be as rapid as on i.v. injection;, whereas the effect on polysynart ic systems was more variable, both facilitation and inhibition being observed. A local decrease of blood-flow in the spinal cord was observed, which considerably outlasted the rather transient systemic blood-pressure changes; and this effect might possibly contribute to the long•lasting effects observed on nervous tmnsmis-sion. A comparative series of experiments on the effects of norepinephrine injections showed that spinal interneurone activity may undergo both facilitory and inhibi- tory changes, from which Skoglund concltided that liberation of norepinephrhte and epinephrine must be taken into account as a secondary factor in central nicotine a~tion. N. C. Bowman and Sangjrvi (1963) reported that, in anesthetir.ed or decerebrate or spinal cats, small doses of nicotine blocked the patellar ar,.) crossed-extensor reflexes to a similar extent. With circulation to the limb clamped, i.v. injection of nicotine still abolished these reflexes, ruGng out the neuromttscular junction and the muscle-spindles as: the site of action. Larger doses of nicotine blocked the flexor reflex, the block being usually preceded by potentiation of the con- tractions and powerful fasciculations of the muscles, which were reduced, but not abolished, by sectioning the sciatic nerve. LebtAev (1861), who sttidied the effect of nicotine on stretch-reflexes of the musculus quadriceps fernorls and the museulus taiceps brxchialis and on the fiexion reflex, con- sidered that the results on the several different t3q,es of preparations indicated a selective inhibiting effect of nicotine on certain ottions of the activating system of the reticular formation of the brainstem. This worker's experiments were performed on eats with sharply pronounced hyper-reflexia. In spinal and decerebellated animals, i.v. injecticn of 0.2-0.3 mg/kg nicotine did not alter stretch-reflexes, although tendon reflexes were inhibited, indicating the possible participation of different motoneurones in proprioceptive reflexes in the same muscle. This same dosage of nicotine inhibited stretch- reffexes in decerebrated animals. Nicotine inhibited the flexion reflex in all three types of preparations. C. \i. Smith and his collaborators have extensively studied the effect of nicotine and other drugs on muscle-spindle afferents of the triceps surae of cats. In de-efferented animals anesthetized n•ithpentobarbital, in t% hirh activitcof afferents from primary and secondary sensory endings in such spindles were recorded ost•illographically frurn dorsal-uoot filaments, close intra-arterial injection of 1.25-10 µg nicotine caused acceleration of aBerent dischatlte, and "explosions" could be produced by 50 pg nicotine (Smith, Xavier and (Irossie, 1964). Investigation of the site of cholinerttic drug action on muscle-spindle aflerents indicated that the pattem of effects of intra-arterially administered ninttine, acetyIcholine, und succinyleholine on primary and sec•ondary afferent endings of the muscle-spindle8 was different, although the tame-c•ounwK ut tu;uun ot we utree agcme, werc ruunu to ire,al+ptn."wmtra the same (Smith and Albuquerqye. 1964). With respect io nicotine, primary afferents were found to be many timr, more reactive than the secondary units, but the differem•c• in response were not linearly relate;rl to conduction velocity. Nicotine was equipotent with suecinvlrholine in d•au.•iul; excitation of primary spindle endings, but was lem than tso aa latent in producing neuromuKC•ular block (.Albuouerquc and Smith, 1964a). No correlatiort was observed in the !tn• tencies of ditnethylphenylpilxvaiinium iodide (D\ll'l'), suerinvlcholine, or nicotine, with respect to their excitatory actions on muscle•spindle aBerents and on autonomic panglia, and their depreaaa:tt actions on extrafwal neuronmsrular tra!:!tmi!tsion. In aaothcr report, .1lbuqucrc,ise and Sntitb (19(14b) pmsented data basE•cd on osvillogratihic trivrrdinp: from 156 functionally isolated single afferent fibers from triceps-surae muscle-spindles, the activity of from 4 to 10 single units being monitored aimultaneous;ly on a single eat. Nicotine in doses as low as 1.25-2.5 pg (administered via a cannula in a branch of the femo~al artery, just above the knee, all other branches of thr femoral artery except that supplying the triceps suraP muiu•le-group being ligated) produced a marked excitation of muscle-spindle stretch. receptors; the larger fibers from primary endings showed a more intensive discharge than those from secondary endings. Extrsfusal neuromuscular block was also assessed by recording the effect of the drug used on the muscle-twitch to,ingle, shock stimuli every 10 sec: nicotine in dosage of 20-320 yg produced a degree of block ranging front about 30 to 7570. In cats in which the spinal cord whs sectioned at C-1 under ether anesthesia, activation of de-efferented tricepp~rrae muscle-spindle aBerents produced by nicotine was eharac• terised w•ith reapect to •)ose-reslron'e relationship, conduction velocity of the a0aent, and effects on the triceps-surac monosyrmptic reflex and dorsal-root reflex (11'hitsel, :1l• buquerque and Smith, 1965). ln preparatrons uenen•aled by section of leg nerves am1 L-4-.S-2 dorsal roots and veri :al roots, except ipsikueral L•0-S•1 dorsal roots and triceps• surse muscle nerves, the intra-arterial or i.v. injection of nicotine elicited an increase in m6tscle-slvndle-afleredts aa tivits•, the magnitude of which was a positive function of the conduction velocity of the aBerent; and also a transient in• cnease (10-20 s+ec) of the monosytrajttic reflex ahd dotsal.uoot reflex, following which there was depression (1-3 min), then a subsequent increase for 10-25 min. In contrast, in de-ef- ferented but o:heraise intact prepai•ations, nicotine produced a sustained depnxeion of the monosynaptic reflex uttd a concomitant increase in the donml;root reflex for periods of 10-90 min. The profound depression of the hnonosyhnptic reflex by nicotine in intact preparations was concluded to be, in part, the consequence of a heteron'naptically-meifiatrd increase in primary a8erent deliolnrieation. Alburyuerqrie and Smith (1965) reported a conqMrativc study of effectR of nicotine, D\IPP, suceinyleholine, and acetylcholine (adminis- tervd by close int ra•arterial injection) on the elect rical activity of 36 afferent nerves and endings of acutely de-e9eurnted triceps-suraemusclesl»ndleFof the cat. C'ompared to nicntine. D\ll'P was leas selective in differentiating fibers front liri- mary endings from those of src•ondary endings; for a given degree of stimulation of aferent discharfte. DINIPP jtad a greater effect on slowh••rondurting afferents than did nicotine. Additionally, the slopes of the do:.r-effect curves with ni'rothtc and surcinylcholine were diatinrth• curvilineer, as corrwpared to the more 6near rzlationshihs obtainerl with Dil I'I'. I)Ml'i' ; Produced bv The Council for Tobacco Researclh-USA, Inc. 000296L,

NEIMOUS SYZA'TE1f 5l Ra.half ss votent as succinyIcholine and 20 dimes more potent than nicotine in producing a block of extratusal neuromuscular trad.<mimon. U. S. G. Clark and Rand (1964) compared the effect on the koee•jerk reflex in ests of i.v. injection of nicotine to that of hiererette-amoke introduced into the lua(ta by fixing a cigarette into one of the open limbs of a'I'-shaped trachea? csumula; the animals were lightly anesthetized with chloralose, and knee-jerk movements were recorded myographicalh• in response to tapping the patellar tendon every 10 sec with a Palmer automatic kneg-jerk hamtner. Injections of 2-5 pg/kg nicotine produced a detectable decrease in reflexly-elirited contractions, and 10 µg/kg could completely abolish con- tractions for about I min. One puff of smoke from an un- Glleml, small-diamet:e~ cigarette (which had been smoked for about one-half its length) produced effects roughly equivalent to 5Pg/4 nicotine in inhibit ing the knee-jerk and in raising binod-pressure, while smoke from filtered cigarettes was less effective, and smoke from a dummy cigarette made of tissue- paper produced no effect on the knee-jerk and no definite rise in blood pressure. O-Erythroidine antagonized the action of nicotine in inhibiting spinal reflexes in doses which atfected neither the pressor response nor the function of the neuro- muscular junction; 1 mg/kg of this substance prevented the action of tobacco-smoke on the knee-jerk without affecting the rise in blood pressure. These workers concluded that the effect of cigarette-smoke on the spinal reflex is due to the nicotine it contains (edntrast Hauser et al. (1958) and Bick- ford (191ft/), above, 150). Clark, Rand and Vanov (1f165) went on to oompare the pharmacological activity of nicotine and related alkaloids' occurring in cigarette-smoke. The threshold dose of nicotine to reduce the knee-jerk reflex in the cat w•es 2-5 ug/kg i.v.; larger (10-50 pg/kg) doses caused, in addition, blockade oi the flexor reflex; but even at the higher doses, transmission at the neuromuscular junction was ur.tm- paired. A number of other alkaloids, known or susperezd to occur in tobacco-smoke, were also tested for their relative ab:lit)• to cause inhibition of the cat knee-jerk reflex. The rels- tive molar potency of these compounds to produce a 50% re- duetion In the reflex wd,s reported to be as fo'.)ows (nicotine a 100): nornicotine, 54; thetanicotine, 0.4; anabasine, 17; myos- mine, 3; nicotyrine, A7; 2:3-dipyridyl, 0.1; 3-methy46-(3- pyrid)•1)-tetrahydro-1:2-oxasine, 3.5; dihydrometanicotine, 0.4; 1'..nethylannhasine, 2; cotinine, 0.05; pyridyl methyl- ketone, 0.1; pyridyl propylketone, 0.1. The relative molar poteicies of these same compounds to produce inhibition of the cat flexor reflex were, respectively (nicotine again = 1t16), 64. 0.6, 33, 3, 10, 0.1, -1.5, 0.8, 5, 0.5, 6.7, and 5. Reduction in the cat knee-jerk reflex caused by nicotine and the other ao tive ali:aloids was prevented by 10-26 Kg/kg strychtdne, 20 pg/kg nivanesin, and 1-S pg/kg b-erythroidine; and it was thcrefore considered likely that the actions were all exerted on the spinal neumnes responsible for regulating reflex activity. In spinal chicks, nicotine in a dose or about 50 NR/ ktz i.v. caused complete blockade of the crossed extensor reflex while wdyslightly affecting the flexor reflex; larger (up to 100 pg/kg) dcses also blocked the flexor reflex hut neuromuscular trans- mir: ion remained uninfllmired. Doses above 100 µa/kg niiro- tine caused contracture of the muscle; and, according to W. C. Ilox•man and Sanghvi (1963), this effect was peripheral in ori¢in. The other active alkaloids had qualitatively the saute c$,rls as nicotine; relative molar )atetmies for inhibition of rhick flexor reflex were given as (nicotine = 100): nornico- lhle. 30: atlaba.Qhle, 33; myrosmine, 3; and nicotyrine, 51. Rela- tive molar )wtencicw for inhibition of chick cro.aed-extensor reflex were reported to be (nicotine again = 100); nornicotine, Ce ; metamcoune, i'La; anatras•roc. Zu; myusmme, 4; auu nicotyrine,10. In dosage of 200µg/kR, strychnine antagonized the effects of nicotine and other ciFarette-smoke alkaloids in blocking the crossed-extensor reflex, whether given before the nicotine•like drug or during its action. 1 icotine injected into the cerebral ventricles of cats through a chronieally-implanted Colliron cannula produced various effe}ts, most strikingly, twitching of the ears (Armitage, Alilton and \forri~.nn, 1965, 1966; G. H. Hall and Reit, 1965). Arnlitage and co-workers (1965), as a result of their experi- merits (see above, 148), suggected that this action of nicotine involved a central cholinergir nrechanism. Using methods de- veloped by Carmichael, Feldberg and Fleischlrauer (J. PhyK- iol. lr3: 354, 1rJ6f), Hall and Reit (1965) shon-ed that this ear recponse does not result from an action on the w•alls of the cerebral ventricles, because it did not occur when nicotine was perfuse.dfrom a lateral ventricle toaqueduct. Acrordingto these latte.r workers, the nicotine has to enter the subaraehnoid space, and the resportse results front an action on structures close to the surface of the cervical cord at C-1 and C-2; ap- plied to this region. I:50,000 or 1:20,000 nicotine produced ear-twitching within 10-60 sec. In a few experiment.- in which these concentrations did not elicit ear-twitching, they facili- tated the pinna reflex (which was also facilitated by i.v. injec- tion in chloralosed animals). Applied in stronger concentra- tions, laying back of the ears occurred. Nicotine applied below C-2 was ineffective. The nicotine does not act b)•exciting the fibers of the first two cervical dorsal roots, since the effect persisted after the) aere sectioned bilaterally; the drug ap- parently acts on structures near the surface of the upper ceni- cal cord, which forms part of the sensory pathvay of the pinna reflex, since trensectirfin at the obex, but not trattsection be- tw•een C.2 and C-3 abolished the ear-twitchinp. R'hen applied to the dorsal surface of the cord at C-1 and C-2, 1:1,000 hexamethonium prevented the ear-twitching, whether applied to this region or ht;ecded intra-ventricularly or i.v. In a patient with progressive ianrilial spastic paraplegia studied by D. b.11'ebster (1964), a drop in spasticity followcd i.v. injection of nicotine and alro the rapid smoking of a rmn- 6lter cigarette, but very little decrease in spasticity waos ob- served when denirotinized cigarettes were strmked by the sub- jeet. (\foll (1926) had previously observed that nicotine reduced muscular rigidity in post-encephalitic Parkinsonism, w hich he ascribed to the paralyzing effect of the drug on the prespimll refles are subserving pla~tic tone (l4h).] 160. I f{afopatho)app (70) (Xo new data.) 161. sPIT•;AL Nt`nvr.N A\D 1<f:RvF:-r:xni\-os (76) For an aecoutd of certain effects of nicotine on autonomir nerve•filmr.~, wr nlNwe, 141. For a dwrription of the artnni nf nicotine on certain invertcbrate nerves, see below•,173. 162. lrritn6ilil,u (76) 163. Sciatfc :1'enr ##) 164. t'')t4er Jlotor \-enva (i7 ) (No nMP data.) 165. Srnsory and .l/otor \-erer-F•ndenga (t7) I.c'mbcrk (1957) Ktudied the influence of drugson cutaneous Produced by The Council for 0002963 Taharrn Research-USA, Inc.

~------ - ..-~-..--..~..: .: _. .. . . ._... _. _,- TOBACCO-EXPERIMENTAL AND CLINICAL STUDIES. SUPPLEMENT I 52 ..1.... ....:.. ...1.i..1. .A......t.......i ..... .. r....... ~~.. :.. ~,..r.......... ...y. . of the rabbit was connected with the bod onlv ihroutch the auricularis magnus nerve. Stimulation of these aBetent fibers elicited a refiex faU in blood pressure and rise in respiration; 10r40 µg nicotine had a stimulating action; repeated injec- tion of the drug resulted in decreased sensitivity, and, finally, paralysis. Diaawnd (1959) reported that close arteriai injectione of nicotine into the region of normal or regenerating sural nerve- endings in rabbits always excited a burst of impulses, which outlasted the injection by many seconds; these were blocked by hexamethonium. 166. ComparaCioe Senaitiaity to Nicotine oJSpinul and Autonomic Nerve-Fibers i78) ]No new data] 167. Hestopaflwlopy (78) Peripheral•nerve lesions were said to have caused wryneck in rats exposed chronically to vapors of nicotine plus methyl alcohol (Petrova, 1963); see below, 1083. 168. MISCELLANEOUS OBSERVATIONS (78) 169. Cerebrospinni Fltdd (78) Lv. injection of nicotine in dogs increased cerebrospinal- fiuid pressure; but, since this increase was aseoc;ated with in- creases in arterial and venous pressures, the chonge was con- sidered to be a purely mechanical effect (Becht, 192D). The secretion of cerebrospinal fluid was not increased by nico- tine; thereiore, there was said to be no need for ascribing the rise in cerebrospinal-Suid pressure to increased formation. 170. ElecdieaR Charge of Neme .8'xfrads (18) [No new data.] 171. Reuielos (78) Bchievelbein (1902) has presented a review of the uewer literature (mostly over the preceding 10 years, but not limited to these) covering the pharmacology of nicotine on the nerv- ous system, central and peripheral, including the autonomic. Mattila (1904), in his brief review of central actions of cholino- mimetics and cholinolyticx, included CNS actions of nicotine and drugs antagonistic to these. Sorgel (1961) briefly reviewed the effects of nicotine on the CNS, and concluded that the action of this dcug as a"ncrve poison" could not be frequentt]y enough emphslicced. A com- prehensive re riew of the actions of nicotine on CNS functions has been published by the compilers (Silvette et e1.,1982;184 references). The several functions reviewed weie:spontaneous activity; conditioned reflexes; learning; higher cerebral func- tions (sensory motor functions; brain potentials; tremor; con- ....i..i.. .nnKnn r..l.•e•. .nnf:no rofols..w.•X• mwion.w. functions (vomiting; resp'uation: vasomotor); cer;ebellar func• tions; spinal functions, including spinal reflexes. For the most part, experiments dealing with smoking or tobacco•amoke were excluded from review. Titis review was somewhat eum- mariaed and updated for the Stockholm Symposium on "Tobacco Alkaloids and Related Compounds" under the title, "Tobacco Alkaldids and Central Nervous Sg•stero Func- tions" (Larson and Silvette,1965b; 66 references). The reader should also consult the important symposium (57 pariki• pants) on "The Effects of Nicotine and Smoking on the Central Nervous Syutenl", held under the auspices of the \ew York Academy of Medicine in April, 1966, and aubsequently published (Ann NY Acad Sci 10, Art 1: 1-333 (Alarcb 15) 1967i. 172. TfIE INVERT9BRATE NERVOUS SYSTEM (19) 173. Eorlhmor»i (79) Sabelli, Marks and Toman (1963) studied drug effects on the two unit spikes of the ephapt ic-fiber system of the isolated ventral cord of the earthworm, Lumbricus ferrestris, using conventional and phase-plane (voltage vs rate-of-change) recording. Nicotine, in concentration of 1 mAt or less, ie listed among threshold-raisipg blockers; nicotine-induced threshold increase was not abollAhed by repetitive supratnaximal etimu- lation. 174. Marine Irorm (79) 175. EcAinoderm (79) (No new data.) 176. Vollasl• (79) Duncan (1961) reported a study of spontaneous actinity in isolated pieces of the pallial nerves of a variety of puhnonate mc,llusks. In concentration of 10-r H, nicotine had an ini• tial excitatory effect o6 the splsnchnic nerve of Lymnces slap• nales, which was followed after I min by a marked, reversible decrease in activity below the initial level. Caanglionic trsuis- miffiion in the abdominal and parietal ganglia was blocked by a 6 X 10-4 111 nicotine solution, but not at a concentration of 8 X 10-4 hl. 177. Cruetwean (79) Spontaneous firing of the crayfish stretch-receptor olkan produced •oy 1:10,000 nicotine was depressed or completely abolished by 80-sec prior application of 1:10 million - 1:10,000 -yaminobutyrio acid (T'Lkuya, 1961). 178. Inaa: „nr) [No new data.) Produced bv Thc Counc:l for Tobacco Research-USA, Inc. 0002964

SKELETAL MUSCLE I 179. Rffliewa Schievelbein (1962) has presented a review of the newer literAture (mostly over the preceding 10 years, but twt limited to flAese), which included the pharmacology of nicotine on muscle. E. Fischer and associates (1960) have also reviewed this subject. The review article by Flrckenstein (1956) is concerned mainly with the work previously reported by his colleagues and himself (94b; l0la), and only brief mention is made to papers by other authore. 180. EFFECT OF NICOTINE Iti 1NTACT ANIMALS (82) 181. Fish (82) 182. Frog (82) 183. Snlonumder (82) (No new data.J 184. Vouse (82) Intiaperitoneal injection of b mg/kg nicotine induced tremors in CF1 male mice (C. A. Day and Yen, 1962; Yen and Day, 19i15). This dose was said to cause tremor in 100% of animals without causing death, and tremors so produced lasted only 1-;i min (Yen and Day, 1965). In CF,-strain mice given 5 mgikg nicot.ine tart.rate i.m., the animals oc- casionally showed tremois and mus^le LaitcheF- with higher (7.5 mg/kg) doses, clonic convulsions appeared (dlaffd and Soncin, 1958). An intravenous injection of 0.4 mg/kg nicotine caused muscle spasm ItremorJ in 100 Po of mice tested (Zeimal, 1964). lionta and co-workers (1960) have described an improved apparatus for measuring the motor activity oi mice, and its application to the quantitative measurement of nicotine-induced tremors. A table of results obtained with their apparatus folloaing intravenous injection of 0.18-0.43 mg/kg nicotine showed graded increases in motor activity with increasing dacage. lnjections of nicotine up to 0.6 mg/kg induced tre, nly, wltereas, with higher doses, the tremors were rapta,; ,.,llnwed by clonic convulsions or even seiBUres of the tonic-extensor type. Hence, for a study of a pure tremor efiect, the nicotine dose in mice did not exceed 0.5-0.6 mg/kg. Further, tremors produced by intravenous nicotine are rather discrete and of short duration (20-30 see). 185. Rat Following injeation of 04 mg/kg into the dorsal tail vein of 78 rats, a fine tremor was observed in all of the animals; this was characterized by a rapid (10 or more per eec) shaking or vibration of restricted estent, particularly noticeable in the extremities (Orcutt, Michaelson and Prytherch, 1963). The nature of the ttemotss was studied by Yen and Day (1i165) in chronic T-1 epinalia.ed rats; nicotine induced tremor only centrally above the T-1 level, and duration was found to be prolonged to 8-10 min in this preparation as compared to 1-3 min in non-operated mice (see abave,184). 186. $ab6if (82) Intravenous injection of I mg/kg nicotine in rabbits caused marked clonic tremors (.a,rotyvnyan and lfaskovskii, 1961). 187. Cat (831 Twitch rebponses in cats were readily obtained by injection of 0.025 mg/kg nicotine into a muscular branch of the femoral artery (Roaskoa•ski, I961). Nicotine suliate in doses up to 0.25 mg/kg , failed to produce twitch respotu,es, but occa- sionally caused a condition ruembiing faec•iculation. In the cat, whether simply anesthetized or previousl,v decerebrated or spinalized, i.v. injection of nicotine induced powerful fasciculations of the hind-lirob muscles, which were reduced but not abolis,hed by sectioning the sciatic nerve (W. C. l3on- man and t5anghvi, 1963). Injection of nicotine into the cerebral ventricles of anes- thetized or conscious cats resulted in taitching of the ears (Arutitage, Milton and \lorrison, 1965, 1966; G H. Hall and Reit,1965). the twitchitqt rvas reported to be continuous and closely resem'.bling repeated activation of the pinna reflex (Armitage et al.); or the ear-twitching was said to have been interrupted irFitially for about 15 wc during which both ears were laid back (Hall and Reit). The ear-twitch response r.as said to have been blocked by hexamethonium only when given intra-ventricularly (Annitatre et al.), but the ear. twitching n•ag alsu said to have been prever.ted when this drug was Injected intraventricularl-r or i.v., Ls well as applied to the dorsal surface of the spinal cord at C-1 and C-2 (Hall and lteit). Xlecamylamure administered either intraver.tricu- larly or i.v. prevented or abolished the ear•tp•itch response, while neostiglnine injected into, or perfused through, the cerebral ventricles potentiated the duration and rate of ear- tuitch in some experiments (.lrmitage et al.). For suggested mechanisms of this nicotine-induced ear-twitch response, see above, 1-18 and 159. 11"hen injected into the caudate nucleus of unanesthetirRri cats chronically implanted with electrode-injection cannulas, earbachol and related choliuergie agents pta4uced tremor of 3-hr duration; in contrast, even at toxic levela, nicotine proved ineffective as a tremorogenic agent (Connor and Baker, 1965). 188. Dog (83) ~No nea• data.) 189. Elfeet ojSmokinp oa 1lusde 7lromor and 7bnua in Jlaa (83) V. R. Rao and Rindani (1962) studied 10 health,v medical student>, aW 1F-70 yow,, who had abstained from arrtolting 83 Produced !»i The Council for 0002965 Tobacco reseai-ch-USA, Inc.

i I L, 54 TC)BACCO-EXPERIAtE:STAL ANl) CLIKICAL l;TU111&A. SUPPLEMENT I o/ IP4Ct' 94 t1Dn1V ,?rinr tn vlertmmvnrranhic readings in the small muscles hf the baotd. Before smoking, the potentials on maximal effQrt were i•hythniic and of regular ampGtude; after smoking 1 cigarette, the amplitude fell and the fre• quency was sloarer, and large bizarre spikes appeared intermit- tently which were larger and lonRer in duration than the ones seen before smoking. These changes subsided in about an hour following Qessation of smoking. The authors suggested that carbon monoxide ~ontributed only slightly to these changes, the mojot effect being from nicotine, both on the myoneural junction and the ganglia. Subseqpentl;•, Rao (1963) auggested that there was a strong possibility that the splitting of the, notmal wave-spike, recorded in electromyo• Qrams after smoking, was due to the action of nicotine on a group of neurdmuscular , junctions; if so, it would indicate a phsrmacological unmasking of heterotrenous neuromuscular junctions. But, he added, the possibility also remained that the delayed component was due to some changes in the muscle itsell. Vartiainen (1jBlt) repon•tted the case of a female with Iwst- eticephalitic tremor, not completely controllable with atro• pine, but in whom the fimoking of a cigarette would cause complete cessataon of the tremor. The effect was observable in 2 min, and complete cessation occurred in 4 min; at 12 min, tremor began anerr; and in " min, the effect of the cigarette had completely disappeared. To obtain a longer lasting effect, the patient would smoke 2-3 cigarettes. A similar effect of tobacco Avas not observed on other patients. In a patient a'vit.h progressive familial spastic paraplegia studied by D. I). Webster (1964), a drop in spasticity tol• lowed i.vi injection of nicotine and also the rapid smoking of a non-61ter eigsrette; but very little decrease in spasticity was observed when denicotinised cigarettes were smoked. H. J. RobertB (1965) has reported a study of 131 patients with spontaneous leg cramps and "restless legs" due to dia• betogenic bypeEinsulinisnn (see below,1254). 190. Uect of Nicotine on F.lectrirnl Nene Stinnelolion of Jf uacte (84) In decerebraRe cats with tibialis anterior muscle prepared for recording contraction.s from sciatic-nerve electrical stimu• latior., close arterial injection of 0.015 rng nicotine eGc:ted a distinct contraktion, followed by slight inhibitiun of sub• sequent aingle electricall,r-induced twitches; 0.15 mg elicited a strong twitch, following which nerve stimulation gave no contraction (()haell, 1946',). Supprevion of the erTect of nerve stimulation wes considered to have resulted from the curare• like action of nikotine. 191. 1Wert of Denen+alion on thc tlfhori of Nicotine on Ntiarle (84) M. \Iorita,l'asuhara and Sugimntn (1902), usinp rahbil., exch<ed and resected abodtt I cm of the fibular nerve at the popliteal tos,ca, and, 2-3 weeks later, recorded fibrillation voltages in the denervated anterior tibial muscle. Injection into the ear-vein of 0.1-0.3 nig/kg nicotine modified the fibril. lation-voltages patteni by producing a burst of discharbms, and, thereafter, complete depression for 5-30 min. After i.v. injection of 8-8 mg/kg d-tubocumrine, the action of nicotine was insufficient to produce either stimulation or depression of the fibrillation vroltagesc and 2 ntR/kg edrophonium chloride (Tensilon) did not increaae the fibn'llation voltages depressed by nicotine. (For the relationship of these findings to the mechanism of action of nicotine, we below, 227.) 192. rjeet of Other Drugs on Nteo¢ine Tremwr (85) The following droga have been reported to inhibit or antago- nize, to a greater or lesser degree, nicotine-induced tremors in animals: atropine; azamethonium; benaMyaine; carami- phen; clrlordiaaepoxide; ehlorisondamine; ehlorpromasine; chlorprothixene; dietbaaine; diethylamide L'\iLA91; dimeca- mine; ethdpropazine; glutethamide; hexomethonium; imipra- mine; lysergic acid diethylamide; mecamylamine; me- probamattn; methdilaaine; morphine; osythiospasmine; perphendsine; phenothiar.ine derivatives; procyclidine; pro- maaine; µroniethaziue; thiospasmine; trihexiphenidyl; tri- methidiniuun. The following compounds have been nclwrted by some investigators to inhibit, by e;till others to have no eflect on, nicotine•induced rrcmors: arecoline; arpenal HCI; butacaine; p-butoxy-p•piperidyl propiophenone, decametho- nium; dicblorpromasine; gallamine triethiodide; meri>rnite; mesphenal; pachycarpine; pentaphene; procsine; tetraethyl- ammonium; and d-tubocurarine. The folloN ing substances reportedly were relatively or completel}• ineffective agah,.a nicotine tremor: caramiphen; chlorprqmasine; phenacetyl- urea; phenobarbital; reserpine; and tetrabcmu:ine. Keostig- mine and dibenamine potentiated nicotine-induced ear- tnitching and tremors, respectively. Details of the experiments on which the above statements were based will now be supplied according to author (in chronological order), rather than according to the drug used, since the majority of workers have tested a number of drugs under the same experimental conditions, whereas the s,ame drug may have been tested by the several workers who hare employed it under non-comparable conditions and in different animal s D hlbom and co-workery (1952) compared the potencies of 24 new l0-alkylaminoaeylphenoth~ines and basic esters of phenothiazine-l0-carboxylic acid to those of cattuniphen (Parpanit), ethopropasine (Lysivane), diethazine (Diparcol), promethasine (Phenergan), and hexatitethonium iodide in antagonising tremor induced by i.v. injection of 0.75 or 1.0 mg/kg nicotir.e bitaruste in male albino rabbits. Three of these-110•(dimethylaminorretyl)-phenothiarine; 10•(diethyl- aminoaeetyl)-pheaothiaziee; and 10-(a-diethyleminolwopio- nyl)-phenotbieaine-had about the same artivity as etho- propasine and caramiphen; and one of the compounds, A-diethylaminoeth.vl-phenothiazine-l0-carbox)•late, was de- cidedl,v superior to ethopropazine and caramiphen. Ethopropazine (Parsidol), methdilasine (Tacryl), and trihex.qthenidyl (.artane) all antagonixed tremor induced in the rabbit by i.v. injection of nicctiue (Lish et al., 19(i0). Ptotection against tremor.• induced in male mice by i.p. injection of 5 mg/kg nicotine was offered by ehlotieondamine, hemmethonium, chlorprothixene, chlorprornasine, me- prolmmate, procyclidine, and h.mtt-hc acid diethylamide (VD-25) (C. A. Day and Yen, 1902). The teat drugs were injected i.p. one-half hour prior to nicotine, tremors being recorded in sptrially.desi,tned activity-cagex. EDe,s arre calculated from dose•rc,ywnse cun•es, and r•erm found to be, respectively, 4.7, 25, 20, 10-15, 100, <100, and 10 mg/k}t, In a rubu•yuent communicatioty H. C. Y. Yen and Day (1965) relwrted that cblorisondamine, hesamcthot>iunt chln- ride, chlorprothixene, chlorpromaahte, meprobamate, pro- cyclidine, and lysergic acid dietln•lamide (4S1)-25) antago- nized not only the tremor induced by this dose of nicotine in niice, but also its ronvulsive action. 13Uie, 5elective lndex (81 = subneurological dose/t.l)„), and Therapeutic Index (TI = LD,o/L•'N) for these drugs were, respectively (ma/ktt. Produced btr The Council for 0 0 0 2 9 6 6 Tobacco pesearch-USA, Inc.

, I SKELETAL MUSCLE l.p./: y.,, J.J, w.); w, 4m (iJ nut aelerrntoea); 'LU, 6a, 4.4; 15, 0.2, 7.8; 100, 0.5, 7.0; 100, 1.0, 1.3; 10, 0.5, ,26.2. The most effective drag was found to be chlorisondamMne, which had a SI value of 5.3 and a TI value of 13.1. Hexamethonium and chlorprothixene possessed some selective acticity, while L~1~-25 inhibited tremor at close to the neurological dose- level, but its TI was as high as 26.2. Dibenamine pbtentiated the tremors produced by nicotine. Imipramine, glulethimide, chlordiazepoxide, promazine, cammiphen, diethylumide 1111L-{9I /1-methyl.d-I-vsergic acid butanolamide tartrate), and morphine sullate were listed as causing diminution or delay in onset of tremors at subneurotoxic dosage; caramiphen was listed as causing diminution of tremors at tieurotoxic dmer. According to Votava (1962), i.v. injection of 0;5 mg/kg atropine in chinchilla rabbits 2 min prior to i.v. injection of I mg/kg nicotine shortened the duration of the nicotine• induced convulsions [muscle tre.morsj; this writer nqte(1 that his r'esult did not contradict that found by Bovet and 1fAngo 1(19!i1) (S7a)), since the latter authors were seeking a dose which would completely prevent nicotine-induced spasms in 50% of rabbits tested. Injection of 0.5 mg/kg diethasine or bEnaetyzine or thiospasmine or oxythiospasmnine 2 min prior to i.v. injection of 1 mg/kg nicotine shortened the dura- tion'of nicotine-induced muscle spasms, but i.v. injection of 1, 2, or 4 ing/kg eblorpromazine or dichlorprornazine 30 min prior to nicotine injection had no effect on the duration of spasdts. Following oral administration of 60 mg/kg phenobarbital to rats, the i.v. injection of 0.5 mg/kg nicotine, which in- dueed mrsale taemor in 100% of unh+eat.r,t animals, induced tremor in only 40% of the ardmak tested (Orcutt, Michael- son and Prytlterch, 1983). Prior oral adminisrration of 280 mg/kg phenacetylurea was ineffective in preventing nicotine tremor, as was 220 mg/kg caramiphen. Prior oral adminiatra- tion of procaine, butacaine, or p-butoxy-p-piperidyl propio- phenone (Dyclonine; P-267) was alsu without efl'ect. Atecolir.e had no effect on nicotine tremor in rabbits (Deni- senko and Pratusevich, 1963, 1984). I.n dd-strain mice of either sea, i.p. injection of nicotine bitartaate induced tremor in 50% of the animals (I'Dec) in dosage of 3.7 mg/kg (ranlre. 3.2-4.2) (Hltjiwara, Yamawaki and Shimamoto,1904). NA'hen administered Lp. prior to tiico- tine, 0.1 mg/kg reserpine increased the TDa without modify- ing the pattarns of tremor; 1.0 and 3.0 mg/kg decreased the TDa. Prior injection of 1.0 rttg/kg tetrabenasine did not tdgnificsntly affect the TDa ; 5 end 50 mg/kg deatmsed the TDet. Phenacetylurea, phenobarbital, meprobamate, benacty- aine, chlorpromazine, perphetmsiae, and ethopropatune all showed a protective effect against nicotine-induced tremor in mice (Otori. 19B4a,b). Zeimal (1964) found ganglionic blocking substances in- jeeted into the lateral brain ventricle of mice to be most effective against the muscle tremor resultit>!r from i.v. injection of 0.4 tag/kg nicotine (ED,a) 10 min later. Ileu- methonium appeared to be a competing antagonist of nico- tine; it prevented spasms, not only from a nicotine dose cans- ing tremor in 100% of control animals, but also from a dose 5 times Rreater (i.e. 2 tng/kg). The ED* for hexamethoniunt ti`•as 0.22 vg/20 gm mouse, and increased by 8.9 times with a 5-fold incrraxo in nicotine dosage. Preliminary data for other aanplionic blocking agentA (dimeaamine; ttaamethoniunt bromide [pentatnine)) showed that these also prevented nicotine tremor in doses of the tnrrte order as that for hexa- S'J methomum. In contrast, tetraethylantmoniuni aind pacb~•- carpine failed to prevent nicotine tremur in dom of 100 µg/Z0 gm and 50 µg/20 gin, respectively. In doses lesa than I µg/20 gm, the curare analol;.., d-tubocurarine And gallamine triethiodide (Flaxedil), did not prevent nicotint• spasms, and, in higher doses, themselve3 caused prolongeil spasrits. The EDa for decamethouium was 5.4 pg/20 gcn mouse; that this value was markedly higher than that for he.ea- methonium, indicated to the atithor that the central choline- receptors excitable by nicotine arc not similar in structulr to the choline-receptors of musc•le, but, rather, have a similar structure to ganglion choline-receptors. With respect to cholinoh•tic substances u•ith a tertiary nitrogen, Zeimal found that the most effective compound against nicutinr' tremor in rabbits was arlmnal hydnx-hloride (tliethylamino- propyl ester of diphenylacetic acid) given by i.m; and i.v. injection; Pentaphen (hydrochloride of the die;)IYlamino- ethyl ester of phenylcyclopentacarbonic acid) was abno:t as effective. On intravenlricular injection, arlbe»ql na, 54 times less effective than hexamethonium; and Pentaphen and quaternary analogs of arpetml (merpenite) and of Pettts- phen (nresphenal) proved to be as ineffective as ari>pnal. In the anesthetized cat, 0.2 mg mecamylaniine adruinir, tered intraventricularly, or 0.2 mg/kg injected i.r., prevented or abolished the ear-twitch response to r:icotina• (Armitage, Milton and Morrison, 1965, 198ti;. \eostigminc irtjehted into, or nerfused through, the cerebral ventricles potentiated the duration and rate of ear-twit.rh in some experiments. \iecamylamim: and heramet.hottium also antaagotiized the ear-tn•itch in the conscious eat (see above, 148). In concentra- tion of 1:1,000, hexamethonium applied to the dorsal surface of the P,rinal cord at C-1 and C-2 prevented nicotine-induced ear-te•itching in the cat, whether applied to this region or injected intraventricularly or i.v. (G. H. Hall and Reit, 190Q (see above, 159). -llecamylamine protected against the tremors induced by the larger doses of nicotine in the rat, while trimethidiniunt aras less effective in this regard (Donuno, 1985s) see above, 121 for experimental details). 193. JJechanisrn of .4tlt;on of )\'etotinc Tremor (86) Most writers ;tave considered that the site of nicotine tremor in the intact animal must be Fomewherc within the central nervous system (88b489a); but, since thiospasmine and oxythiospasmine are assumed to have purely peripheral effects, and Votava (1902) found that these substances shortened the duration of nicotine-induced tremor, this particular writer concluded that the peripheral action of nicotine played a certain role in the tnechaniam of these tremors. The reader may also consult the reviews,by Silvette and associates (1982, pp. 148-150) and P. S. Larson and Sih•ette (1.9fi5b, pp. 112-113). 194. Ejfert of .1'irotinc on .llnsrfe Trcmor Ftrom Other Causea (89) In rabbits under pentobarbital anesthesia, in which shi%rr- ing had been induced b,• cooling, injections of 140-525 pg/kg nicotine regularly stoppal the shivering Olott, 1963). The rate and depth of breathing (in 4 vagotomized animals) a•as also increased; administrations of 15 ma/kg hexatnethnnium abolished the respirator)• stimulatinn elicited bi nicotine, and nhivPring then continued uninterruptedly. In cats, injection Into a muscular branch ot the femoral artery of nitrotirn, in dowa alKrve 0.05 tna/ktt ocraxirnrelly Produced by The Council for 0 0 029 6; Tobacco Research-USA, Irrc.

F.-. - . •~- I I 50 TObACCU-EJCPERIMENTAL AND CLINICAL STUUIE.c. SUPPLEMENT I caused transient blockade of control acetylcboline responsea (Rngsknw-ki after nicotine, or vice verm, evoked tremor and convulsions, just as though no drug had hitherto been given which acted on the central cholinergic system (Denisenko and i'ratuse- vich, 1963,1964). Bdskovii: and co-workers (1964) reported that they had found it possible, using tremoriue (1,4-dip)-rrolidino-2- butyn), to produce a tremor in both decapitated sud non- decapitated nata, which could be stopped by i.v. injection of 0.25 mg/kg mcotiae or atropine. This dose of nicotine was said to transform the rest tremor produced by dremorine into an intention tremor. P. Stem (1964) reported that i.v. injection of 0.25 tng/kg nicotine in mire transfodnied the Etntic tremor produced by 25 mg/kg tremorine i.µ. into an intention tremor; for the supposed mechnnism ot ti:i efTect, see above,146. 195. Tonic Rigufilk Dur to .1'irotine (89) (:Qo new data.) 196. F',$ed of A'icotine on ADusrlea of tke Intart Fou•1 (89) In chicks, minimal effective dosea of nicotine (1-2 pg/10 gm body-weight i.v.) produced 9arcid paralysis; larger (5-10 µg/10 gtn) doses caused a spastic paralysis, and this spasticity phoduced by nicotine was alweys followed by a further period of flaccid paralysis during which death occurred, or front which recove» took place. W. C. Bowman and 8ana•hvi (1963), who reported these ea7ieriments, considered it unlikely that the 8accid paralysis produced by the smallest effective dose of niro8,ine could be due to depressed excitability of the mturle, since larger doses, given immediately aftetwae+ds, caused c->tttracture. In the anesthetized hen, nicotine caused a sustained 'a•ontracture of the gamroenemius muscle on i.v. or close arterial in±ection. With small (50-100 µg) doses, the ms?cinuil taitches (eliciW bv electrical stimulation) fojlowing the contracture wete not depressed below the pre-inje.ction level, but with larger doses, the subsequent twitches remained depressed after the contracture. /For a corttpatitmn with the cat, see alarrve,187.j See also below, 225, Effect of \icotine on Isolated Avian hiuscle. 197. EFFECf OF KICOTI.`:E AND OTHER SAIORE- CONSTITCENTS ON ISOL.\TED NtUSCLE (90) Carbon monoxide impaired the ability of frog striated muscle to perform tetanic eattQrection, but tobaaco-smoke, from which carbon monoxide had been removed by a dry heutoglobin filter, largely lost its myol)•tic properties (Danyss and Dmochoa-ski, 1959). 198. Cencendalion/oerl RelafionaAip (90) 199. E6eet of Alaktiple Uoses (91) 200. Nirotine Contrarttu+e and F'sbriUnry 74aitrUny (91) 201. Reaponse of t'arfotks Parts of the .lfurrle F'i6er to ,ti irotinc (91) 202. Reaponae of N'irofinited alusrle to fflecfrirol Sfiarulalimn (92) (No new data.) 203. E8ed on the Chnynnzie of Alusde (93) Laptcque (1930) ttad briefly summarized in a review anirlT previous, work by himself and others on the effect of nicotine on muscle clawnaxie and the influence there on of certain physical-chemical factors. Apparently, there has been tio recent interest in this particular subject. 204. Effect on Deneraated Dt urrle (9f ) 205. Or(rgen Conau»rption During A'irotine Conow•ture (94) 206. Action-Potentiafr (9I) [To new data.1 297. Membrane Potenliole (94) Henze (Pflogers Arch. 8Z- 451, 1902) studied the etfect on the membrane potential of frog muscle of a number of pharmacological agents, including nicotine, causing muscle contraction. According to Fleckenstein (1956), this was the first time these drugs were reported to be depolarixing agents; this was the beginning of the electropharmacology of muscle. Nicotine applied to the end-plate regions of muscle-fibers of the adductor longus muscle of the Australian tree-toad, Hyla aurea, produced an immediate contracture, accompanied by a negative potent.ial, which was maintained for approsi- hnatel)• the same time (ICuffier, 1946). The electroplax, or electric plate of electric fishes, is the iultimate component of clos•ely-packed prisms of modified Enuscle tissue, separated by connective titicue, and abundantly supplied with nerves. The effect of nicotine on the monocellu- lar electrdplax preparations of the electric eel, EteRrophorus electrieus, has been studied by liartels and Podleski (t964). At pH 7.6, nicotine in concentration of 5 X 10'' U blocked the action-potentiral reveraibly within 5 min; with increasing nicotine concentrations, a step-wise depolarination of the raembmne took place. Increasing the pH from 62 to 9.0 decreased the potency of nicotine by a factor of 2-3, and, in addition, increased the thne for reaching the equilibrium potential by a factor of 5-10. Curare inhibited the effect of nicotine; since both compounds act at the synapse only, the dtode of rD,ction was competitive. Clam luoison (a powerful marine hiotoxin), even at 20 times its active eoncentration, did not influence the nicotine effect. 208. R(feM of Temperatum on r1-icdine t,'orttracture (94) 209. Elferf of Stretrkinp and of Load on Nicotine Controettue (95) 210. Effe•rl of F'atfgue on Xirotine Conbarlare (95) 211. 1'iestoration of F'i.atetion of Arieotiaized 9lusrle (95) [ho new data.) 212. fbmparison of the a'ievtine Ejlrrt on Jl nacle uifl tJrat q( its Deritntirea (95) Shitnamoto and co-workers (195ti) tested nicotine mono- methiodide~ nicotine isomethiodide, and nicotine dintethio- dide on sdatic-gastrocnemius prepamtions of rats and on ex- tirpated rectus abdominis muscle of frogs, and stated that the first of these showed a considerable increase in the ncuro- muscular effects, o•:er that of nicotine bitartrate, while the other two rhmpomtds bad no nirotine-like aetion. The imallest dose of nicotine producing a clear contraction of the isolated rectus muscle of Rana tenrlroraria erea found to Produced br The Cot!ncil for Tobacco liesearch-USA, Inc. 0002968

ti. SKELETAL MUSCLE 57 be 0.1 µg/ntl (Mattila, 1983a, b). The activities of nornico- uue ano ethytnormcottne were about one-eixth that of nicotine; allylnornicotine and benro)•lnornicotine were, respectively, 175 and 900 times weaker than nicotine; carba- minoylnornieotine and acetylnornicotine were, respectively, 1000 and 3200 times weaker than nicotine. The action of nicotine could not be blocked by nornicotine or its derivatives. Erdtman, liaglid, Nellinga and von Euler (1963) reported the synthesia of all possible lsomers obtained when the pyr- rolidine ring of the nicotine molecule is opened in different positions. Tested on frog rectu.-abdominis muscle, all dis- played markedly lesser activities than nicotine, and, in some instxnces, no activity at all.,Taking the potency of ni.wtine as 1, comparative activities for nornirotine and its N-alkyl. substituted derivatives, nicotine, N-ethyinornicotine, and N-propylnornicotine, on frog muscle were, respectively, 0.5, 1, 0.07, and 0.015. These workers also included relative potencies on the same test ssstem of mme model compounds, in which only a part of the pyrrolidine unit of nicotine was present; again, all activities were markedly reduced or ab. aent. Haglid and 1Pelfings (1963a) reported preparation of a number of N-substituted 3-picolylamines, together with their benzyl-, thenyl•, furfuryl., 2-pyrrolylmethyl-, and 34n- dolylmethyl-analogues, together with their biological ac- tivities relat,ive to nicotine on frog rectus-abdominis muscle; again, relative potencies in all instances were markedl,v re- duced or absent. These same workers prepared a number of N.substituted aminoslkylpSridines and phenylalkylamines, and found that relative potencies, compared to nicotine on frog muscle, were in all instances markedly reduced or absent (Haglid and U'elUngs, 1903b). A number of 2- and 3-sub. stituted 1-methylpymolidines, together with some N-sub- stituted pyrrolidines, were prepared and tested for biological activity on frog rertus-abdominis muscle; relative to nicotine, the potencies in all instances tvere markedly reduced or alMent, or even reduced to a relaxing effect (Haglid and Nellings, 1988c). Finally, Haglid (1065) prepared all possible isomeric "secanicotines" derived from nicotine by cleavage of the pyrrolidine ring, as well as certain derivatives of these, and he presented attucture-activity relations based on activities on frog rectus muscle. All these synthetic analogues ot nico- tine were also tested on isolated mammalian intestine with similar results; see below, 611. Nornitmtine, twabssine, and metanc.otine caused ear. taitch when injected intraventriculady into the conscious cat, although thtxe t:otnpounds were less effective than nicotine (Armitage, Milton and Morrison, 1905, 1966) (see above,148). The isolated frog-rectus muscle was caused to contract by 8 of a number of cigarette-smoke alkaloids tested by M. 8. G. Clark, Rand and Vanov (1905). Relative potencies were determined by interpolation on the log dose-response 6nes or by giving concentrations to the rectus which gave mntrec- tioas bridging or matching the trsponse to I µg/ml nicotine. Taking nicotine as 100, the relative molar potency of the o0er alkaloids were found to be: nornirotine, 61; anabasine, 28: m,vosmine, 3; nicotyrine, 0.4; &methyl-0•(3.pyridyl)- tetrabydro-1:2ozaaiae, 2; N•methylanabasine, 3; and p,vridyl methylketone, 0.3. Barlow and Hamilton (1905) compared the activities of the (-)- and (+)-iaomersof nicotine onfrog rectue-abdominis mtucle, and found a ratio of 10. (Thb finding was exptrsre<I In equipotent molar ratios in tchich, lf the number is greater than 1, it indicates that the (+)-ioomeris less active than the (-)dsomer.) Thtse wrorkern noted that an accurate nmy of the two isomers w&4 not feasible. since tho tiwmo .vnx desensitiaed for a consid'ierable time after the addition of the drug, particularly after dosea of (+)-nicotiMe which caused any appreciable contract . Based on 4 results on cat tiblalis muscle (block), a ratio 0 3.1 * 0.5 was obtained. 213. Composison of ~ the Atneele Effect of Nitane toitA that nINier Drugs (96) At. F. Cuthbert (19&ta reported relative pbt.encies to nico- tine for tyramine m~~hiodide, dopamine , methobromide, and noreltinephrine metlhochluride on frog rectus-abdominis muscle. The nicotine-like compound, dimethyllbbenylpipetttzin- ium iodide (DJ1PP), caused ear-twitch when injected in- traventridilarly into the conscious cat. but eras less effeet.ive iu tltia respect than nicot)ne (Armitage, Milton and Aforrison, 1965, 1060). 214. Effect of pH on A'icatine Controclvre (96) The contracture and 3. fold increase in'aCa uptake induced by 2.5 m.df nicotine in rog sartorius muscle was found to be inhibited by lowering, he pH of the Ringeu's bathing solu- tion from 8.4 to 7.4 (G; B. Weiss, 1966e, c). Additionally, the uptake of "C-labeled, nicotine into the muscle was found to be more than doubled by increasing the pH of the Ringer's solution by this value. (She also the studies of Hamilton and Barlow, below, 227.) 215. tnftuenoe of otner Drugs on Nirotine Com,tracftpe (96) 216. Ganglionic/Neuroittuscuilar Blocking Agents (98) (Xo new dinta) 1-17. l.ohal Aneetheties (97). (h'o new data.) 218. Quinane (97). (No new data.) 219. Veratrine (07). (1\o new data.) 220. Parasympathomin.etic Drugs (98). (No new data.) 221. Parasympathetic $locking Agents (O8). (No new data.) 222. Sympathomimetih Drugs/Blocking Agents (98), (:Qo new data.) 223. lnorgPanic Salas; Ions (98). The inAnanrr of the potassium ion (K*) on nicotine contracture and "Ca uptake of isolated frog sartorius muscle has been investigated by G. B. n'eiss (1966a, b). 224. Alisceblaneous (98). The influence of EDTA (ethylene- diaminetetraacetic acid) on nicotine eontracS.ure and uCa efflux has also been studied by G. B. Weiss (196Ga, c). 225. :4oian a/usde Fleckenstein (1950) has published (from a ptpper by Cinrel and co-workers (Arch. exper. path. pharm. L"l6.^ 103, 1952)) a table of substances capable of producing a contraction effect in avian muscle through anodal polatization; in this table, nicotine is included. Child and Zahnis (1980) have described a nen• biolo~ical method for the assay of depolarieing aub- atanres, using the isolated semispinalia eervicis muscle of 3-10-day-old chicks, and they reported that, on this prepata- tdon, nicotine had less than one-fiftieth the activity of deca- Produced bv The Council for 0002969 Tobacco Researcit-USA Inc.

58 TOBACCO-EXPERIAiEtiTAL AND CLIhICAL STUDIOS. SUPPf.E]tf6.\T I methonium. On the isolated-nerve-sterno-traehealis-muscle preparation of the chicken, nicotine caused a contracture response of the muscle and a dose-dependent degree of neuro• muscular block; the concentration for production of a 50% maximel contracture was given by Carlyle (1962) as 1.59 t 0.29gX10"". It may be mentioned here that Barlow and Hamilton (1962a) tesW a number of isomers and homologues of nico- tine for their ability to act like acetylcholine in causing con- tracture of the chick biVenter-cervicis muscle; for details, the reader must be referred to the original paper. These workers (Barlow and Hamilton, 1965) also compared the activities of the (-)- and (+)-isomers of nicotine on the chick biventer-cervicis preparativn (contracture), and, ui 6 experiments, obtained a ratio of 4.60 t 0.07. [This finding was expressed in equipotent molar ratios in which, if the number is greater than 1,, it indicates that the (+)-isomer is less active than the (-)-isomer.) 226. Afantneolian 11uaccle (98) Verhey and Voorhoeve, (1963) studied activation of group Ia and group II musele-sp?ndle afferents by certain cholincrgic drugs in anesthetized csts, in which one hind-limb had been denervated with the exception of the nerve to the gastroc- nemiue-soleus muscle; laminectomy was performed, and single fiixrs originating in the muscle were isolated in the L-7 S•1 dorsal roots, and action-potentials from the single fibers were monopolarly led off and recorded. Ventral roots were cut in such a manner that the distal end could be -.timu. latrd. Conduction velocity was measured; a velocity of 72 m/azc conventionally separated grvup Is from group ll afferenta In this preparation, close arterial injection of nicotine activated ls afferents to the same e..tent as did suceinyleholine. Based on I results, a(+)-niebtine/(-)-nicotine ratio of `s.l :k 0.5 was obtaine:l by Barlow and Hamilton (1ti6S) on cat tibialis muscle (blocl•). (See 225, above, for significance of this ratio.J X F. Cuthbert (1964) reported relative potencies to nico• tine for tymmine metl.iodide, dopamine methobromide, and norepinephrine tnethochloride on cat sciatic-nern•e- tibiaas•muscle preparations. 227. lledAaninn of Action of the Nicotine F(fect on .11 resr•fe (99) ,zapanR (1953) has elaborated a theory of the mode of action of acetylcholine, which he extended to a hypothesis on the mode of action of other biologically active subitanees. He postulated that the receptor protein upon, which acetyi- choline sets is identical to cholirte estetuFe (f'hF, a non-specific astetnse). Hence, the reaction is as follows: released aretyl- choline combinea with tissue ChE, acts upon it (stimulation), and is hydrolyzed to inactive compounds in the next fraction of time; the receptor protein, i.e. ChE, is ready to react with the next molecule of acetylcholine (ACh). :lcetylcholine esterere (AChI:) is a"specidc type"; it hydrol,vzes only ACh; the receptor protein of the muscle end-plate constitutes AChE. (1'his statement apparently applies to all receptor structures ulwn which ACh acta.)1t'ith respect to nicotine, the following is postulated: According to the receptive.enzyme theory, cell receptors for carbon-dioxide,sensitive structures would be identk•al to carbonic anhydrase (CA). This h.•pnt.he- sis seetned attractive to its author, since c•vanides are knottro to be reverstible inhibitors o`. CA, and, at the same +inte, stimulatrrs of chemoreceptors. The most potenit stunulator of chemorrceptors is said to be lobeline. Actually, in-vitro studies showed that lobelino inhibited CA, the Ia value being at 10-' .11 inhibitor concentration. Nicotine, which is a strong stimulator of chenboreccptota at low concentmtio:r-, but inhibitory at high concentrations, most probably acts on ChE; for the paralyzing dose of nicotine on receptoni completely inhibited AChE in vitro. Therefore, nicotine, in spite of its numerous pharma•oloocal similariticx with lobeline, can hardly be expected to inhibit CA. Actually, experiments have shown that it dues not. C. 11. Smith and colleagues, who studied the effect or drugs on afferent endings and nerves o) triceps urcae miuclc. apinflle- in Tinal or at>u~thetized cat.. reported that nicotine caused acceleration or •`explosions" of a8erer't dia•harge (Smith, Xavier and (Drossie, 1964); primary afferents were many times more reactive to nicotin,e than the secondary units (Smith and Albuquerque, 19G1); and, while nicotine was equipotent with succim•lcholine ui causing excitation of printar•y muscle-spindle endings, it was much less potent in producing neuromuscular block (Albuquerque and Smith, 1964a). There was no correlation in the, potencies of dimeth}•I- phenyl piperazituum iodide (Dyll'P), succinylcholine, or nicotine, with respect to their excitatory actaons on mur'cle• spindle aBerents and autonomic ganglie, and their deprec:ant actions on extrafusal neuromuscular transmission (Albuquer- que and Smitb, l96U). Albuquerque and Smith (19&1b) presented data based on osciliugraphic recordings from 156 functionall,i• isolated single aiferent fibers from triceps strrar muscle-spindles of cats, the activity of from 4 to 10 single units being monitored simultaneouslr on a single animal. Ticotine in doses as low as 1.25-2.8 pg (administered via cannula in a branch of femoral artery;, just above the knee, all other brenches of the femoral artery except that suppl.-vinR the triceps surae mmle-group being ligated), produrnd a marUed excitation of muscle-spindle stretch receptors; the larger fibers from primary endings showed a more intensive discharge than those from secondary endings. );atrafwsal neuromusculsr block was also ax4exsed by recording the effect of the drugs used on the musc•le twitch to single•±3hock stimuli every 10 ser: nicotine in dosage of 20-320 µtt produced a drgnre of block ranging from about 30 to 75~: . ISee above, 159.) Structure-activity relat.ionships of nicotine and rrlated com•wunds have been extensively-and, to the non-specialist, abstrusely-broestigated, particularly by J.'P. Hamilton and Barlow and Pelikan. According to Hamilton (1962), He,r suggested in 1952 that the peripheral nicodne-like activity of compounds such as choline phenyl ether might be due to the presence in the molecule of a cationic head, separated from a partial positive charge by a i<oitable distance. It is known that in nicotine there are par•tial positive charges at the 2. 9, and 6 positions, and that, at body tetuperature and pH, nicotine exists nxbuly in Qie form of pyrndidinum ion; consequently, Barlow (1960) suqgestexl that the same hylwthcaiq holds for nicotine itself. Resuhs of experiments (see above, S20) peHormed by Hamilton (l9¢i'l) indirated that there is a quantitative relation' between activity and degree of ionisatiun; and these and other resulis lent support to Barlow's hypothesis. Barlow and Hamilton (11162b) studied the effects of pH on the activity of nicotine and nicotine monomethiodide on the mt-dinphratrm ptrparation (see 520), and reµarded their findings as substantiating the hithcrtu a+nmed view that it i.% the unh•alent nicnthlhtm ion, rather tlu,n the un-ionisnl base, which acts tit the neun,• mutxular junction. A further diseu,ccinn of sttveturaaetivit,r Produced bv The Coulcil for 0 0 0 z 970 Tobacco Research-USA, Inc.

, i SKELEI'AL MUSCLE relationships of some isomers and analogues of nicotine on ,D gj.ou u, uYUVN u,1u a.wauiwu (1982a), to which publication the reader must be referned. The effects of pH (7.04 * 0.084 and 8.86 * 0.072) on the activity of nicotine, relative to that of nicotine monomethio- dide, were studied by Hamilton (1963) on the isolated frog- rectus-abdominis prepatation. Nicotine monomethiodide was found to be fully ionized at all pHn, wheness, with uicotine, there was about 7 ttmea more nicotinium ion at the lower pH than at the higher one. Findings on the relative ability of these two compounds to produce contracture of the rectus abdominis muscle at these pHs substantiated the view that It is the univalent nicotinium ion, rather than the un-ionized base, which acts at the neuromuscular junction. Subsequently, Barlow (1965) extensively revieu•ed and analyzed work which has hee.r. done in attempts to correlate chemical structure with nicotane•lilte activity at ganglia and at the neurvmuscu- lar junction. Pelikan, Lipper and Carten (1964) investigated the ap. parent "affinity" and "intrinsic actavity" of nicotine; the authors' summary of their work is as follows: "Isotonic contractions of the uneserinieed frog rectus abdomitdie were produced in vitro by randomized doses of nicotine (N), nornicotine (NN), ttnabasine (AA), choline chloride (C) and aeetylcholine (ACh). Concentration-effect (C.E.) curves for ACh and for the single test agent studied in each expetirnent were fitted to the data by the method of least squa~tes. In- trinsic activity (I.A.) was measured as the as)lttptotic value of the dependent variable in the equation best fitting the dsta, apparent affinity (A.A.), as the reciprocal of the micro- molar concentration which produced a response of 50% of this asymptotic value. Of 53 sets of C.E. data, 45 were fitted by logarithmic equations, only 8 by Clark's rectanRttlar hyper- bola. A. A. of each test agent was computed relative to the A. A. of ACh found for the seme experiment, as were,similar ratios of I.A. and of EDeo (computed from log coucentration. probit curves). The order of mean relative A. A. was: N» AN > NN > ACh = 1.0 > > > C; of inean relative EDao, exactly the reverse;.of mean relative LA.: N > AA' > NN e C > ACh A 1.0. High apparent affinity of N depended on the methyl substituent on the pyrrolidine ring. Intrinsic activity of N did not depend on this substituent." It should be mentioned that, at the conclusion of his phar- maeological investigation of some pyrrolidine N-substituted nomicotine derivative.c, blattila ()00~, b) discussed the re- lationship between their chemical structure and cholinergic action. E\yrernnentS by Alunad and Lewis (1961), who used frog sartorius muscle and radioisotopes of calcium and twta&sium (for details, see below, 236), suppxted to these author* t.hat, in the presence of nicotine, calcium may become more mobile, md is, perhaps, displaced from combination with a earrier or receptor, and that this may mmlt in breakdown of harriers which retain potassium within the cell and sodium outside. As re-phrasel by Ahmad and Lewis (1962), when depolarizing or contracture-producing drups, such as nicotine, are used, calcium ions may become more mobile, being dissociated from combination with a rarricr, or displaced from a binding rite on the cell mefnbmne; this may break down barriers which retain, against the concentration gradients, )mtaRaimn bns within and toodium ions without• the cell. It may be perti- nenl here to refer the reader to the discuRaion of the nmclm- nixm of the nicotir,e effect on toad ventricular nmscle and mdioralcium movement by Nayler (1963), below, 300. 59 228. TAe Erdet Site ojthe NisMine Acfaon on :.+4 11" .ii " \iiw11 Df. Alorita, Yasuhara and Sugimoto (1902) considered that nicotine and nieqtine-like compounds acted on the a.cetylcholine, receptor located on the muscle end-plate or muscle membrane, which had become hypensensitive to acetylcholine in consequence of chronic denervalion; these workers also presumed that the successivedecreaPe of fibriUa- tion voltages would correspond to the fatigue of the excita- bi6ty of the acetylcholine receptor (aee above, 191). Beani and co.workers (1964), using guinea-pig-phrenic-nerve-dia- phragm preparations, found that nicotine did not modify acetylcholine release, which led them to the conclusion that the neuromuscular blocking action of nicotine has only a postjunctional origin. Itiee Beani and Bianchi (19Wa. b, c), below, 52D.] 229. EFFEC'q' OF fV1COTINE ON CHEMISTRY AND DtETABOLISi11 OF MUSCLE (101) 230. Cfycopen (101) 231. Lactfe Aetd (101) (No new data.] 232. tYealine (101) See below, 233. 233. Phosphates; Phosphoros-Containing Compounds (102) Paper chromatograph+y and radiophosphorus (-I'-labeled ortbophnsphate) were used by Fleckenstem, Janke and Davies (1956) to determine the individual turnover rates of the three P atoms of adenosine triphosphate and of creatine phosphate on pairs of frog rectus abdominis muscles. The incorporation of nP into adenosine triphosphate and creatine phosphate was reduced to one-third to one•fourtb of the normal during the contracture resulting from the addition of 1:2500 nico. tine tartrate to the n.dio,active medium. Similar results were obtained using acetylcholine and succinylcholine. The inhibi. tion of the uP-uptake in contractured musdes seemed to be a con.-4quence of the fic.sioq of ereatine phosphate leading to an inctrased phosphate gradient and to a net phosphate eHfux. Therefore, them drugs did not inhibit the ttl' uptake if the extra-cellular orthophosphate concentration .vas itMmsstd up to the intracellular level. In no cam was more uP-lahcled orthophosphate incorporated into adenosine triphoaphate or creatine phosphate during couttactures, as compared to rest- ing controls. 234. Sadiwn Nicotine significantly increasecf sodium uptake by frog sartorius muscle (Almiad and Lewis, 1961. 1962); for details, see belmr, 236. 233. Potoasium (102) Ahmad and Lewis (1961, 1962) were able to demonstrate an increased Iwtassiurrh release from frog sartorius muscle, along with a decreased potassium uptake (see below, 236). 236. Caleium L'sing Isolated sartorius muscle from the frog (Rana lem- horarin), Almnad and Lewis (1961) found that 1 mglml nicotine caused a very i;iNnifieant increase in calcium-07 up- take (P = >0.001), ralc,iam-a7 release, and /xttassium--f2 : o acco Research.vSA, Inc. P Trod"ccd by Tile Cour.cil for o 0 0 2 9-71 b

==I - - =. __..._~._.__ 60 TOBACCO-K7CPERIDfENTAL AND CLIli 1CAL STUDIF.S. StJPP1..EafE.\T 1 release. Nicotine also markedly de.ct+easeq potasoum-ye. ar- tske (P =>0.001), and Eignificdntby increased sodium•24 uptake (P ->0.02). These same results were also obtained with dosages of nicotine from 0.2 to 2 mg/ml (Ahmad and Lewis, 1962). For the possible signiticance of these results with respect to the mechanism of acttdn of nicotine on skeletal muscle, see above, 227. In this connection, it may be meErtioned that Nayler (1963) ddemonstrated that nicotine enbanced both the uptake and release of calcium-45 from toad vedtricular muscle; see below, 300. 237. HISTOPATHOLOGY: HISTOCifEMISTRY 238-A. Esperimeatol 13anprrne R. B. Lewis and 11oen (1953) exposed the depilated hind Id g of male sibino rabbits to a cold alcohol bath at -12° C f~or• 30 min, and compared the exCeat of muscle gangrene in rticotine-ttestsd animals to that of controls; in none of the experiments was there a significantly greater lors of tissue in the nicotine-treated rabbits (for experiatental details, see below, 408). 238-B. Pofoniunt-210 Confent It has been reported that poloniuin-210 concentrations bt penas muscle of smokers were higher than in the cornxpotuiing tissue of non-mtokers (Ferri and Iiaratta, 1U86a; Baratta and Ferri, 1966); but Ferri and I3aae.tta (1968b) stated -pecif- ically that postmor'em samples of pr•oas mmrle from 4 non- smokers and 4 smokers avetttged 0.08 =i: 0.025 micnocuries nOPo pr l0(t gm fresh tissue in each group. jAlso see below, 1t36.) 239. EFFECT OF NICOTIw AND SS1olUNG OV MUSCLE WORK (102) 240. Prop (102) (No new data.J 241. E2at (102) Rats poisoned by carbon monc•Ncitie exhlbited a decrease in ph)'sical e(itrdency (swimming test), end this was intensified by simultaueous ta•r:atment with , pyridine compounds in otherwise notxtoaic doses (Danysz and Dmochowski, 19.R9). 242. Cal (102) Pyridine, picoline, collidine, and carbon monoxide had a similat myolytic effect on cat's muscle (llanyse and Dmo- chow-aki,1959). Nicotine, even in fatal doses, did not suppress the ability of the muscle to perform tetanic contraction. Although single contractions were not suppressed by earbon- monoxide poisoning, the ability to perform tetanic contrac- taon was Impaired. Tobaeco-smoke, from which curlson mon. oxide was removed by a dry hemoglobin filter, largely lost its myolytic properties, from which it was concluded that the myrolytie action of tobacco-strpoke depended tvainly on carbon monoxide and pyridine compounds in the smoke. Hirvonen and Sonnensebein (1981) nsported that nicotine, infected or infused intra-arteriaUy in cats, depressed muscle force Igastrocnemius-soleus muscles), whether muscle blood. flow was incressed, decreased, or utichanged; they considered that the depression was due to neuromuscular block. ]ntra. venoualy.injerted nicotine could enhance muscle force as a result of increa,Red flow secondary to elevation of arterial pressure. n.o FB..r nlR.nnWnn nn Unatle Il Ork in Dfan (103) According to Iiirvonen, Paavilainen and Peltonen (1961a), cigarette-htnoktng definitely reduced the working capw•ity, but, according to HernberK (191i.1), th4re was no siindfx'rout difference between heavy ciltarrtte-umokers and non.smokrr» in their c.iNsacity for phy3ical work. In each experiwmt, the amount of work was measured on a bici cle ergometer; dctails of both investigations sre giveu below, 1183. 244. EFFECT OF NICOTINE ON 1A'VERTEBNA'AE 'NtUSCLE (104) The sections below comprehend Ihe available data on both striated dstd smooth musele of invertebrates; for a discumion u( ti,e effe t of nicotine on invertPbmte cardiac muBcle, see below, 2$6-290, 341, and 379. 24,i. Coelenterate (104) [1\o new dataJ 246. Plalyhelntindt (104) Strips of body-wall musculature of cat tapeworm (Taenio taenfoejormia) trslwnded to nicotine with a slow tonus de- creasE, which reached ity mammum in 2-4 min (1'eaAmten and Yar¢iainen, 1958). The threshold of response Iqr belween concentrations of 1 and 10 pg/mi, and was usually about 5 times that for "denen•ated" preparatione. Pretreatment with bexamethouium did not antagonize nicotine action. 247. ,Yemniketmialls (104) (No nkw data.) 248. Annelid (104) Scrotqnnt (5•hydroeyt*-ptamine) reduced the contraction of the dorsal musele of the leech produced by nirntiue, and accelerated the relaxation of the muscle when Ihe nicotine was washed out (Schwain, 1961). 249. Vollusk (105) The interior retractor muscle of the byssus of .11yti(ua edulia (edible mussel) t"sonded to nicotine by a contraction (Cambridge, 1961). On thix same preparation, nicotine in roncentratioas below 10'' .1l potentiatcd the action of acetyl- choline; above 10-' 11, nicotine evoked depolariaation and prolonged contraction (TwatoK, 1959). Tachyphylaxis de- veloped with repeated alsplicationa of, or continued "king in, nicotine, and the aceq•lcholine-lit:e activity reanvl. Loss of activity was associated with an aceeylcholine.blor)dng action. According to 1. Singh (1963), barium produceo a typical pennattent tonic contraction of the anterior bymus tettactor of Uytiltts edsdie, which is not produced by any other sub• stanrn, and hence is easy to recognize; the cuntraction is usuall.y immediate, but sometimes occurs only after a knrg latent period (suclt as an hour), .h hich augResta that the mere presence of barium ions in the interspaces is not audcient to produce oontraction. \1'hen the muscle is lying quiescent in barium solution, then )>amge of an electric current, sudden stretch, removal of calcium, or addition of neetylcholine or cyanide, produces a htiiical barium contraction. Rince these agents are know n to delwlarise muscle fibers and incrgs.`c their pemieability, the author r•ugtte,,ati) that barium, in order to ptm(uce escitation, entera an mia•r compartment of the muscle fi!sera, and that the rect-ptorz, lar the action of Jrugs Produced by The Ccuncil for 0002972 ToL4cj Pjescaj,ch-GSA, inc.

BKELETAL MUSCLE 61 and ions Are not sitoated on the cell surface, but sre intrs- (Holotheaia) hsve signs of both aicotinic and muscarinir roq.do. lNb.... ...i.».d...: .. ~,..1.. r.~.t..~:.. .`:....~ . .t._ . :.... ~. .:u:.ri:.ww n,,, ap~u nhich excite the muscle were said to act like barium. y to~be highly sensiiive~to nicotine. 250. Fwdinodena (lUS) 251, Crnetatem+ (103) According to Magazinik and Fntentov (1983), the eholino- 252. lnsed (10S) receptore df smooth locomotive museles of some invertebretes (No new data.) I Produced by The Council fft o 2 9 7 ~ Tobacco research-USA, Inc.

BLOOD 253'. liRT77IROCYTES (106) 254. Eryfhrocytes: An(mnle (106) Nicol and Cordingley (1964) found no significant, changes in tbe blood picture of mice receiving nicotine by stomach- tube ot subcutaneous injection in such dosage that the animals appeared to be unaffected by the drug (for a fuller account of this study, see below, 268. Phagoeytes). According to the English summary available, Petrova (1963) found that exposure of rats to methyl alcohol and nicotifie vapors (said to simulate the natural working environ- ment in tobacco-fermentation plants) revealed a tetidency of the red blood count towards hyperchromic anemitd; experi- ments pAtb nicotine alone were also performed, but the results mete not reported in English, cnd the coinbined treatment was said to have presented the more severe clinical picture. 255. EBect of Smoking on the Erythrocytes (106) 256. Sedimenletfon Rale (107) (No new data.l 257. Red-Cdl Fragility (107) According to T. Sato, Suzuki and 1•likqymma (1962), the pater-inroluble but acstone-soluble fraction of cigarette- smoke condensate hemolyzed red blood cells suapended in saline; this activity , decreased by one-third in 24 hours at room temperatnte, and was susceptib:e to aeration, but not to SH neagents. 258. Red-CPll Penreabiffly to Nicotine (107 ) Schievelbein and $chirren (1964) made intravenous bijec- tion into rabbits at a rate of 0.66 mg/kg os; nicotine free base per minute, and drew blood immediately following respiratory arrest; the blood wa's then fractionated for analyr,es of nico- tine cantent, The sutn of the nicotine contents in eryth:roc;hte.c. leukoeytes, platelets; and plasma amounted to about 10% of the injected dose. 259. Cytoehernistry Eryihrocyte free-fatty-acid reslwnses to cigarette-smoking and glueose-loading were determined by Soloff, $chn•arta and Baldwin (1903) in healthy and diabetic subjects; apparently, free-fatty-acid patterns rertained unaffected (for a fuller account of this study, see below, 696-8. Fat Aleta6oliani). 260. Remogfobfn (107b) H. Blackburn and associates (1960) reported that hemo- globin tended to be higher in smokers, but not significantly Fo. Cardus, Luft and Beck (1983), who r.tieasured total circulat- F2 ing hen.oglobin by n6eans qf the carbon-monoxide rebreathinR nlethod in ?V men And 21 women who had refrained from smoking for at least 12 hours, found the mean total hemo- globin in male smokers to jie SV.9 gin compared to i 64.0 gm in male non-smokers (p Q<0.1). In female tunokera, how- ever, the value au 498.9 compared to 510.3 gin in female non-5mokers (p =<0.5). The esperimental error was said to be considerably greater in smokers (+l.l cio) than in non- smokers (2.JcJo); in any event, the data given do not prrmit of calculations on a body-weight basis. liristoflersen (1964) metisured alkali-resistant hemoglobin in the blood of 100 non-pregnant women and in thatt of 377 pregnant and parturient patients. Since an increased content of carbon monoxide in the blood (caused, for example, by smoking) can give rise to d slight increase in concentration or alkali•reQistant hemoglobin', the author analyr.ed the majority of the patient material with rcry*ct to smoking habits, and found values for smokers to be mignificantly higher than thoe for non-smokrrs in the third and fifth periods (in both groups, the values were significanth lower in the fifth period than in the third). Differences in alkali-mutant hemoglobin between smokers and non-smokers were not correlated with the birth results (abortion; pnernatuhe birth; stillbirth). Through study of oxygen diasaciation curves, Astrup (1904) fcund that, compared to normal subject-, the blood of pa- tients with tbromboangiitir obliterans (TA()) possessed a,n abnornaliy high afCinity for oxygen. Affinity of hemoglobin for oxygen increases when tree SH-groups are blocked; and the author cited experimental drork supporting the theory that the SH/SS equilibrium in blood from TAO patients is easily pushed 'ui the direction of 88-formation, which happens dur- ing tobacco-smoking, ahen the oxygen affinity considerably increa.~es, the enhanced oxygen affinity disappearing when the patient stops smoking. (In normal rubjects, oxygen aitfinity is uninfluenced by smoking.) Further speculation as to a mec)t- anism for the increaed affinity of hemoglobin for oxygen in TAO patients smoking tobiacco may be found in The Lancet (R: 11(i4, 1964) in an Annotation on .Ustr.tp's article. 261. C¢rboryfiemrogfobirt (107) The average carbon-mqnoxide content of the tnnuke fnm, 15 brands of cigarettes tested by \lumporrer, Lewis and Touey (1982a., b) ranged from 3.0 to 5.0 per cent. Practically no retention of t^0 occurred after putling cigarette-saioke, while on atnoking with inhalation, there was about 95 v retention (Bokhoven and Niessen, 1961). For a given consumption of tobacco, blood ('O content v:-as rela- tively higher in subjects who inhaled the smoke (Parent, 1902). &hf+crher (1984) has sugge.etcd the depth of inhalation, and thus (according to him) the resulting individual danger of developing cancer, can be evaluatcd by determining the car- bo.<tihemoglobin (IibCY1) of the blood. Produced bf The Council for 0002974 Tobacco Cescarclr-USA, Inc.

I. . Acrnrding to Chevalier, Krumholz and Ross (1963a), in- hxlnt iou of 0.5% CO for 2.5-3.5 min resulted in s Hb(Y) leve) eqptivalent to that obtained iu a group of smokers. In a group of 68 men employed at a vehicle-inspection moter in a large U. S, mid-western city, the mean HbCO level of 54 smokers was 3.8% and of 14 non-smokers, 3.4170; in mmparison, the mean HbCO level of 19 smokers employed at a t-nited States Publyc Health Service (IJSPHS) field- ,tation waq 2.9% and of 6 non-smokers similarly employed, (Hofreuter, Catcott, and Xintaras, 1962). When the rigarette-smokers were divided into two groups on the basis of the total of cigarettes smoked during the day prior to blood- ,otdtpling, the mean HbCO level of smokers of 10 or more riaarettes wa, 4.3%, compared to a mean level of 3.5% for ,otd,kers of le,s than 10 cigarettes in the vehicie-inspection- cutrr emitloyees; in the IiSPHS field-station employees, the mcan HbCO levels of smokers of 10 or more cigarettes was 5.2';, while the mean for smokers of less than 10 was 2.4%. Desoille, Truffert, Lebbe and Parent (1962) also found the CO coment in the blood of smokers to be a function of the number of cigarettes smoked per d5y; the mean CO content reached 2 tnl/100 ml blood in smokers of 40 cigarettes per day. The average CO content of blood drawn in the morning was found to be higher for smokers than for non-smokers (1.1 % vs 0.2~0); blood drawn from smokers in the evening showed a CO of content 1.7-1.9% (Parent, 1962). Ringold and w-wrorkers (1962) oonfirmed the fact that rqtarette-smoking was a major factor in determining the HbCO level in a population; pipe- and cigar-snokers, on the eonirary, showed l•• tie increase in HbCO. Acrording to Parent (1962) also, blnxod CO content was higher in cigaretWstnokers than in pipe- or cigar-smokers. langmann (1964) drew attention to increases in the CO con- tent of hemoglobin--already 8 io among cigarette-smokers- in urban surroundings. Several recent studies have been concerned with CO levels in the blood of smoking and non,smoking motLers and their Wants. Haddon, Nesbitt and Garcia (1961) found the CO cm,rentrations in venour blood of pregnant smokers to be statistitmlly significantly higher than in the blood of non- smokers; this series comprised 50 wo-uen seen consecutively in a pre-natal clinic. In aa additional series of 26 cases, CO intirentration in paired unibilical•vein and maternal blood- r<f,eranens obtained at term were found to be approximately equal, whereas CO concentrations per 100 gm of hrmoglobin arre lower in cord than in paired maternal specimens. Differ- entr.t between smokers and non-smokers in the reduced oxy- Rrn-carrying capacity In the umbilical-cord and maternal blaK;, respeetively, were airo statistically sigaificant. Heron (1962) too found CO blood concentration to be higher in toaternal and fetal bloods On patients who smoked. Young and Pugh (1963) determined the C0 content of fetal and msternal blood from 19 full-term parturient women (16 of rdnm had normal delivertes); 0 of the patientR were said to lutr rmoked 10-20 cigarettes per day, but there nais no tdiable information about inhaling. At normal deliveries, the ('0 trmot ent of umbilicsl-vein blood was found to be 0.52 vol %, in infants of mothers who sntoked, and 0.36 vol % in those of nemmtoking mothers, compared to 0.33 and 0.28 vol %, re,lrrtively, in the maternal venous blood. The non,signifi- tgnt difference between the blood CO content of mothers who mtl-ked and those who did not wae thought to be probably telated to the tiwe which had elapsed between the last ritarette and the collection of blood-sample. According to Msnteii (1964), smoking and its associated higher HbCb - --U : --------. B1.00U 03 levels in the mother and the fetus, leads to a depression of rarhonir-anhvrirx,-r nrfieitv in tho nn.rt hlnni- r,- rt... ;..e portaure of thi.; depm-ion was said to be unknown. With respect to precautions which must be taken to lessen any error resulting from relatively high inrtial values of HbCO in smokers (199b), Baten, Christie and Van•is (1960), who were inlerested in determining pulmonary capillary blood-volume and membrane-diffusion component by meas- urement of lung-diffusing capacity for CO at different alveolar oxygen tensions, found, in the case of heavy smokers with a mJatively high concentration of CO it• the blood before the experiment was begun, that la:ge random variations in calcu- lated pulmonary capillary blood-volume may occur. It would be interesting to know whether a preliminary period of oxy gen breathing, as described by Dahlstrdm and co-workers (1958) (109b), would allow the tests described by Bates and associates to be used even on heavy smokers, as was found to be the cate with SjBstrand's (1948) method for the estimation of total hemoglobia (109U). A critical r6sumE of chronic CO poisoning has been pub- lished by Pfrender (1962). This subject, with special reference to tobacco-smoking, has also been reviewed by Van Prooaxlij (1960, pp. 22-26 (24 references)). 262. Blood Groups B. H. Cohen and Thomas (1962) have reported a study involving 1,005 white males and 393 Negro males, in which the distribu:.ion of the ABO and Rh blood-groups were classi- fied according to smoking habits. Among the wbite males, there appeared to be a signifirant deficiency of group-B individuals among heavy cigarette-smokers, and an excess of gs+oup.B persons and of Rh-negative individualc among occa- sional smokers, and an excess of group-B persons among non- smokers; but among Negro maies, significant differences in blood-groups were not found in any of the smoking compari- sons. The British l/edunl Journal (7: 932, 1982) was critical of this relwrt; and at least two groups of arorkers have pub- lished conflicting findings. Iu a random sampleof over l,00D miners and ex-miners, aged 35-64 yes,ry in the mining valley of Rhondda Fach in South 11'ales,l. T. T. Higgins and asso- ciates (1982, 1963/ found that neither ABO nor Rh blood- groups (or secretory status) was significanWy related to sm,oking habits. An inquiry into the smoking habits of 6,038 male and 2,348 female blwd-donors by Bourke and 0'Riordan (1964) failed to reveal any associations between the type of smoking habit, or the amount of pipe- or cigarette-tobacco consumed, and a particular ABO or Rh blood-group; the ABO blood-group distribut ion of these blood-donors did not differ significantly from the known distribution in Eire. Higgins and co-nvrkers (1962) suggested that the findings of Cohen and Tbomae (1902) might have been due to chance. (A probability of 1:1,000 should be requited before an ap- parent association between blood-groups and dir.ease (or smoking) is accepted (Fraer Roberta, Proc. Roy. Soc. Med. 48: 143, 1955).) The :1110 rewlts of Higgins and colleagues were said to be, neverthclcss, in the same direction as those reported by Cohen and Thomaq; and the former workers (Higgins et al., 1963) felt that further studies were needed to IncreaFe the accuracy of the iwoled estimate, which, at ptes- ent, suggests that the ,vnall number of men who have never smoked Is half ac large at,tuin in group-B as in other grnups. The Brt7ish llcziiml Journal (lur. cit.) also suggested that further research is needed before the association between cigarette-smoking and tduod-groups can be regarded as fum. Produced bv rho Council for Tobacco R(;SeaICh -U$A, Inc. 0002975

, 6-1 TOBACCO-EXPkR1.%IB%TAL ANA CLINICAL rTtil)lE,>. SUPPLEMEtiT 1 I 263. R.EUCOCti-rtss (110) Z61. Total and Drfferentfal Leucoeylr Counl in Animala (I10) In the study of Nicol and Cordingley (1964) already re- ferred to, doses of nicotine which appeared to have no effect on mice also failed to produce significant changes in the blood picturc. In rats actually poisoned with nicotine, Veress t1902) reported that the propcrtion of monocytes increirsed very rapidly. A comparison of such animals with nicotine-poisoned thymectomized rats indicated that the appearance of mono- nuclear cells in the latter group was delayed, and their num- ber decreased by 50%. In rata chronically exposed to nicotine and methyl-alooho) vapo¢s, the leucocyte count tended towards slight leucopenia, with neutrophilia etid a shift to the left (Petrr,va, 1953). According to lnitchkov and co vrorkcre (1960), intravenoun injection of 0.02-0.04 mg/kg nicotine into decerebrate cats caused an appreciable decrease in peripheral-blood eosl •)phils, not observed in animals with denervated carotid sinums. In one rabbit exposed repeatedly to tobacco-smoke, the blood picture was said to have been characterized by a leuko- penis and granulocytopenia, and increases in eosinophils and basophils (Minuth, 1959). 265. Total and D{Berentiol Leucocyte Count in Uan (I11) A case of eosinopbilia, apparently related to cigarette- smoking, has been reported by Sehoen and Pizer (19fr1); the patient was a 35-year-old female 7ho smoked 2-2.5 packs a day, with blood eosinophil counts up to 1700 per cmm. On two itrdependentt occasions, the patient had a prompt decrease in blood e.osinopbils to "normal levels" of 150-400 per cmm a ithin 5 days du.~ing periods of no smoking; this normal level persisted during the 2-week period of no smoking; and when smoking was resumed, the eor•inophilia returned after 3 weeks. The authors suggested that this phenomenon might have reptesented some aotigen-antibody (allergic) response to cigarette- or tobaceo-smoking or to a constituent of either (for eosinopbilia as a sign of allergic disease, see (fi47b))- 266. Cytochemistry Nicotino was found by Schieve)bein and 11°erle (1962) to relense h.stamine from mast-cells of rabbit f`;ood ;for a fuller account of these experiments, see beioa, 272. Cytoehemistry of TArontbocytes). Calder, Curtis and Fore (1963) detetmined ascorbic-acid levels in leucocytes (including thrombocytes) of 91 non-smok- ers, 83 smokers of 14 cigarettes or less daily, and 31 smokers of 15 or more daily; they reported finding values of, respectively, 29.1 :k 0.85 (S.E.), 24.8 * 0.84, and 21.8 * 1.50/+g per 10' cells. Plasma ascorbic acid alsp decreased progressively with the degree of smoking. 267. .111fotilily of l.eucocytes (112) (No new dataj 268. Plwgocytosia (112) In tests on 96 mice and 24 oontrols, Nicol and Cordingley (1964) foun9 that nicotine had little or no effect on the phagocytic activity of the reticulo-endothelial system, as measured by the carbon-clearing index. The authorr noted that the animals (and their blood picture) appeared to be unaffected by the drug, which was given by stomach-tube or subcutaneousinjections 269. sttnounocn•Es (112t 270. TAromboryte Count (112) Smoking has been variously nsborded to increase or de• crease the thrombocyte count, or to be without effect on il. Grassi and Caltabiano (1956) were said to have fuuad an increaeed platelet count after the smoking of I cigarette (Blackburn et al., 1959). Ambtus and Mink (1964) aho rr ported finding an increased platelet count after the smukng of I cigarette; and, while the increase was not FtatWically .ig nificant, the authors considered it at least compatible w-itI, thr increa.red blood .vagulability observed (see below, 27,i Coogelalion of Blood). On the other hand, \1ik.ic (19GI), testing 50 smokers, found that the smoking of 2 nue-filter cigamTte~ for 12 min rncultwl in a thromlxuvte derrca.w uf 100,0i10-150,000 per cmm; and Niltt:n (1959), in studte= on 2 smokers and 2 non-srnokers, reported d that smoking I cigarillo was without effect on thrombocytest however, in a patient with Rayitaud's disease, smoking I cigariUo wac followed by depression of thrombocytes in the peripheral blood. 271. EjJecla of Samding on Propertie8 of T)irornbocytca (I12s Mustard and 211urphy (1963) studied the effect of rmokinq on platelet survival in 7 phite male subjects 8tnder metabolic- ward conditions during two periods, in one of which they were allowed to smoke and in the other in which they were not. Platelet survival was found to be significantly shorter, and platelet tumover correspondingly greater, ahen the sub• jects were smoking than during non-smoking period}. Platelet clumping-time was prolonged by 20 % when the subjects ceased smoking; this difference was just Fignificant at the 5' ~ level, but the autbomconceded the pornibility that this might have been an artifaet arising as a*esult of the multiplicity of the comparisons. IThis psirer was the subje:,t of comment in the British .llodioal Journal (1: 833-t)34, 1963).1 In the study by'lustard and .1lurphy (1903) dercribtrl above, no significant differences in p)atelet adhesive index were found between periods of smoking and non-smoking. However, Ambrus and Mink (1964), who studied the effect of cigarette-smoking on blood coagulation ?n non,smokets, found a statistically non-SigniScaNt hxreaa: in the average blood-platelet counc, but a significant (0.05 < p< 0.10) in- crease in platelet adhe}iveness averaging +ti1.4 f 41.We of the hase-line (range, -13 to +300). 272. Cyfoeltemiatry The subject of terotonin (5-bj-droxylryptamine; SHT) storage and release by blood plateleta under the in8uence of nicotine has been extensively investigated by Schievelbcin and his colleagues. At pH 7.3, incubation of preparations of rabbit blood-platelets (obtained by differential centrifugation) with increasbtg concentrations of nieotine (0.05-3.0 tttg rncu- tine per 3 ml platelet su-henMnn,l, re•nltMl jn the relenrr of increasing amounts of serotonin; about 58',0 of the serotunin in the platelets was released by the highest concentration of nicotine (Suhievelbein and t4crle, 19t12m). 11ith inctca ing nicotine concentration, the molar ratio of nicotine to lib- ereted serotonin decreased, from ti87:1 aitlt 0.5 mg added nicotine to 295:1 tvith 3 mg nimtitte. Helease of eerotonin by nicotine was found to be p1il-dependent: at pH 7.0 and below, no release occurred; with increasing pH up to 7.9, increasing release was observed, but an opthrnum w-a,s not established, owing to an effect of pH per ac o:t tlte alkaline aide amountinR to about 20% of the verotonin released by nicotine. Semtunin f t Produc, rl bv Thn Council for 0002976 = Tobacco liesearcl,-USA, Inc~ ~

I relea.red by nicotine increaced with increasing incubation- eimp. Of several enwvrne inhihitnra fewwi nnlv 111M11 Nf -adium aside caused a aGght (25%) inhibition of r-erotonin release by nicotine; sodium Buoride, trilon B(the sodium salt of ethylenediamine•tetra-acetic acid), 2,4-dinitrophenol, and \larFiGd (isopropylisoniootinic acid hydraeid) were w•;thout effect. Schievelbem (1965a) cans-dered that these and later investigations showed that nicotine probably increases the Irr:ueabilit}• of the platelet membrane for 5-HT. \ieotine was also found to have an inhibiting effect on the uptake of eerotonin added to suspensions of platelets (Schievelbein and Werle, 1962a). Nicotine released histamine from blood plate- lets as well as from mast-cells; however, the nicotine concen• tration required was about 10 times greater than for com- parable release of terotonin. Like ni,wtine, lubeline liberated serotonin from rabbit blood-platelets, and was found to be twice as pottnt as nioo- tine in equimolar concentrations (Schievelbein and Werle, 1962b). Release of serotonin by nicotine could be otserved at 20°C; was scarcely measurably increased at 37°; and was only rapidly acceletated above 40'. In contrast, lobeline failed to release serotonin at 20"C; began to do so at 25"; and increased release proceeded rectifinearly to 40"C. Whereas nicotine produced no t;etotonin release at pH 7 and below•, lobeline released 36% of the maximum amount at pH 6.5, and in- creasing release occurred with increasing pH up to a maximum at 7.5. As with nicotine, release by lobeline increased linearly with increasing incubatron-time, up to a maRirnum of 85% in 120 min. In combination, the release of serotonin equaled the sum of the individual ac•°[ons of the two alkaloids. Following intravenous administration of nicotine or lobeline to anesthe- tized dogs, seratonin was liberated from body-stores, with eonseqnent t.emporary increase of free serotonin in the plasma and storage of the amine in blood platelets, followed by deple- tion of the serotonin in the platelets by the circulating alkaloid (Schievelhein, Werle and Eekert, 1968). Oral administration of nicotine had the same effect as intravenous injection; but oral administration of lobeline led only to serotonin stormge in the blood platclcts (this rerotonin was presumably lib• erated from the gastrointestinal tract). The toxicity of intra- venously injected nioot ine was found to be decreased when the number of thromboc) ies was augmented by administration of platelet stuqrensions, possibly due to storage in part of nicotine-released biogenic amines in the platelets (Schievel- bein and Srhirren, 1964). In a recent symposium, Schievel- bein (1965a) summarized and integrated his previourh• pub- lighed studies on the release of terotonin by nicotine. Regarding the possibility that nicotine may release epineph- rine from blood-platelet stoM st!e Singh antt Oester (1964a), beloa, 274. Coogtrfaleon of Blood It should be recalled at this point that, in their survey of asmrbic-acid levels in leueocytes of smokers and non-smokers, Calder, Curtis and Fore (1963) noted that the values they reported for leucocyte,c included blood platelets a. urll. 273. coeanzv-rrop or guoon (112) 274. Erperimeada in Vitro (112) In a series of paperF, J. Shtgh and Oester (1961; 1962; 19W b, c) investigat.ed the effect of nicotine on the coagulation of human blood in vttro under a variety of experimental condi- tions. In ooncentr:-t.ions of I X 10-' M. I x 10'1 .11, 1 X 10`' ,l/, and 1 X 10-+ 11, nicotine lowered the coagulation• time of human blood in vitro (Singl- and Oester, 1961). Nico- tine in concentration of 10-' J/ shortened the coagulation time hP 2747. while rnmrntrntinn° nf 111-4 1l1-+ 1!t-° nn,l 10'7 11 r<hoatened it, mpectively, by -13.4, -7.9. -6.3. and -8.0% (S ngh, and Oe.aer, 1984a). IIt shuuld be particularly noted that cobcentrations of nicotine producing effects in these studie.h were tar above '4mtoldng" do,agec; thui.k, 10'r V nicotine equals 162 mg per liter, which is about 162 tinies the estimated lethal concentration for tnan.) In other experiments, maximum shortening of 18.8i•o was said to occur with con- centrations of 16.2 µg nico+ine per ml of blood, higher (162 pg/ml) concentrations resulted in less shortening as did smaller concentration.s than the maximal (1.62 and 0.162 pg/ml terulterir in 8.0 and 2.4;'o shortening, re.aiectively) (Singh and Oei;ter, 1964b). Coagulation-time of plasma was shortened by 22.9% by l0'' 31 nicotine, and by 11.3: 4.8, and 2.5% by 10-1, ju-b, and lU'° .11 niootine, respectively 15ingh and Oester, 196Aa). In dosage of 0.1 ml of 10° if, 10'' 31, and 10-1.11, nicotine retarded the prothrombin-time of human plasma in vitro, while 0.1 nil of 10-' 11 and 10' 1 .11 accelerated it (Singh and Oester, 1962). Coagulation•time of plasma was shortened by 22.9% by 10°' -11 nicotine, and by 11.3, 4.8, and 2.5% by 10-4,10'6, and 10'6 11 nicotone, re.qpectively; in concentration of 10-311, nicotine reduced prothrombin time by 7.7% (Singh and OeRter, 1964a). Nicotine in concentration of 10°.11, 10'' J1, and l0'= 1! prolonged luothroinbin time; concentrations of 10'3 J1 slightly accelerated it; concentra- tions of 1", 10-6, and 10r-' 31 had no effect (Singh and Oe-cter, 1964c). In concentrations of 10-4, 10-6, and 10-', epinephrine and norepinephrine decreased coagulation-time of human blood in vitro (Singh and Oester, 1961). Pipe-oxan in concentration of 1:5,000 completely blocked the effect of 10'11epbtephrineon whole-blood coagulrtion•time,but had no influence on the norepinephru-e effect, and only partially blocked theeffectof 10`' .11 nicotine (Singh and Oester 1964a, b). Epinephrine and norepinephrine in contrntmtionR exert- ing maximal effect on normal blood (10 al:d 0.1 µg, respec- tively), exerted practically no effect on blood containing heparin (Siagh And Oester, 1969b). Heparin, in concentrations of 0.001 mg/ml and 0.005 mg/ml, prolonged coagulation-time of whole blood,(Singh and Oester, 1961); however, nicotine shortened the clotting time of blood containing this amount of heparin (Sin$h and Oessrr, 1964b). Heparin in oonrentra• tion of 0.0001 mgl0.5 ml pla.bna retarded prothro-nbin time; and this retardii-g effect was uariially rever.•ed by 11T'. 10-'7, and 10'' 31 nicotine; nicotine in concentration of 10-1 if also partially reversed the retardingeffect of beparin (0.001 mg/0.5 ml) on prothroinbin time, but the other concentrations of niaotine were devoid of this effect (Singh and Oester, 1962). In comparisons of t-eparate rersus combined actio-is of heparin and nicotine on prothrombin time of human plasma in vitro, there was a suggestion that certain concentratiorK of nicotine exerted an antagonistic action to certain concentrations of beparin (Singh and Oester, 1964c). Results of ntudie.j on the effect of nicotine on pla.ma mcalci6catinn time were inter• preted by Singh and Oestrr (1964c) as being similar to the effect of nicotine on prothrombin time; no correlation was found between the effects of nicotine on plaaua recalcification time and pmthrombin time, and total lipidxora/d-lilxopmtein ratio. With respect to the mechanism through which nicotine exerts its influence on coagulation in vitro, the above experi- ment+. using antagonistic drugy led Singh and Oester (1961) to infer that the nicotine action did not appear to be exercised through the relcase of epinephrine and norepinephrine, and that the nicotine effect did not appear to be dependent upon the presence of catecholamines (Singh and tkster, 1961b); Produced by The Ce~.incil foro 0 uZ 9~ 7 Tobacco Research-USA, Inc.

i l 66 TOBACCO-Ey:PE1tI1fE1TAL AR1) CLA\1CaL FTUI)18.S. C+UPPLE1fEXT 1 but the authors did raise the possibility that nicotine may I • .L.....~.u...... .. .. .rw ,......... ......_ ,w...t,.. ...... Oester. 1964a). It appeared to these authors that nimtinc shortens blood coagulation•time by inactivating or neutraliz- ing or antagonizing the anti-coagulant effects of heparin or heparin•like compounds (Singh and Oester, 1961, 196fb); at least in certain concentrations (Singh and Oesler, 196•lc), aud that this effect does not appear to be dependent upon the presence'of cstecholamiues (Singh and Oester, 1961, 1964b). H. J. P',•hite, Gore and Larkey (1903) confirmed the findings of Singh and Oester (1962) that smaH arnounts of nicotine acceler9ted, while large amnunts of the drug prolonged, prothrorobin time; and the acceleration effect w-aa reversed by heparin. Nicotine (10-20 pg in fibrinogcn) accelerated thrombin time, and thi- effect w'ag reverRed by Lrior inruha• tion of nicotine with heparin. Thrombhr, however, when incubated with 20 pg nicotine, gave prolonged clotting. These authors eaggested that a direct antagonism exists between nicotine and heparin, and that nicotine may react with other clotting fnctors, possibly in relation to their respeet ive charges. In furthet studies of the effect of heparin and nicotine on the convelsiom of prothrombin, Alarin and White (1964) found that heparin depressed the formation of plasma thromboplw~- tin, prob'ably by inhibiting PTC (plasma thromboplastin component; Christmag factor). The serum of patients and animals, treated with high doses of heparin, contains, in addition to large amounts of prot.hrombin, an agent which accekrates conversion of protbrombin to thrombin in the presence of tissue thromboplastin; this agent is related to accelerators of the stable-factor group. (This pher.omenon is similar to that described by Quick in thrombocytopenic or hemophilic blood.) Nicotine inhibited the anti-coagulant effect of lteparin, and, 'vi addition, was found to accelerate the prothrom6in time of factor VIlI-deficient 1)lawna in a quanti- tative relationship, which wa.c not the case when nicotine was added to factor A1I- or factor IX-deficient pla.vna. 2?S- F:.rperimenfs in Viro (112) Singh and n'ensel lretiorted by Singh and t).,.-ster (1961)) found that nicotine lowered coagulation-time in vitro, with concomitant release of epinephrine. However, In vitro eqperi- mentc by Singh and Oester (1961, 1964b), described above, suggested to the latter workerr that the effect of nicotine on coagulation-time drlec not necessarily or exclusively involve the release of epinephrine. On the other hand, D. G. A'enzel and Singh (1962) reported experunents, the results of which were what might be anticipated, if nicotine exerted its uction through catecholamine release. As measured by the Lee-n'bite method, intravenous administration of 0.01 or 0.02 mg/kg nicotine to rabbits effected significant reductions in the time for blood to coagulate. Epinephrine induced a biphasic re- Fponse, w•ith signi6cant reductions resulting ftmn small (0.01 and 0.02 mg/kg) do:rs and an increase from a larger (0.08 nrg/kg) dose, while intermediate t0.04 and 9.06 mg/kg) doses did not significantly affect coagulation•tin•re. The combined treatment with 0.01 mg/kg nicotine and 0.04 or 0.06 mg/kg epinephrine increased coagulation-time over that of the r.maller doses of each drug alone. Also, administration of 0.25 mg/kg piperoxan 10 min before 0.01 mg/kg of either nicotine or epinephrine blocked the increased rate of coagulation. 11'eauel and co-workers found, in rabbits, that nicotine administration and a hy7iercholesterolemic diet, individually and in combination, caused decrea.ces in the coagulation-time as well aa in systolic blood pressure ard thermal circulation index (Il'enzel, Kamel and Turner, 1960; Wenzel et al., 1961). Coagulation-time was also progressively decreased in rabbit• ninnrrnn rr •Ln.)nnl•i mnr) nt.ntnMnn.l:. •A: diet, ldus injection of ergonovinenmaleate into the marginal er<r•vcbl at 4-week intervals- (11'enzel et'al., 1961). ln further experiments ou rnbbit% divided intc groups receiving for 24 d•eelu either nicotine, a hyperchole¢terolemic diet (H('U), nicotine plus HCD; caffeine, caffeine plus nicutinc, caffcine lilu.+ HCD, caffebie plus HCI ) plus nicotine; ouabaiu, ouatwin plus nicotine, ouabain plus HCD, oua6in plu.+ NCI) plu> nicotine, coagulation-times were consistenth• reiluced by all thratn)ents, although only nicotine or the hy~xrchele~tero• lemir diet-with or without caffeine or ouabain--cau.nl a significast (p < 0.05) lowering (Wenzel, \IacCaraM• and Idutledge, 1964). H. ltlsrkhnrn, Jr. and rro-workerx (1959) found no sievoifi• cant acute effect uf cigarette .-unoking upon the blood coagula• tion, as meawred by reealcified plasrna "Styren" time. Twn sets of control studies were made, one with 16 adult rrhizo• phrenic men in a metabolic w•ard, the other with 8 young university students; all were habitual smokers who had refrained from eating or srnoking overnight. Half of the Rub• jects on each experimental day chain .sunoked 2 standani• btand, regular-length, non-fJter cigarettes; those who were smokers on the first day became oontrols on the second, in a cyu.-vver-contmlled procedure. The authors stated that Gra~i and Caltabiatto (1956) had reported shorteuina of recakified plamm tir.ie, augmentation of prothrombin activity aptd utilization, shortened clot retraction time, slal increased platelet count, after the emiokint; of I cigarette. A nornrsl Weed'sig and clotting time was found by Alikaic (1961) in 50 tirnukens smokiag 2 non-61ter cigarettes over a period of 12 nfin. Likiewica (1064) studied the effect of cigarette-amokuyt on the coagulatiou-time of blood and recalcified plasma. and on the prothrombut and thronrbin time of pla.cma, in 10 healthy rigarette-wnokers (6 men, 4 women), aged 20-27 years; the subjects were fa,ting, aud control experimenlr were carried out al the same individuaLq on different days without srhuking. Smoking of 3 cigarette, in 30 minutes, or 10 cigarettes in 2.5 hours, mwlted in reduction of blood coaaulatron-time; the .hnriening was said to have been mote marked in the latta:r smoking procedure, sometima- below the phc.ioloµie limits. The thmmboelastograph Is an apparatus which detcnninrK 4ratlhnalh• the time at which a blood-rlot starts to tnnn, the rate at which it fonn+ nnce it haa started, and its 4+m- tlnned (e14sile utreil)tth and PltWtlrit\•. H\' use of this dM'nY'. Humitz nnd 11'aktorf (1960) mcvrdrd thromboela-to- gram:s on 10 nnrh:al fasting subjects (b men, 5 uYnncn), before and after the smoking of I standa)d-lenatb non-filter cigarette. There was uo change in the time in which the clot r•tartiM tu form; there was, however, an increase in the te+lrilc StrMl[!th of the clot, as well as in the rate of clot formation. and invariably an increase in the MA/k value, which averaged (ML1, mra:uivd in tuill'uuetenf, varic~ dit.•ctly as Ihe Rensile strength of the clot; k, measured in minutes, varies inr•erfely as the tate of clot formation.) From these re.wlts, the authors concluded that tobacco•smoking does not in8u- once the time at which a clot startc to form, but does hrcrea-e the tensde su•engtl,i and the rate of formation of blood-chot<. tJit the other hand, unequivocal effects of cigarette-smoking orti the thromboelastogram, or on prothrombin-time, recalcifi- cation time, and enti-thrombin activity, were riot oh.rervcd by Gibelli and co-workers (1964), w•ho tested 6 young normal subjects before and after a fatty meal, with or without WnnkiuR cigarettes (18 ciqarettes at an averagr frequency of Produced I),! The Council for 0002978 Tobacco research-USr'1, Inc.

1i1AOb 6e 3 per hour). It was noted that bleeding time decreased more marpeuiy wbeu wuuawy, N•ns xuperunpusYw uu tue iauy tueai. Ambrus and Mink (1964) reported the result of cigarette- Ftnoking on blood coagulation in 18 healthy medical students (noti-smokers); the subject,a smoked 1 standard non-filter cigarette in 5 minutes with inhaling, and the several mea4ttre- ments were made on venous blood samples drawn before and 6 minutes after smoking. Means, standard errors, and ranges of the per-cent changes from the pre-smoking control values were as follows: whole-blood ooagulation-time, tnin -3.14 --L- b.75 (range, -24 to +16); recalcified plasma coagulation- time;, sec -7.64 zh 4.43 (range, -41 to +9); partial thrombo- plastin time, sea -1.43 f 4.3 (range, -I7 to +19); thrombo- plastin generation (yield x rate), -3.31 :& 7.67 (range, -34 to +61); tbromboelestogram r + k, min +LAS + 10.51 (range, -33 to +118); tensilestrengthof clot, +1.00 :h 3.93 (range, -13 to +l7); bioassay of serum thrombotic accelera- tor, ml/kg +32.5 t 43.23 (range, -90 to +400); platelet count, per cnun +2.00 + 3.34 (range, -13 to + 16); platelet stickiness, % of base-line +84.4 _+ 41.95 (mngc, -13 to +30b). Of these, the increase in platelet adhesiveness atone was said to be statistically significant; although not statisti- sally significant, whole-blood ooagulation-time, rera)cified pIasma ooagulstion.t,irne, platelet count, partial thromboplas- din time, thromboplastin generation time, and tensile strength of the clot, were considered to show changes compatible with Increased coagulability. During 8 days of abstinence fiom smoking, 5 smokers in good health showed an increase in venous c)ottang-t.ime from an initiaa14.5 rain to 7 min after the first 2 days, 8 min after 3 days; and 11 min after 6 days (Fiske, Fox and Carusog)u, 1962). After 5.5 weeks on a daily regime of vitamin A, niacin, And riboflavin (for the results of this therapy in diseased ratbjecta, see below,1456. Peripheol drterr.aadaosfa, Af aaoge- nfent), during w},ich 1.5 packs of cigarettea were smoked daily, clotting time was 14 min; e4er 2 weeks on placebop and con- tinued smoking, clotting ttme had fallen to 10 min. In the 7 subjeMs studied by Mustard and Murphy (1963) and de- fir-ribed above under 271. Throraborytrs, there were no signifi- cant differences in whole-blood clotting-time, prothmmbin time,, or plasma thromboplastin time, between periods of snoking and non-smoking. Under the title, "Smoking and Blood Clotting", the $ritfs)e A/e3iced JourYtal (1: 833-834, 1063) commented on this end related studies. Rotenman and Friedman (1960) found that women with aei overt behavior pattern characterized by excessive "dtive" exhibited signi6eantU• faster blood clotting than women lack- ing such environmental and behariorial qualities; but these differences were not ascribable to differences in smoking habits. Rosenman and co-workers (1964) have also reported comprehensive studies of overt behavior pattern, coronary heart-disease, and blood coagulation (among other param- eters) in a large number of "well" men; these findings are diwrlbed in tnure detail brlna, )397. Firultiroga in Cormtary F'rnrt.Diaeaac. 275. 3tISCE41dlNE0Utt OBSERVATIONa 277. Blood Protelns (113) According to Aiearora, Komorl•rarna and Ohno (1960), a single injection of 5 mg/kg nicotine to rabbits resulted in a decrease in total serum protein, the lowest value being reached 6-9 hours after the injection; there were no changes, however, in the proportions of each protein fraction. Total circulating plagn'a and blood, w tell as extrarellular fluid (thioryattate "space"), a)so decreased. Following repeated nlcotine adrninir•- I rauwI U/ rauuul•, NerWa WUUmni N'a6 KaW to have rernanletl within normal values (Veress and Rengei, 1959). Rabbit.a which received daily injections of catk•ine plus iticotine showed no quantitative or qualitative deviations from control valutM in serum proteins (Cr•ochra-L}•sanowica, Gotski and Kedra, 1959). N7tile rabbits given daily intrarenoths injections of nicotine shoned no blood biochemical change.c, animals re- ceivihig cholesterol by mouth, in addition, were said to hare showed a slight decrease in serum albumins And an uncrease in globulins; however, cholesterol feeding alone had the sante result (Czochra-Lysanou•icz, Gorski and Kedra, 1960). In dogt:, plasma fibrinogen concentrations decreased folloa•- ing chronic infusions of nicotine (L. Feinberg et al., 1961). Ar?nrding to Tipiekell and rn•a»rkern (1og?), C-resctire protein (CRP) is an abnormal serum protein which is usually found in the sera of patients with most in8ammatory or invasive neoplastic disease.`, and which is never found in the sera of healthy subjects. A second serologic abnormality is an abnormal serofiocculant (SF) for the ethyl ester of cboladienic acid; this is less aell-defined, but is frequently found in the sera of patients with neoplastic or non-neoplastic di-eases, and is found in the sera of less than 7~'0 of putatively hea)thy subjects. These workers studied the relationship of these two abnormal serologic findings to smoking habits in an embula- tory population of 963 female- and 549 tuales over 25 years of age. When the effect of age was held constant,, the cot•relation between the incidence of positive SF and CRP tests and in- creased cigarette consumption were significant in both male and female groups; when the effect of age was not held con- stant; a significant difference was observed between the frequency of positive SF tests among males who did not smoke and those who smosed cigarettex, regardless of the number smrked. The only significant difference in frequency of CRP tests in males was observed betweeu nonsmokers and those smoking more than one pack of cigarettes a day. 278. Fibrinolysis Recalling reports that nicotinic acid and epinephrine pro- duced fibrinolytic activity in oiuo, L. Feinberg pnd co-workers (1961) decided to investigate niontine (which has sympatho- mimetic properties and is structurally similar to nicotinic acid 1but which is nevertheless not metabolically similar)) for such activity. The p)aana of 9 dogs receiving chrorhic infusions of nicotine was studied for fibrinolytic activity before and after the daily infusions, as follows: I. the lysix tim, ot a standard fibrin clot by unJiluted plarow; 2. the lysis time of a standard fibrin clot by plastma diluted 1:10 and 1:20 in imidarale- saline buBer; 3. the euglobulin l.zcis times; 4. the anti- thrombolytic lysis times againA rerial dilutions of strepto- kintse-activated fibrinoh•sin; 5. the plasma fibrinogen concentration. The 1y* times were found to be shorter in all iustanee+ after the infusion of nicotine; the planna fibrinogen concentrations also decrea<ed; and the authora therefore felt that nicotine htfivecf intravenouRly in doga 14 capable of producing fibrinolytic activity In their plaama. On the other hand, Pohnla, Yen ar.d Shighcr (1962) reported that intra• venous injection of 0.1 mg/kg nicotine in this speeieg had no effect on blood-plavna fibrinolytic ac•ivity. Gibelli and associates (11964) studied Bbrinol;•siu aa well as blood coagulation, in a group of 6 normal )~ov:.d subjects, before and after a fatty meal, with or without smoking 18 cigarettes at an average frequency of 3 per hour; and they reimrted that fibrinolytic activity (plarmin and fdemmhi,qtrn) Produced by The Council for 0002979 Tobacco Rescarch-USA, Inco

t)8 TnBACCO--EXPERIMENTAL AN I) CLIT ICAL STUDIES. SUPPLEMENT I drnrrswl momw marlt.edlv when amokfn¢ was ounerimnosed upon the fatty meai. , Lackney and Broate-Stewart (personal obeervations cited by Bront,2-$tecrart, 1961), in studies on subjects with and without iechemic heart-disease, found that fibrinolysis in the cigarette-sonokers of gnch group was more tapid than in non- smokers, although the differences were not significant. 279. Serwa Sfa6ilfyy (113) 200. Blood Pepsinopen (113) 281. Vaaomnen•ictor Substance in Blood (1 13) (No new dataJ 282. Fluorescene Su6efueca in Blood Unghvm and eo-workere (1982) noted that they had to modify the analytical method used in determining whole- blood cBf.eeholaminea because fluorescent compounds in cigarette-xonoke interfered with the detemoination of cate- cholaminea in the blood; these fluorescent compounds were neither bound to blood proteins nor to cells, for they were also presedt in oell- and protein-free blood-filtrates. The nature of these fluonesoent materials hae been suggested b,r Unghvary, , Hovanya and Farkia (1962-0); Qomnarln and eoopoletin kvere thought to represent the greater proportion of fluorescent tneteriaia in tobacco. These latter workers found that, 5 min ofler smoking a cigarette, a gjeenish-yellou fluorescence could be demonatrated in blood at peak int,ensity; subsequently, t6e,degree of fluoreycence decayed, and Ihe entire phenomenon ceased after 90-BO min. The amount of absorbed fltlon~ceht substances was said to depend markedly upon the type of am©king, the quality of the tobacco, and the quantity s>inoked. (A procedure for assaying floAOreacent ma. teriale in blood was described ; the fluorescing tobacoo-oompo. aents produced the same greenisb-yellose fl8roreseence as estecholamines, but, by use of an iodine solutio>D, the catechol• amines fluoreacence, could be destroged without influencing continuation of thc tobacco fluotesceuce.) 283. sertsws Scbievelbeln (1962) has presented a revie~r of the new literature (mostly over the preceding 10 )esra, but not limited to theFe), which includes the effects of nicotine on blood. Produced bv The Council for Tobacco Researcb-USA, InC,

V CARDIOVASCULAR SYSTEM I I 284. AlVIAfA1.S (114) The cardiovascular e8ects of nicotine are exceedingly com- ples (114a). Van Proosdij (1980, pp. 21-22) has succinctly pointed out that the ultimate reaction to nicotine represents the algebraic aum of the various, sometimes conflicting, cardiovascular effects of this substance, which even the pharmecologists cannot by any means always foretell. 2115. n>J+nrnart: (114) 286. segmeatcd Worm (114) [No nea data.] 287. AloUnek (114) Addition of 2 drops of 1:40D niootine caused diastolic arrest of Straub preparations of isolated htarts of the sea- hare, Aplyaia /tmocina (f . Heymana, 1929). On the heart of apfyaia dactylomela, nicotine (0.3 mg in a 20-m) bath) caused inhibition and standstill, tollowed by a strong stiraula- tion and increase of the frequency of the beat (von Euler, Chaves and Teodosio, 1952). Asano (1962a, b) reported that concentrations of nicotine less than 1 pg/ml had had no effect on the isolated ventricle of the clam, Meretri.z lusmio. Rtiding, but that 10 µg/nl caused a deceease in the systolic ampl'uude of besf.s. This response became marked at 50 pg/m1 nicotine, and was fol- lou•ed by some irregular strong contractions with long in- ternals, and gradual retxirn to regular rhythm and amplitude; regular beats after tecovery were higher than before nicotlni- sation. At 100 pg/m) concentration, ventricular movement was immediately arrested in diastalic state, and, following a period of quiescence, irregular beats (including strong contractions) appeared, followed by high and regular beats. At I mfe/mi nicotine, sintilar arrests otanrbed, followed by only the strong contractions vrith long intervals between. At concentrations higher than 5 mg/ml, movement was stopped in a systolic arrest. In repetitive nieotinisations, the isolated ventricle did not respond to nicotine at concentra- tiens less than 100 pg/mi, but showed the strong contractions or the systolic arrest at concentrations gnatcr than I mg/ml. 288. Cruateceaa (115) 289. Insed (117) 190. Tuxicote (117) (No new data.) 291. Cydostomea On the perfused heart of the lamprey, Lompelra ,Auuniotilia, 0.5 µg/mi nicotine caused marked acceleration (Augustinsson et al., 1956). In stronger concentration, nicotine produ¢ed, in additinn, pronounced increase of tone, irregular rhythrn, and a tendency for the heart to stop in A kind of systolic flutter or Sbrillation. A concentration of 300 pg/mi nicotine accelerated the rate of the perfused heart of the river lampzey. Enfoaphenus japon{oa, fot' I or 2 txc, folWhed by returtl to normal; therEafter, increasing the concentration had no addi- tional effect (Otoril, 1953). 292. Fiah (118) Jullien and Fipplinger (1951), who studied the effeca of nicotine and other dnrge on the hearts of certain marine fish, reported tltat nicotine augmented the frequency, and diatin- ished the amplitude, of awntraMions of the auricle. Applied to the ventricle, nicotibe produced the opposite effects. Nicotine inverted the tonotropic effect of the vagus, which remained excitable, subsequent excitation of the nerve pro- ducing a matked increase in tonus instead of the usual fall; and the inversion persisted, even after an application of acetykholine; then atropdne, succeeding nieotine. 293. Solamander (118) [No new data.] 294. Frop (118) 295. Intact Animal (1d8). Tocco (1923) treated trogs with nicotine, and then made a histological study of the myo- fibrils, observing more or less intense rnndifmt.tions of the saroplasm and chromatic substance (119,a)• Additional hitY'ological findings were presented by Tocco-Toeco (1924). 2%. Isolated Heart (120). GaAoamki (1905) reported that I c/c nicotine hydrochloride caused initial inhibition, followed by stimulation, of exaised frog-heart, bdtt that 2% and stronger concentrations had an over-all weakening eflectt a-Oxybeneylphosphinous acid (Phoselit) exerted a nega- t.ive, followed by a positive, inotropic effect on fresh frog. hearts, both of which could be completcly suppressed by nicotine hydrogen tartrate (l5cheibe and Niemer, 1954). 297. Excfeed Cardiac Muscle (122). On isolated itng- ventricle, the p-adrenergic blocking agent, nethalide~ in concentration of 1I,g/mI (which had a slight deptessant action), almost abolished the positive inotropic actionb of ephinephrine, norepinephrine, and isoproterenol, and also prevented the response to nirotine (Dhalla and hicl.ain, 1984). 298. Alechaniem of the Cardiac EH"ect of Nicotine (123). Results of the experiments described immediately above lec Dhalht and J1cLain (1904) to conclude that the positive iuo- 69 Produced by The Council for Tobacco Researci~•USA, Inc.

70 TOBACCO-EXPERIAfENTAL AND CLINICAL STUI)IfS. SUPPLEMENT I fmnin o/ra•fc nf nirMinn (wml f1w MhPr nnherAnre:.) aEY' mediated through the activation of 0-adrenergic receptors 'in the frog heart. 299. Lymph Hearts (123). [No new data.) 300. Toad (123) Application of 10 µg nicotine tartrate to the perfused heart of the toad (Byfo eulgmis fornunrs) produced transient de- pression of the heart-rate and no subsequent accelerAtion (Deguchi et al., 1063). 2,4-Thiatolidinedione caused inhibition of both ratc and force of contraction of the isolated perfused toad-heart, both before as well as after nicotinization and atropinir.htion of the heart (1'awab and Afustafa, 19tft). On the excised toad siho•auricle preparat.ion, I pg/ml nicotine caused a marked decresse in the amplitude of the contraction, followed by a slight increase; and this was coni- pletely blocked by 10 fag/ndi thiamine (I. Yamamoto, 1963). Nayler (1963) studied the effect of nicotine on cardiac- muscle contractions and radiucalcium movement, using in- tact ventricles and strips of ventricular muscle From the toad. Bufo nwrinus; both preparations were stimulated with rer- tanguiar supra•threshold pulses of 10 msec duration at a rate of 6 pulses per min delivered from a Tektronix square•n•are gererator. Perfusion of intact ventricles with 0.01 m.11 rucci- tine in Tyrode's solution evoked a small but well-de,fined positive inotropic reslwnse, which was well maintained, provided the perfusion was continued in the presence of nicotine, but when nicotine-frec Tyrode's solution was re- iotroduced, the contractions gradually returned to thecr pre-nicotine size. The magnitude of the positive inotcopic response varied according to final concentrations of nicotine over the range of 0.01-0.1 mJl. Concentrations of nicntine which evoked a positive inotropic reQponse in vent*iclea perfused with primary Tyrode's (containing 0.65 m 1/ Ca+*) sometimeR caused partial roiatracture when added to the kame ventricle,~ during perfusion with Tyrode's containing 1.3 or 2.6 mdl Ca**. Both iwat•>3timulation potentiation and the classical stautase phenomedron (treppe) were readily deimori- atrable in ventricles perfused with primary Tyrode's, but these phenomena were abolished during }w:•tusion with pri- mary Tyrode's containing nicotine in excess of 0.63 ntJl. Nicotine in concentrations of, or in excess of, 0.2 m.11 evoked reproducible partial or complete contmetures, both in quiescent and actively contracting ventricles; the amplitudc and duration of these contractions were not influenced by the presence or absence of electrical stimulation, and they gradually decayed on perhdsion with nfcotine-free Tynbde's solution. Developrnent of contracture by 0.2 m1f nicotine was only partially dependent on extra-cellular Ca**, the amplitude developed in Ca++-free Tyrode's being onh slightly smaller than that develol*d when 0.65, 1.3, or 211, m V Ca** was present. TheBe contractures gradually decayed when the Ca**-free Tyrode's containing 0.2 m.tl nicotine was replaced by nicotine-free Ca**-free Tyrode's, and such a preparation repeatedl,c responded to electrical stimulation, despite the absence of any added extra-rellular Ca+*. Nico- tine in coneenarations of, or in excess of, 0.2 tnJ/ evoked contracturex in ventricles previvusly perfused for at leae;t 30 min with Ca**-free'h•rode's containing 0.5-1 m1l eth,rlene- dinmbtetetra-acetlc acid (EDTA). ti'hen perfusfon wa. con- tinued in Ca**•fine Tyrode's containing I m1/ Er)TA, and rontractums evoked b)• 0.2 ml1 nicotine every 15-20 min, the amplitude of the eontractures slowly declined, so dhat, after 2 hours perfusion, the contrarture height was reduced by half; re-perfusing these ventricles with '1'y-rode's eontatn- ing 0.65 m.V Ca*'' fully restored their ability to respnnd to nicotine in the absence of extra-cellular Ca++. Substitution of,%Or' or 13r" for Cl' in the primary Tyrode'a solution did not affect the amplitude of the contractures induced by 0.2 m.U nicotine. Sucrose in 585 mJl concentration induced submaximal contractures in ventricles which had beebr pre• vioust)• perfused with 0.65 m.U Ca**-Tyrode's, and 0.2 mt/ nicotine au)ttttented the sucrose-induced contracture. Con- trarture. evoked by perfusion with modified Tyrode's eon- t.aining 0.65 m 1/ Caa'~' and 100 01 KCI decayed spon• taneouly, but contractures similarly evoked in the same ventricles, but in presence of 0.2 m.11 nicotine, were main- taiurd, and showed no trudt•u,•y tomar3h Nuch dci:a). Nicotine-induced contractures, whethrrdereloped in the pre.c. ence or absence of extm-cellular Ca*+, were rapidh• rei ersed b)•theintroduction of nirotine-free T)•rodr's containing 4 mM ED7•A; and this procedure similarly revenced those rontrac- tures induced by nicotine in ventricles previously perfused with Ca'H'-free Tyrode's containing 0.5 m.l/ EDTA. Further experiments using "Ca demonstrated that nicotine enhanced both the uptake and release of "Ca from the muscle. Relating this to the other findings, Nayler pointed out that, since nirotine evoked contracture even in the absence of extra• cellular Ca**, it seemed unlikt:y that the increased uptake of "Ca, which occurred in the presence of 0.2 m 3l nicot ine underlies the effect of nicotine on the contractile process; the author considered it more likely that the increased re- lease of "Ca brougat about by nicotine reflected that process whereby nicotine affects the contractile ptoces.q probably the release of some of the inttscellularly-bound Ca*'', an action which probably establishes an increased intracrllular level of Ca**, and which therefore may be associated with the augmented mechanical response. However, the prompt re- versal of the effects of nicotine by GDT.t, was said to suggest, either that these intracellular.re4a:rd calri,tn ions accumu- kite at the surface of the ceils, where they participate in subse.luent contractions; or, alternatively, that the primary effect of nicotine may be more on the mobilization of calcium ions at the cell surface, rather then inttacellularly. In a later review-article on calcium e!:c•bange in cardiac muscle, Nayler (IOfi7) expressed the view that available evidence Indicates that the positive inotropir effect of nicotine probably reflects an increased myopla.cmic Ca*i'. rThe reader is referred to the experiments reported by Ahmad and Lea is (1061,1f162) on the effects of nicotine on radiocalciom uptake and release by frog sartorius muscle (see abr,ve, 227 and 236), and also to experiments reported by Gross and liahr (1984) on the action of nicotine on isolated vascular smooth muscle (see below, 884).j 901. Lymph Hearts. [No new data.) 302. TurUc (124) - In experimentss described by Van dcr Linden (1934), the head of a turtle isolated from the trunk except for intact vagi was perfused wi-.h fluid containing nicotine, which, in suficient do.cane (0.06-0.1 mg), alFO caustxd arrest of the heart, followed by a long period of bradycardia, indicating excitation of the eardio-inhibitory center. Larger (0.2-0.5 mg) doses resulted in intense and prolonged tsxcitation, fol• loxrd by paralyFis, of the cardio-inhibitory center. When tested on exrised atria of the 1•:astern Painted Tur- tle, Cryremys pircta pirto, in aerated muscle-chambers, niro- ooozssz Produced bv T,ic ('ouncil fot Tobacco Research•USA, Inc. i I

_;9.a..- _'._.___.___.--- ,-___. CARllIOt'ASCULAR SYSTEM tinr in optimal doses (1 pg/mI) produced "the clar:sical re- ., n"~, vnddnn wnd wlmnvt :..vtnn~wnnm,v dnnn.winw n( ancpiitude, and a ntarked but more gridual slo+t~ing of the beat, followed by a gradual return to normal ampPGtude and rate (M. T. Dimond,1959). The pattern of response to various doses of nicotine ass more erratic than to those of s)inpatho• mimetic drugs, both because of tachyphylaxis and because of var3ing sensitivity of individual specimens at low dosage let'eL,. Iuotropic and chronotropic inhibition occurred more often with strong doses. Treatment aith hexamethonium abolished the action of nicotine. 303. Snake (125) (\o new data) 304. Bird (125) 305. 1larnnwls (125) 306. Rabbit (125). It may be of interest to mention here that heart-rate was either reduced or unchanged in even aon- sensitized rabbits injected i.v. with tobacco moc9rc virus (Chopm, 1964). 307. Cat (126). [\o new data.) 308. Dog (126). I.v. infusion of 15 µg nicotine/kg/min in- creased the mean heart-mte of dogs from 66 to 111 per miu (Forte et al., 1960). The chronotropic effects of intrh-aortic administration of 10 pg/kg nicotine were similar to those of an i.v. injection of 2 µg/kg epinephrine (E. F. Woods and Richardson, 1955). Lv. injection of 0.1 mg/kg nicotine resulted in bradycardia followed by tachycardia (Yoshikata, 19&f). Lv. injection of 2 mg/kg nicotine into anesthetized dogs with both vagi cut caused bradycsrdia (Clttcser, 1900). Similar injection of 0.4-0.6 mg/kg leptodactylhna, a naturally-occurring base possessing cholinergic and adrenergic eilecta of the 7icotine type, had the same effect. Recording electrocardiogtsms in dogs using a cross-circula- tSon technique (for details, see below, 824), Solti and ro- trorkers (1900s) observed an increase in heart-rate in both acceptor and donor animals folloiring injection of nicotine into the acceptor dog. 309. Other Riamntals. In chloralosed sheep, intrn-carotid injection of nicotine elevated blood pressure and produced a bmd-,rardia (Van Damme, 1942). Also in sheep under cblora- la.e anesthesre, injection of 0.1 mg/kg nicotine augmented the bmdycardia; at higher doses, slowing was not manifested during the hypertensive phase (13runaud and Navarro, 1953). 310. Factora .1lod;fying (he E,pert of A'frotwte (128) 811. Anerthesia. In doga chronically prepared so that the observations could be made in the unanesthetiecd state, the results of 50 injections of nicotine (in 8 experiments) showed that about four times the dose effective in the conscious state Wsa nerepssry to produce ramparable cardiova.ccular e8ects (chronotropie; heart contractile force; arterial blood pressure) in the anestheticed state (E. F. Woods and Richardson, 1955). 312. Nerve Sttrrtulation and/or Seetion (128). In 3 of 8 experiments on sheep, application of 11% nicotine to the right >;teliate ganglion completely abolixhed the ratdio-arrelemtor 71 response to, stimulation of the right ceh-iral R\Tlpathetic nwn wnd . tbn .nmw:n:.... 9 nn....n/: rbm .n .. .. n+..• nn/.• panially aholished (11'aitrs, 1957a). )l,a other exLerimentP N'aitea (1957b) ueed nieotinr in extablishing ganglionic re- lays of efterent cardiac nerves in the sheeli.) Injection of 0.1-0.3 mg/kg nicotine, i.v. or into the right ventricle of cats and dogs, rewlied in ab initial fall in heart- rate and blood pressure (Cakasaki, Yokoo and \agasaki, 1959). Bilateral vagotomy just below the hilas of the lung did not affect this picture; after bilateral vagotomy obore the hilus, the fall in heart-rate and blood pressure uas sGghtly less. Yoshikata (1964) gave dogs 0.1 mg/kg nicotine i.v., and observed reactions in the heart-kate both before and after transection of the vagus, carotid Rinys, and spinal cord, in order to study the reflex ntechanism; foe an interpretation of his findings, see below, 339. Hashidnoto and cw-workers (1964) also studied carotid chemoreceptor-reflex effects on heart-rate in dogs, and found that 0.02-0;1 png nicotine caused temlarary arrest of atrial beat, and this ttrs)rotune was abol- ished by cutting both vagi; instead, an incimse of atrial rate occurred. When a large (0.5 mg) amount of nicotine was given, the acceleration lasted longer (for details of these experiments, see below 313 and 335). 313. Cardiovaecular Reflexes (129). In dogs under mor- phine-chloralose anesthesia, 0.3 ml/kg of acetic acid injected into the carotid sinus and glomus blocked the bradycardia and Ityperpnea of a subsequent injection of 10 µgft nicotine (P6rssAss, Such and F6rss6ss.Gibisser, 1957a). In adult dogs given 0.1 mg/kg nicotine i.v., the observed reactions in heart-rate, blood pressure, and electrocardiogram before and after transection of the vagus, carotid sinus, and spinal cord, were interpreted to indirate that the bradycardia and succeeding bloocl-pressure fall were excited panly by ra,diac reflex through the vagus, whiie the blood-pressure rise and tach.-cardia were said to be attributable to the action of nicotine per ae (Yoshikata, l9&f). Hashimoto and co-workers (1964) studied carotid chemoreceptor-rhfl,rx effects on corv- nary floN• and heart-rate in male dogs under chloralose anes- thesia, using innervated Langendorff heart preparations in ventricular fibrillation with (mnpletely separated cross- circulations to the head and to the heart; drugs were adminis- tered by rapid injection into the blood perfusing the head. Nicotine in dosage of 0.02-0.1 mg caused temporary arrest of tt,e atrial beat, which suon reappeared, and tended to overshoot the control rate. Coronary blood.flow began to increase during atrial arrest, and became maximal when the atrial rate was returning to normal or above. When the ca- rotid area was completew denert•ated, neither deceleration nor acceleration of auricular rate was pt'otlueed by nicotine, and there was no change In coronary flow. Comroe and Mortimer (1963, 1964) determined the td{ecta of separate stimulation of the aortic and of the carotid chemo- receptorn by injecting low comwntratiann of f.rniium e,rsnide, nicotine bitartmte (4-10 irg/kg), phenyldiguanide, and serotonin into the ascending aorta of dogs under rhloralose anesthesia, with delay-lmths of coiled plastic tubes inserted in each cotnmon-carotid artery; thtMv co?Is prevented chemi. cals from reaching the carotid body utttil 15-78 tmc after they reached the aortic chemorereptors. In a typical result shown for nicotine in an animal breathing spontaneously, hlymrimea, hypertension, and tachycardia began arithin 2 sec (aortic-body re`lwnse); 00 eec after the injection, greater hyWrfinea occurred, accompanied by bradyrartiia and hJ7w- tem=ion (carotid-bodv rest+aiwel• in a few inatancea non. Produced by Th n Council for 0002983 Tobacco Research-USA, Inc.

ir: 72 T'JBACCO-FJiPERISIEIQTAL AND CLINICAL STUDIEi. SUPPLE1fENT 1 typical carotid-body (increase in heart-rate and blood pres• cUre/ tlWl aurllc-UUUy kuetn:Lee W uraut-,m,r ww u.wu pressure) neponses occurred (Comroe and Mortimer, 19161). In general, the cardiovascular responses were similar, whether the dogs breathed spontaneously or were ventilated artifi- cially, although initial tacbycardia occurred Eess frequently following atimulation of the aortic chemoreceptoru during constant artificial ventilation. In their study of the hemo- dynamic effects of stimulation of aortic- and earotid-body chemoreceptota, S. Stern, Ferguson and Rapaport (1964) injected Ir20,pg/kg nicotine bitartrate into the hase of the ascending aorta of anesthetised (pentobarbital), artificially ventilated, open-chest dogs. With respect to heartrrate, bradycacdia occuned after combined stimulation of the aortic and carotid chemoreceptors; however, when brady- cardia was prevented by atropinin.ation, no significant change in pulmonary blood-flow occurred (for the cobaplete account of these experirnents, see below, 395). According to Bevan and eo•workers, nicotine has a stimu- lant action on the pulmonary-artery reflexogenic zone, sbnilar to the action of lobeline, and leading to brady,cardia in man (Bevan and Murray, 1963) and in cats (I3evan, Atutpby and Hardy,19s4). In dogs anesthetized with urethane and morphine, both circulatory and respiratory reflexes resulting from i.v. injec- tion of 0.1-0.2 mg/kg nicotine tvere blocked by teUaethyl- ammonium, heanmetbunium, and procaine; the respiratory reflex was more difficult to block than the circulatory refle:. (Takasaid at al.,1959a). Procaine instilled into the pericardial soe blocked the circulatory re8exes normally caused by nico- tine (Takeseki et al., 1959b). ln the depressor reaction oc- curring in anesthetized cats in response to pericardial administration of nicotine (acting on pericardial chemotrcep- tors), a deceleration of cardiac rhythm always occurred, whereas the strength of cardiac contractions either diminished, increased, or remained unchanged; the eSerent route of this reEex lay in the vagus (9'rachenk.o, 1962); for details, see below, 350-C. 814. Relat.ionship between Heart-Rat•e and Blood-Pree• eure Changes (130). In dogs anesthetized with urethane and mo-phine, i.v. injection of 0.1-0.2 mg/kg nicotine re• sulted -ui a fall in blood.pre,ssure and heart-rate, followed by hypertension (Takasaki et al., 1959a). Injeet,ion of 0.1-0.3 mg/kg nicotine, i.v. or into the right ventricle of cats and dogs, resulted in a fall in blood-presure and heart-rate; the blood-pressure fall was followed by a tecondary rise (Taka- aalti, Yokoo and Nagasaki, 1959). 815. lVed of Other Drugs in tAe Niootinind A nimal (130) Following repeated doses of spartelne (averaging 40 mg/kg) to anesthetized animals under artificial teppiralion, a slight pressor effect, accompanied by bmdycardia, developed (Lu, 1952). Complete nirotinization failed to prevent this in dogs; in atropinised cats, however, nicothtisation considerably re- duced the bradycardia produced by sparteint•, although it did not abolish it. 816. Uect of Niootine joUoaing the Admiitiabation of Other Drugs (131) Bradycardia resulting from intra-aortfo injection of nico- tine in dogs was preventev by atropiniaation of the animals (S. Stern, Ferguson and Rapaport, 19@4). Following administration of hexamethoni,tm, nicotine bradycardia in rabbits was greatly reduced (Alott, 1963), and waN prevented in anesthetir•ed, vagotomissd dog,} (Cliis• 01.1, I JW/. 311. Flfeet of Nieotine in .-tdrrnalerfomired Animo/a (133) The chronotropic effects of nicotin~e in anesthetised, olmi• chest, bilaterally vagotomiztd dogs R•ere latleely alwlirhnl by adrenslectomy (E. F. Woods and Richardson, 19a5). In control dogs under pentobarbital anesthesia, nicotine provoked some cardiac acceleration; but, in adrenalcMomiscd anirnals, the drug elicited a sipni6cant deceleration in the stage of early adrenal failure; in late failure, nicotine induced an insignificantly smaller acceleratiodi than waa observed in control animals (F. K. Brown and Remintrton, 1955). 318. lnf.uenre o! Prerioa r Hta•t Demogs ;133? 819. EBeet of 3lultiple Aoera of \'icofine (133) 320. Heo•rt-Rott Jollouing Chronic ,1 itotine Administraiion (133) INo new data.) 321. Jtisceflaneous lnjfuenees on the Effect of Nicotine (133) .•tge. Increases in heart-rate and blood pressure were pro- duced by nicotine in young dogs, as well as in adult ou%, but bradycardia and blood-pressure fall in the former were slight (Saito, 1964). 322. Cardiac Arrhytlu„ios following Administration of Nicotine or To6accaSnzoh (133) In anesthetized dogs maintained ttnder artificial respira• tion, i.v. injectaon of 0.5-1 mg nicotine tartrate produced occasional arrhythmias, for which an increase in vagus tone was said to be probably responsible (Papp and Solti, 1959). The changes were preveated by atropine, but arparently not by dibenamine. However, in crisa-circulation experi• ments in dogs, nicotine tachycardia' did not develop after the adminictration of dibenamine (Salti et al., 1960a). ln experiments on dogs anesthetized with 16% eyclopro• pane and receiving repeated injectioris of 0.5 mg/ltg nicotine at 30-min intervals, ventrictilar ectopic beats were produced by the first three injections in all casea; hox•ever, on the fourth or succeeding injection of nicotine, the animals failed to develop arrh~Khmias, although the pressor response had only slightly decreased (Drill and Ha)s, 1951). Thus, repeated injection of nicotine alone may exert some blocking effect. It may be noted that, although SY-30 I11'-(2-(2•bl• phenylyloxy)ethyl)-N-(2-chloroethyl)butylamine HCIj pre- vented arrhythmias following nicotitbe, an increase in blood pressure was always obtained. The blocking effect of SY-2tt (,V-(2.bromoethyl)-N.ethyl-l-naphthedenemethylamine Hlir) and SY-21 j,V-(2-chloroethy))-\-ethyl•9.8uorettylamine HCI) on nicotine-induced eanlinc arrhythmias was generalh• aw • ciated with a reversal of blood-preacurc response. It may be mentioned that eardiac irregularities occurred occasionally in even non.sensitited rabbits injected i.v. uith tobacco mosaic virus (Chopra,1984). 323. EfecVrotordiogrnpAit ond Beltietocardiogrophir Changes (135) 824. Dog (135). Changes in the electrocardiogram (EKG) of dogs given intra-eoronary arterial injections of 0.2-2.2 µR/kg nicotine, previously reported by J. 11'. West, Gusman aud Bellet (1958) (130a), have been reported again by Bellet, Produccd bv The Cou;;c:I for 0002984 Tobacco Rescarch-liSFI, Inc.

CAIIIrW1'AaCULAIt SYSTEXI West and Guziumu (1960). These EKG effects were com- pletely blocked by tetraethyhtnunopium. Dogs receiving wcoLure 'uiwj uaw i.u.- iu a eww-a'wuryuwi, aqueuun veutcte daih- for 6 w•eeks (0.5 mg/kg/day for 4 weeks, increased to 1.0 mg for 2 tveeks) showed no EKG abnotmalities (chest leads) (Kershbaum, $cilet and Khorsandian, 1965). 1'app and Solti (1959) studied the influence of dibenamine on EKG changes induced by i.v., injerdion of 0.5-I mg nico- tine tartrate in dnesthetir.ed dogs under artificial respiration. Nicotine alone i.nduced changes in the T-wave aud in the ST-segment, as well as occasional arrhy-thtttias; and these changess were eohsidered to have been due to an increase of syinpathetic tone produced in the heatt aud circulation, and not by a constricting effect on the coronary tvscels, since coronary resistance diminished or remained unchanged in most cases, and no direct relatiotlship was observed between the severity of the EKG changes and the changes in coronary resistance. Out of 7 cases, the nirotine-induced EKG changes were hrevented by i.v. infusion of 7 mg/kg dibenamine, in 6 cases with respect to ST-segment changes and, in 5 cases with respect to T-wrave alterations. In 5 of these cases, there was a drop in heart-rate following the injection of nicotine. In a subsequent series of experiments, Solti and co-workers (1960a) investigated, by means of crosa-circulation techniques, the role of the central nervous system in the EKG and circula- tory changes caused by nicotine. '1'he circulation to the head of an acceptor dog was baolat.ed from the systemic circulation, and perfused by :;rom-anastomosis with the circulation from a donor animal; thua, nicotine, injected into the aKetiat branch lexding to the acceptor animal's head reached a high concentratiut in the brain, and, passing through it, entered the circulation of the donor dog. lnjection of 0.5 mg nicotine tart rate into the arterial branch of the acceptor's head caused acute hypertension and increase of heart-rate in both animaLc, but venous pressure and cerebral blood•flow were practically unaffected. The EKG showed acute changes in both animals; in addition to tachycardia and eatrdsystolie arrhythmia, significant alterations occurred iro the T-wave and the ST- se.gment. In some rasas, markedly inverted T and ea<ttvanely depressed ST were noted; in others, very high peaked T- svaveR, reminisrnnt of "Fratirktangs-7"'. After injection of dibenamine into :he trunk of the acceptor dog, tachycardia and increased blood pressure did not develop in it following administration of nicotine; but, in such animals, disturbarrces of conduction, marked brad)rardia, and arrhythmia were common; these changes suggested an inctease of vagal tone. ,lfter administering dibenamine and performing bilatcral cervical vagototny, no changes in EKG and hearbrate ap- peared following injection of nicotine. The authors concluded that the EKG and circulatory changes caused by nicotine were, at least in part, central nervous in origin, probably due to centrsl s>mpathetic stimulation and consequent increased epinephrine output. Released antidiuretic hormone (ADH) could not have been a factor in the acceptor dogs, becatr.m of the isolation of the braitt from the syretemic circulation; furthermore, neither antidiuresis nor reduction in glotnerular flltration-rate occurred in these anitnala According to Kien and Sherrod (1960), the EKG ntani- festations of cigarette-smoke inhalation or nicotine tuirninis- tration to open-cheat pentobarbita6sed dogs were not corre- lated, either tetnpotal(v or in magnitude, with the chtutges in eardiac work. Further analysis revealed a precipitous fall in csrdiac otygen-utilisation, which immediately preceded the EKG change8; and the discrepancy between apparent oxygen requirements and actual oxygen utilization (possibl,v being a 73 transient metabolic effect of the kigarette-snwke or nicotine) appeared to initiate the E1iG diiturbancen. truruascw and t+est ttatnqi, oj mtaoe a study ot the balirstu- cardiographic (13CG) effects e/ieited in dogs by i.v. and inti'a-coronary injections of nicotine, as well as by cigarette- stnmke inhalation, in order to ehtridate the basic pharmaro- logfr pattern of response to the drug with respect to the ultts- low frequency ballistocardiogrant (CLF-I3C(). 7-he animals were lightly anesthetized nith morphine and a mixture of Dial-urethane and \embutal; rt•spiration was stopped by the administration of decamrthonium bromide, while ade- qua,de ventilation waK maintanied by a respiratory purup; and EKG and right-atrial and femoral arterial presstrres werr recorded concurrentl%• with t'LF-IICG. Intra-coronary ndministration of 0.2 pg/kte nitotine [or its congener, DUPI' (d'unethylphenylpiperaziniwn imdide)), was follun•ed by a marked impru.•ement of the belli..tic tracing, while heart- ratF, EKG, and systemic arterial pressure remained un- changed. Lv. administration bf 5 µF/kg nicotine produced a diphasic response, characterized by an initial systemic arterial-pressure increment, which induced deterioration on the ballistic tracing, following by a normotensive phase, with a concomitant improvement in the tracing. This picture could be faithfully reproduced bP• inducing the animals to inhale cigarette-smoke through thc intake of the respiratory purrdp. 325. Cat, EKG changes in anesthetized cats developed itn. mediately after i.v. injection of 0.05 mg/kg nicotine, and indicated disturbances of colonsry circulation (Kareva, 1963a, b). In most cases, the aT-segment formed the as- eending line of a giant T-wave; sometimes, the ST-segment fell below the isoelect.ric line, in which case the T-wave was negative or diphasic. Arrhythi,nias were observed in some cares. At the time of the most .ttiarked EKG changes, the increase in coronary vascular tune resulting from nicotine injection was ma~imal (see bel,ow~ 394). lu cats, the EKG effects of nicotine were blo:ked by trimethidinium (I:. Yamamoto and Domino, 19F.A). 826. itabbit (136). Mori (1960) injected 12 rabbits with increasing doses of nicotine dailr for 32 days, the doses used being in the toxic range and causing the anitnals to lose weight; yet, in EI(G taken every 4 days during the experiment, no constant and progressive modification occurred in aay lead to make the author suspect a direct or indirect harmful effect of nicotine on the myrorardium. In a few cholesterol- fed rabbits, which had demonstrably developed coronary atherosclerosis, injection of nicotine caused depression of the ST-segment, similar to that induced by etgonovine (Travell, Rinaler and Karp, 19C0); 327. Guinea Pig (137). (Ko new deta.) 828. Rat (138?. In view of the fihrdings of Grewal, Lu and Alhnark (1960, 1962) that injection of nicotine into rats led to the release of approximatel) 4 tn(' of po~,sterior-pituitary hormone per ml of blood, these workers studied the possible consequences of such concenttation's of the hormone on the rat heart (Grew-al, Lu and allmark, 1962). In rats under urethane anesthesia, i.v. injection of 50 mt'/kg (corresponding to about 1-2 ml;/ml of blood) caused a marked elevation of the ST-segment of the EKG, suggestinR myrocardial anoxia, possibly due to coronary rnnRtrictiqn. (Coronan• flow in the isolated petfused rat-hean was reduced by 25 % following Produced bv The Council fot z s s 5 Tobacco Researc(i-USA, In~ 0002985

(r, I 74 TOBACCO-EXPEIt131EN'TAL AX1) CLI\ICAL STUDI&.S. SUPPLE!•IE%T i an injection of 4 mU of the hormone.) hiut compare Solti and _..~..... nnnn,.. .t.~.... vn4...,...1:.,.. H...-1:4.A:b....d if-not imlwssibility, of nicotine-induced aDH rejeasc• being responsible for EKG changes observed in cross-rirculation experiments on dogs.J 329. Cardiac Outpu!/Force/11'orlt: Stroke l'olunie; Sl yonardial Efficiereey (138) In 23 experiments on 11 anesthetized dogA, in which nico- tine was infused i.v. at a rate of 0.02 mg/kg/tniti, a tagnifi- cant (p < 0.05) Increase in cardiac output was observed (R. K. Larson and Murray, 1963). This study w•as extended to a total of 26 experiments on 13 dogs, w ith the same findings relrorted (Larson, Fukuda and Murray, 1965). S. Stern, Ferguson and Rapaport (1964) simultanwoiv=•ly measunxf pressures in the pulmonary artery, left atrium, and femoral arteries, and the stroke volunc~ of the right ventricle, from which they calculated pulmonary and systemic vascular resistances; for an account of these experiments, see below, 395. In anesthetized, open-chest, and bilaterally vAgotomized doFa, intra-aortic administration of 10 µg/kg nicotine pro- duced heart contractile-force responses approximately equiv- alent to those following i.v. injection of 2 µg/!cg epinepbrine; these effects of nicotine were largely abolished by adrenal- ectomy (E. F. Woods and Richardson, 1955). Increments in heart contractile force produced by adrenal stimulation, nomwUy proportionate to catecholamine levels, %yere not significantly altered fogoning prior treatment of the animals with reserpine (l3agwe1l, Woods and Richardson, 1960). The increases in heart eontraetile force follon•ing intra-aortic injertion of small quantities of nicotine in dngs were also thought by Gatgottnis and Aycock (1961) to be due to direct stimulation of the adrenal medullae. The increase in cardiac contractile force resulting from nicotine injection was sig- nificantly blocked by the prior administration of selected doses of monoamine-oxidase (MAO) inhibitors, but not by the non-\1A0 inhibitor, isoniasid (Gatgounis and Aycork, 1963); for details of this study, see below, 762. Gatgounis, Richardson snd Clayton (1964) determined, in anesthetized, open-chest, vagototnized dogs, the relationship between changes in heart force, blood pressure, and adrerial-venous- plastaa catecholamine levels produced by nicotine injected before and after the MAO inhiltitor, Jl3 518 (pheniprazine; d-pheuylisopropylh.•drasine; Catron). The mead changes in heart force w nicotine were 143 * 38 Rm bPfore, and 78 d: 30 gm after, itqectiou of 5 mg/itg pheniprarine; Alood-pres- suure changes and catecholamine levels were also reduced. According to Leaders and Long (i902c), an increa.oe in the myrocatdial force of contraction in dogs could be elicited with one-thirtieth of the dose of nicotine necessary to obtain coronary perfusion-pressure reslwnses (see also below, 339 and 394). Left ventricular mechanical efficiency (work) was un- changed by nicotine infusions into anesthetiaed dogs (Forte et a)., 1980) (previously reported by Schmitthenner et al., (19fi7 )(138a)). 13elletand co-workers (1960) found a significant increase in cardiac output at %vork in normal (anesthetized) doge given femoral-vein infusions of 20 µg/kg/min nicotine. Nicotine increased cardiac work markedly (J. 11'. West, )3ellet et al., 1982; liellet et al., 1902) through an btcrease in the mean arterial bloryl Imssure and rardiac output (ljellet et a).,1982). The degree of this increase was said to be shnilar in uonnal dogs and in dogs with chronic coronary insufBciency (Nellet et al., 1962); but it aa alsu said that, in animals with experimental coronary insufficienc)~, cardiac otitl'rut ,,,,d w•nr4 wnrn inrrrromd in• nicntino inius;inn to a ltrcr extent than in normal anitnal. (Ifellct ert al., 1960)- f`or details of these experiments, see below, 391. The EhG ti.:ani• festations of nicotine admiiustration or cigarette-smokc inhalation were not correlated, either teml,wrally or in imag• nitude, with the changes in cardiac work (laen and Shetnnl, 1960). Regardless of the observed degree of change in the level of cardiac work, there was a brief but marked incqraw in the oxygen saturation of the coronary venous blood. Arterial-blood ox;•tien-saturation did not 'change. Equating thue parameters with changes in coronhn•-blood-Bow red vealed a precipitous fall in cardiac osygetj utilieat'ton which immediately preceded the F.KG changes. Details of many of tht: experiments reported above are given under 391. Coronary Circnlolunr, bela~. 330. Cfrcrrlation-Time (138) Circulatory effea•trt, measured by detemunation of the rat-tail fluoreRCence-time following i.p. injection of 600 mg/kg sodium fluorescein, were determuned by Wenzel, ingionna and Grundeman (19(il) on Sp*ue-Uatrley , rats subjected to one of the following treatments for period of 28 days (Suorescence-times in see at the end of 28 days): 1. control (no treatment), 75 f 4.6; 2. nicotine (1.14 mg/kg/day in the drinking-water), 195 at 13.1; 3. epinephrine (2 mg/kg in peanut oil s.c. on alternate days), 388 f 8.7; 4. ergondt-ine malcate (0.5 mg/kg/day i.p.), 396 ot 10.2; 5. nicotine pluK etgonovine (dosages as above), 550 f 9.5; 6. epincphrhte plus ert„ronovine (dosages as abo%e), 553 * 23.3. All tbmt• ments differed froni the control by p- <Q002. Changar in fluorescrnce-time at 14 days were slightly lcsc than half of those obtaining at 28 dkys, illustrating the cumulath•e nature of the -ffects. 331. Calecholantine Content of the )feart of N'irntine-Treuted aninafa/Organa (138) b: contrast to the findings of DeSchaelxlr)ver and Preziosi (1959) (138b), Sharman, Vanov and Vogt (1962) reported that i.p. injection of 0.1 mg/l:g nicotine I and 3 hours before sacrifice did not significantly change the norepirephrine content of heart or spleen of mice from that of control values. The content of epinephrine, norepinephrine, doparnim, and DOPA in the heart of rats receiving 20 mg/kg nirotine i.p. was deternined by h'agai '%1963a, b) at various stagdx of nicotine convulsions; DOPA was said to have increareil at the tonic-com•u)sion phase, but the other catecholamines remained unchanged. In acutely adrenalertomised rats, norepinephrine in the heart increased during this Esnie phase, and the other catecholamines retnained unchanged. Westfall (1985a, b) administered nicotine to Sprague-Datckc,v ratq in dosage o1 I mg/kg/day i.p., and determined norepi- nephriue and epinephrine content of the heart at intervala for S days; norepinephrine content uf the heart showed nu chanae, while there n•as a decrease in the epinephrine content on days 3 and S. A closer study of the time-reRltonFe pattern was undertaken with two dosages of nicotine (0.5 and 1.0 mg/kg, i.p.), the rat: bcing sacrificed for tissue anahrer at 5, 15, 60, 180, and, in sonte cases, 14-f0 min (24 hours) later. With both doses, there was an immediate decrease in the norepinephrine content of the heart mcasurable in 5 pnin, and reaching a maxinium in 15 min, with return to a level slightly greater than the control at 60 min. 1loaimum dc- creases were to 79.7 and 68.9 0 of controls for the 0.1 and 1.0 tng/kg doses, respectively. Produced bv The Gouncil for Tobacco Research-USA, Inc. 0002986

CA11D101•ASCULAR SYSTEM 75 lisrmon, Alasuoka and Clark (1963) gave mice, rats, and .• {n • Z.• inM:nnn r.~ ntnn.i.+n nn•ntn;rt nnri rel>hried that~ the catecholamine and serotonin content in the beart remained unaffected. In mice sacrificed 60 min following i.p. injection of 0.1 or 0.5 mB/kg nicotine, or 0.1 mgAkpt every hour for 4 hours, no definite effect was Found in the total-catecholamine levels of the heart (Hansson, Masuoka and Clark, 1964; Hansson, 1965). A sbnilar lack of effect on tot,dl-catechoiamine levels in the heart was observed in rats or guinea pigs sacrificed 60 min following i.p. injection of 0.1 rag/kg nicotSne. Adrenalectomised tsts did not differ froitt btormal animals in their response to nicotine. In dogs under pentobarbital anesthesia, infusion, of nico- tine in doses of 2 mg/kg over a period of l hour (i:. Kako, Chrysohou and Bing, 19CA), or i.v. injection of 1 mg/kg nicotine (Kako, Chrysohou and Bing, 1961), caused a slight, but statistically insignificant increase in myocardial-cate- cholamine concentration. In animals pretreated with iproni- a:id and DOPA, however, nicotine caused a significant 'm- crease in myocardial catecholamines; and Kako, Chrysohou and Bhtg (1961) considered it likely that stimulation of the adrenal medulla and sympathetic nerve-endings by nicotine elevated catecholamine levels in the blood, with subsequent storage in the heart and other organs. \luaeholl (1961) found no effect of nicotine on the cata cholathine content of cat atria; but Swaitte, Perlmutter and Ellis (1964) reported that nicotine released a relaxing sub• stanre---a cateeholamine, probably norepinephrine-from Langendortf preparations of the isolated cat-heart. (ln a few experiments, rabbit heart was substituted for cat heart.) Nicotine (25-100 pg injected into the aortic camiul&) caused cardiac stimulation, and released a substance into the per- fusate which produced a rela%ation of isolated rabbiGintestine (used ps the assay tissue). Pretreatment of the heart with 100 gg dichloroi.coproterenol blocked the effects of nicotine on the heart, but not its release of the relaaing substance; 1-5 µg/ml, cocaine reduced or completely eliminated the effect of nicotine on the heart, as well as the release of the relaxing substance. With hearts from reserpine-pretreated cats, nicotine caused only equivocal effects ou the heart; and no significant release of relaxing substance. For other chemical changes in cardiac tissues, see below, 33g. 332. Norepenephrine Uptake 6y Cardiac T:;ssuea Leaders and Long (1902a, b) demonstrated that there was no evidence of norepinephrine uptake by isolat-d cat atria afte p:Itglioimctnmy and nerve degeneration, but uptake was suggested in atria from control animals, and from tho.se pretreated with reserpine (for a further account of these eq)eriments, see below, 337-8). 338. Histopotlrolog,u o,/Uee Hrort Jollousng t lnonft A'ttifi.ne or 9bbacco.Srnol•e .-1dm.int.sEratfon (l39) Expu?riments by Wenael, Turner and Kissil (1959b) on rabbits suggested an interaction between nicotine and cho- lesterol to produce cardiac neerosis )for detaiL, see (191a-b)). Subsequent exVetituents by R•ensel and his associates (1961) revealed an absence of cardiac necracis in rabbits receiving 1.14 nig/kg/day of nicotine in their drinking-water and 1% cholesterol and 5% cottonseed oil in their diet for 24 weeks, in apparent contradiction to their earlier findings. .lreas of cardiac necrosis were also absent i:i animals receiving nicotine Hlone, cholesterol alone, or a basic diet without either. On the other hand, areas of cardiac necrcasie were present in rahhi.e ra,r=vino rhr hvnnmhnfae/ominmir ,iio1 nlno inivrrinn of 0.05 mg/kg ergonovine maleatc i.v. at 4-week intervals, as well as in animals receiving nicotine plus cholesterol plus ergonovine treatment. Accordingly, it was felt that the combined effects of nicotine, ergonovine, and cholesterol were necessary for the observed damage to the myocardium. Similar e%periments, in which 5 mg/kg caffeine or 0.025 mg/kg ~.uabain were injected i.v. once a week, alone or in conjunction with nicotine and/or the hypercholesterolcmic diet, were said to have produced a few myrocardial lesions, although histopathotogic study of the hearts did not allow of any clear-cut conclusions (Wensel, MacCarthy and Rut- ledge, 1964). Nensel and Stark (1965) produced myocardial necrosis in female Sprague-Dawley rats by the following regimen: daily injection of deaametluasone and de3oxycurtiro- sterone acetate, and gavage with a solution of monobasic sodium phosphate for 5 days, followed by 7.5 hours of restraint on the 6th day. Separate groups given the above• described treatment were concomitantly administered 0.14, 0.57, and 2.28 mg/kg/day of nicotine in the drinking-water; groups of animals receiving the latter dosage of nicotine were also given 1 mg/kg hheneisine or 12•3 mg/kg mer- amylamine daily, or pretreated with I mg reserpine ier animal per day 3 or 7 days prior to, and also during, the steroid-salt.stress treatment. The two highcr doses of nicotine significantly (p < 0.01) increased the average lesion severity, the per-cent incidence of lesions, and the per-cent mortality. Myocardial potassium was significantly decreased (p < 0.01 and 0.05). Treatment with mecamylamine or pbenelaine did not reduce the response to the steroid-salt-stress treatment, but significantly (p < 0.01) reduced the severity of the response when nicotine was added to the regimen. Seven days of pretreatment with reserpine did not change the response to nicotine, whereas a 3-day pretreatment increased (p < 0.01) the severity of myocardial lesions. White mice ea-Iwsed to either light or heavy concentrations of cigarette-smoke fo• 10 min daily for periods up to 703 days were said to show necrotic changes in the heart, as• sociated wi,h coronary disease (Komesytitski, 1958). Lupu and Velican (1901b, 1962) found, in the myocardium of guinea pigs subjected to chronic cigarette-smoke caposurc, certain very marked modifications in the histological and histochemical picture (for details, see below, 1090). 334. Heart•Ltmg Preparatfmt (139) In eaymrimcnts with the Starling heart.lnng preparation o/ the dog, J. H. liurn and Walker (1954) found that the injection of 20 mg hexamethonimn abolished the action on the heart.-rate of 5 mg nicotine acid tartrate; however, when 30 mg hesamethonium were given, and followed by physo. stigmine in concentration of 1.2 X 19-I, .11, the injectionof the same dose of nicotine caused inhibition. The authors postulated that, either thc presence o1 ph•isostigmine relieved the hevamethunium block, or that, in the presence of heu• methonium, nicotine still exerted a slight but undetected action on vagal ganglia, and that the previously sub-effective amounts of acetylcholine liberated at the Iroxt-ganglionie endings became effective in the presence of physostigmine. A second observation by these workers concerned aa action of nicotine which resembled that of physostiRmine. Since nicotine stimulates receptors sensitive to acetylcholine, it ia likely to have some power to combine with, and therefore inhibit, cholineaterase. In the e..•)rerimmnt in which 20 ing hesamethonium were injected, aod in which b mg nh•otine Produced bv The Council for 0002987 Tobacco Research-USA, Inc.

76 TOBACCO-EXPERIDiENTAL A\D CLINICAL STUDIEg. RCPPLEAlE1"P I ~ acid tartrate were then found to be rnactive, a turuier w~ec- tion of 10 mg nicotine acid tartrate was made (the total nicotine acid tartrate present in a volume of 700 ml wae at this point 18.4 mg). This last i.ijection ot nicotine caused a slow drop in the heart.•rate from 142 to 118 in 6 min, and the rate was maintained at the low level. The inhibitory effect of I µg acetylcholine was then greater than before the Idrge dose of nicotine was given. Thus, it appeared that the ]gage amount of nicotine had had a pbysostigmine•like effect. SSS. laotafed, Perfused .Uammalihn Heart (140) On the perfused cat- or rabbit•heart, 1% nicotine HC) caused initial acceleration and strengthening of contractile energy, followed by gradual xloning of the beat to below control rates (1{akowski, 1905). Similar effects were observed on atropinized hearts perfused with nicotine. Other workers (e.g. Reinert, 1959) have also reported that nicotine had a positive inotropic and chronotropic action on isolated eat- and rabbit-hearts. In the Langendorif preparation of the atropinized rabbit.-heart, 0.2 mg nicotine caused an increaCe in contractility and frequency of about the order of that resulting from 0.005 mg epinephrine ()t{ukovets, 1!)62). S. Middleton, fL H. Middleton and Toba (1919) nreported that, in the atropinized, isolated cat-heart, perfused with T yrvde's solution, the excitation of the cervical vagus induced positive inotropic and chronotropic effects on both atria and ventricles, and these effeets were abolished by nicotine in concentrations of " ntl/liter. Nicotine per se caused an initial period of increased activity, which passed off rather rapid:y; excitation of the past-ganglionic sympathetic pro- duced a cardio-stimulating effect as intense as before nicow tiniaation. F. Hoffmann, S. Middleton, Talesnik and NTi)liam- son (1952; compared the reactions of ieolatad-vagus taeati preparations of normal hearts, to hearts in which cardiac sympathetic fibers or cardiac vagus fibers had been caused to degenerate through operative procedures carried out 12-16 da,-s previously. Nicotine injected into the abrtic cannula cbse to the heart in doses of b and 20,rg regularll• induced in atropinised hearts (7 normally innervated; 6 vagatomised; 5 sympathectomised) the welbknown stimu- lating e6ectof Increased amplitude a.-td frequency of ventaic- ular contractions. In normally innervated and "vagoto- miaed" hearts, the magnitude of the response to nicotine was similar; in "sympathectomised" preparations, hoarever, the drug exerted a rather slight effect on the amplitude of the cont.ractdons, a phenomenon characteristic of the responses of the "sympathectomised" hearts to epinephrine and aee- tylcholine. Hashimoto and ro-e-orkers (1964) used the innervated Langendorff preparation of dog-heart in ventricular fibril- lat imt with cotnpletely, separated cross circulations to the had and the heart to study carotid-reflex effects on heart-rate and coronary flow (see also above, 313, aud below, 391). Rapid injection of 0.02-0.1 mg nicotine into the blood per- fusing the head caused temporary arrest of the at.tial beat., which soon reappeared, and tended to overshoot the control rate. Coronary flow began to increase during atrial arrest, and became tnmimal when the atrial rate aas returning to normal or above. When the carotid area was completely de:nervated, neither deceleration nor acceleration of auricular rate was produced by nicotine, and there was no chanRe in coronary flow. After administration of I tng atropine to the isolated heart, or after cutting both vagi, the initial response of deceleration of atriel rate artd increase of corotutry flow ,,:. 2 .t ...;01 mfo woA dprrpsse of coronary flow occurred. When a latge (0.5 mg) amount of nicotine was given, the acceleration lasted longer, and the decrease of coronary flow was reversed to a Iong-lasting increase. Travell, Rinaler and Kauh (1980) reviewed their earlier finding (Rinaler, Travell and Kaq) (1957); Travell (1957) (141a)1 that nicotine caused an initial decrease in flow in perfused hearts (modified Langendorlf preparation) fmm both control and cholestet'+i)I-fed rabbils, but a subsequent rise in flow in only the normal hearts, vasoconstriction was greater for the nomtal than' for the atherosclerotic hearts. Mannsioni (1960) reported that the norepinephrine an- tagonist, dichloroisoproterenol (DCI) in concentration of 0.01 mg/ml abolished the bi'phasie uvtiun (inhihition folkmed by stimulation) of 0.05 mglmi nicotine on the isolated per- fused hesrt of the Ruinea pig. Swaine, Perlrnutter and Ellis (1961) found that pretreatrnent of isolated cat-heart (IAjtit- endorff preparation) with 100 µg DCI blocked the effects of 25-100 tng nicotine on the heart, but not nicotine•induced release of norepinephrine (r.ee also above, 331). These effects were also reduced or completely eliminated by 1-5 µg/ma cocaine; and with hearts from reserpine-pretmated cats, nicotine caused only equivocal effects on the heart, and no significant catecholami»e release. In such hearts, following infusiott of levarterenol (/-norepinephrine), nicotine did not induce catecholamine release, and only occasionally were there sitrns of cardiac stimulation. Isolated rabbit--as well as isolated cat-hearts, were also stimulated by this dose of nicotine. In 1sngendorS preparations of the isolated hearts of rats, guinea pigs, rabbits, eats, and dogs, the effect of acetylcholine and vagal et+mu)ation was enhanced by g-strophanthin, and this enhancement could be blocked by perfusion with hexatnethonium or with 2-4 pglmi nicotine, arguing for a ganglionic action of the g/ycoside (Pen;v and Reinert, 1954b). On the rame typ- of preparation of isolated rabbit-heart, serotonin showed rome cra.:ted tachyphylaaia with nicotine with respect to its inotropic atvl chronotropic effects; beneyl- 1-dimethyl-2,bserotonin (IiAS) antagoni:ed the stimulant action of nicotine (Jacob and 1'oite.Bet•ierre, 1t1B0). Ar rhythmias induced by BAS were not antagonized by nicotine. Harkavy and Perlman (1960, 11164) reported that, in perfusion experiments with the isolated heart of an im- munitcd rabbit, homologous tobacro.ealract caused a sig- niGcant deeresse in contractile force, some decrease in heart- rate, and consistent decrease in coronary flow. (For an account of the immunir.aUon procedure followed, see below, 1156.) No such reactions aplwared in 7 control auintals. Two immunized rabbits were said to have been resistant to the perfusion of nicotine in doses as high as 200 pg Since the immunization was to virtually nicotinedtre tobacco-extracts, and sinre, in addition, nicotine is not demonstrably antiF,rnir, it is difficnlt to perceive the mechindsm bky which 2 tobacco- immunised rabbits were especially resistant to the cardiae perfusion ot nicotine. It 4 tempting to suspect that tht~le tNro particular animals may simply have been out on one of the limbs of the bell-shaped frequency-distriLution-cun•e, and that a sut6cient number of "imnuniud rabbits" would have estabUshed the true shape of this curve. Kukovets (1961, 1962) correlated the increasr in con- tractility produced by nicotine on LangendorQ pretuaratiima of the rabbit heart with phosphorylase activity of the heart muscle; see below, 338. Produccd by Tile Council for 0002988 Tobacco Research-USA, Inc.

CARDIOVASCULAR SYSThxtt 77 936. Embryonic Heart (142) •~.wuuu xne iuuuu uy W. v. to exhibit marked positive inotropic activity at a ooncen- trBtion of 20 µg/ml, or greater, on the non-innervated heart of the 4-dayold chick-embyo. An initial transient positive chronotropic effect was usually observed, followed by a decrease in rate. Higher concentrations produced negative chronotropic elgecte. Innervation (e.g., 8-12-day-old embryos) did not sigtti6eantly alter the cardio-stimulant response to nicotine. Dichioroisoproterenol, which blocked the effects of nicotine on the heart of the 4-day-old chick-embryo, abol- ished the cardio-stimulant responses to nicotine, but not those to bariann chloride or ouabain. Trimelhyl 12-f2,6- xylyloan•) propyl) ammonium chloride (a congener of choline 2,&rylyl ether), which interferes with release and/or syn- thexis of the adrenergie mediator, blocked the cardiac re- sponse to nicotine, but not that to epinephrine. Reserpine markedly reduced the catecbolamine concentration of the 15•dsy-old embryonic beart, and significantly decreased the positive inottapic response to nicotine of the heart of the b-day-old embryo. Significant amounts of catecholaminu, as demonstrated by spectrophotometric methods, were present in the 4-day-d1d embryonic chick-heart• The authors con- cluded that the positive inotropic and chronotropic effects of nicotine are not dependent on the presence of sympathetie nerve•eadings in the beart; and they further suggested that there exists between the post-ganglionic syanpathetic nerve- endings and the adrenergic receptora in the heart, certain structures which contain epinephrine-like material(s). Nico- tine, and similar agents, cause a release of this material, which in turn effects the positive inotropic and chronotropic tesponses. 337. lsolated Cardiac Tissues (142) 887-A. S-A Node. Amory and West (1962) studied the responses of the isolated sino-atrial node of rabbits; sus- pended in an organ-bath, the preparation showed regular spontaneoas contractions, and when 2 pg/ml nicotine were added to the batb, inhibition, followed by an acceleration in pacemaker nltes, occurred. The same dose applied to a reserpinised node produced no positive chronotropism, iatplyting that the acceleration was probably due to release of norepinephrine. The negative chronotropism was en- hanced by 0•5 pg/ml eserine; and the negative chronotropic effert could be blocked by 0.5 µg/ml atropine, when ac- ecleration alone was observed. The potentiation, and its antagonism by atropine, point to an effect mediated by scetylcholine. In the presence of 20 pg/ml bexamethonium, the action of nicotine was completely suppressed. 357-8. laolated Auricle. Nicotine in concentration of 10 vglntl (Lindmar,1952) or of 1-300 µg/ml (Forster attd Hoefke, 19m) had no effert on rat. auricle4. On guinea-l.ig auriclc.z, 5 pg/ml nicotine increased the rate and force of contmction (Lindmar, 1962); 13 µg/ml had a pronounced effect, regularly increasing amplitude and rate of beat (L. 7Yendelenburg, 1960); 50 pg/ml produced a clmlincrgic Itiltibitory and an adrenr*gic stimulaton• effect (Pepeu, Nlannaioni and Giotti, 1959). Kic•otine consistently produced a biphasic chrunotropic reqponse on rabbit atria, tna•cimum slon•ing occurring in I-:i min, marimum acceleration in 5-S min (Leaders and Long, 19ti2h). On cat atrie, 27 p;t/mi nicotine caused accelt-stion, pnYrdcd by tmnsient deceleration (U. Trendelenburg,1960); but it has also been reported that 1-2700 pg/ml had no chrono- troaic or inotronic effect on isolated auricles of this species (Forster and Hoefke,1960). The stimulant action of nicotinb on eat atria wae still evident 12-14 days following removal of both steliete ganglia, iand this action was abolished by hexamethonium (Ginzel And Kottega l= l:otugoda) 1954). As a preliminary to discussing the effect of other drugs bn nicotine action on isolated auricle, by author, it may be helpful to list alphabdfcolly the various drugs employed by R.lte several investigatots, and indicate in the most summary fashion, and by symbols, their experimental findings, with 0 signifying that the prior drug had very little or no effect on ihe regular inotropic and/or chronotropic actions of nicotine bn this tissue; - signifying that the nicotine effect was in hibited or abolisLed lu a greater or leooer degree, and + signifying that nicotine action, or some phase of it, had been enhanced or Iwtentiated. Obviously, these symbolic results are the function of drug-concentration and/or experimental conditions, and, in many instances, +, 0, and - n•ill be seen to be dose-dependent when the details below have been studied. Ajmaline, -(Bardolini 'et al., 1903); amino•2- metbyl-6•beptane tartrate (.(.L. 1~4(1), - (Timsit, 1961); d-amphetamine, - (llnteit, 1961); atropine, -(Lfvy and :1lichel•Ker, 1956d, e; Schmitt, 1956; 1'epeu et al., 1959; U. Trendelenbutg, 1960; Jlattilp, 196.3a, b) or +(L4vy and Aiichel•Ber, 1956e; GlasSer, 1960; Azarnoff and liurn, 1961); bretylium, -(Asarnoff artd Bum, 1961; H. Boyd, Chang and Rand, 1961); chiorisondamine, -(1'hilippu and SchO• matw, 1962); choline 2,6-xyly1 ether, - (U. Trendelenburg, 1960; Schoepke and Shideman, 1961 ); cocaine, -(U. Trende- lenburg, 1960; Pbilippu and Selpr7mann, 1962; \lattila, 1963s, b); dichloroisoproterenol (DCI), - (\lannaioni, 1960; U. Tmatdelenburg, 1960; Lindmar, 1982; Mattila, 1963a, b); dimetbylphenylpipetasiniw» (DUPP), - (Sonrin and Nlaflii, 1956)p epbedrine, - (Mattila, 1963m, b); epi- nephrine, + (Pepeu et a1,195f1); eserin., 0(Ikvy and \lichel- Ber, 1956e) or +(Iksy and Alichel-,Ber, 1956d; I'epeu et al•, 1959); ethylnotiilcotiae, -(\Iattila, 1963s, b); gallamine triethiodide, 0(Livy and \lichel•13er, 195tid); troanethidine, 0(Kadaielatva, 19ft2) or -(Kadsiefar•e, 1962; Philippu and SchOmamn, 1962); hemicholiiuum, - (Leaders and Long, 1962n); hexamethonium, - (LM•y and \lichel•lter, 19,.46d; U. Trendelenburg,1960; Vehioaky and Clark, 1960; Lindtnar, 1962; Jlattila, 1963s, b); hydrasino•2-ntethyl-tihefrtane oxelate (D. 1301) and h•vdrazitio-2•oetane sulfate (D. 1514), - (Titnsit, 1901); iproniazid, -%l'epRu et al., 1981); lysergic acid diethylamide (LSD), 0(Ikvy b<nd \lichcl-lier, ll95&1); I•methionine, + (Sugimoto et al., 10(34); monunminc•c.~dase ('.%LaO) inhibitors, -(1'epeu et a1., 1961); morphine, - (U. Trendelenburg, 1960); norepinephtine, + (Asnrnoff and Burn, 1961); nornicotine, -(Aia(tila, 1963a, b); penta- methoniutn, - (Ldvyaod AIpcbel•Bcr, 1956d); phcnipraaine (Cat.ron). 0(Sugimoto et al., 1964) Or - (Pepeu et al.. 1961; Timsit, 1961); procaine, 0(H. Bo,vd, Chang and Rand, 1961) or -(LAvy and \fiehe)-Ber; 1956d, e; Boyd, Chana and Rand, 1961); pyrilaminc mAleate, - (U. Trendelenburg, 1960); pyroltallol, -(Sugunoto et al., 1964); quinidine, - (Bartolini et al., 1963); reserpkne, -(J. H. Burn and Rand, 195Sd; Pepeu et al., 1959; Soncin and llaR, 1959; J. J. Reuse and Bourgain,1960; liuffoni and Giotti,1961; Schoepke and Shideman, 19(31; liadsielawa, 1962; Leaders and Long, 1962a; Lindmar, 1962; \lattila, 1963a, b; Sugimoto et al., 1964); serotonin, 0(Lfcy and Uicht•l-ller, 1957) or -(Lttr•y Produced b,.f The Council for Tobacco Researcli-USA, Inc. Uoo298J

T. 78 TOBACCO-E.CPERI.NfENTAL AND CLINICAL STUDIES. SUPP1.EbfEA'T I mann, 1962); tetraethylammonium •.~ ~ - -.. . , - (Velnosky and Clark, 1960; Mattila, 1903a, b); thiamine, -(Mattila, 1963a, b); tripelennamin2, - (U. Trendelenburg, 1900); d•tubocura- rine, -(Ldvy and Micbe)-Ber, 1950d); veratratnine, - (Lindmar, 1962). Previous nicotinisation of isolated auricle failed to block the stimulant action on this tisaue of amino-2-methvl-8- heptane tararate (J.L. 1340) or of d-amphetamine (Tirnsit, 1961); epinephrine (Lkvy and \fichel-Ber, 1958d, 19b7); hydrasino.-methyl-6-heptane oaalate (D. 2524) or by- drasino-?.octane sulfate (D. 1514) (Timsit, 1961); 4-metbyl- 2-aminopyridine and isomers (Schoepke and Shideman, 1961) and pyridine and certain metln•1 derivatives (Schoepke and Shidernan, 1962). Phenipmzine (Catron) itself did not afiect the contraction of isolated atria, but on atria pretreated wtith nicotine, pheniprasine caused marked stimulation (Sugimoto et al., 1964); the positive inotropic and chronotropie action of pheniprazine was not suppressed by paral,vsant concen- trations of nicotine (1"unsit, 1901). The stimulant effects of the following drugs were reported to have been blocked by prior application of nicotine to the test tissue: acetyleholine, acetyl-d-methylcholine, buty rylcholine, histamine, and sero- tonin (Lr?vy and Yfichel-Ber, 1956d, 1957) and tetramethyl- smmonium (Scboepke and Shideman, 1901). The first phase of DNIPP action on isolated auricles was blocked by prior .administaation of nicotine; the secon,d phase was only slightly tedueed by it (8oncin and Aiaffii, 1959). Details of experimental conditions, dosage, and results, and their authors' discussion of their findings, follow in chronological order: Lhvy and Afichel-13er (1958d) reported the effects of a num- her of drugs modifying the action of nicotine on guinea-pig and rabbit auricles. Eserine, on guinea-pig auricles, potentiated the negative inotropic and chronotropic effects of nicotine, but not the positive ones; on rabbit auricles, it considerably potentiat.ed the initial inotropic and chronotropic effects of nicotine. Atropine, on rabbit auriclea, abolished the depressor phase of nicotine on the eserinized as well ac on the non- eserinized auricle. d-Tubocurarine supprt-ssed the stimulant action of nicotine on guinea-pig aiuricles. Ilexaroethonium inhibited the depressor effects of nicotine and suppressed the stimulant action of nicotine on rabbit auricles; on guinea. pig auricles, this drug abolished the t,egative inotropic and chronntropic effects of nicotine. Pentamethonium rnhibited both the depressant and stimulant action of nicotine on rabbit auricles. Gallamine triethiodide did not modify the stimulant action of nicotine on guinea-pig or rabbit aurieles. Procaine suppressed the effects of tticotihe on both guinea- pig and rabbit auricles; 5 µg/mi procaine diminished or suppressed the Ixrsitive inotropic and chronotropic effects of nicotine on rabbit auricles (LFv,r and \iichel-Ber, 1956e). Lyreqtir acid diethylamide (I SD) did not alter the atimulat+t action of nicotine ou rabbit auricle (Lisvy and Michel-i3cr, 1956d)• Serotoniu (5-h~dro.ytn~~tamine; 5-HT) blocked the action of nicotine on rabbit auricles; incidentally, a eoncem tration of 0.075 µg/mi nicotine h,rdrocbloride did not prevent development of taeh.•ph-i•la%is to serotonin on thiq tissue. In concentration of 25 pg/ml, nicotine hydrochloride had a marked biphasio action on guinea-pig aurirlex; and, at the end of the etimulant pha.ce, serotmnin w9s inactive, although the stimulant. effect of epinephrine e•as still apparent. Added at the moment when the stimulant effect of nicotine on r abl : auriclre ceased. serotonin or histamine had no effect, a•hih• epinephrine continued to have lrositive inotropic and chrono- .:~ ~: . ..... - ....~•:::d .... ~~V.'.-.~::. .u...u..::..~ :: •c inotropic effects, and the subsequent positive chronotropie and inutrupic effects, of nicotine on rabbit auricle, serotonith or acet;-Icholine or acetyl-p-methylchqline had no effect on rhythm or amplitude. The stimulant effect of butyn•Icholine on the atropinized rabbit-auricle was also suppressed by this concentration of nicotine (Lhvy and 1Jichel-Ber, 1957). Pelmu, \lanniaoni and Giotti (19,59) reported that 50 pg/mI nicotine produced a cholinergic inhibitory, and a s)mpathergic stimulatory, effect on gpinea-pig auricle. The cbolinergic effect, which progressively decreased to thg point of disappearing, could be restored by pretreatment with 0.1-0.01 pg/ml epinephrine or 0.1 µg/ml eserine, while it was suppressed by 0.1-0.01 pg/ml atropine. The stinro. lating effect of nicotine constantly disappeared io surieA% treated with reserpine, in a period of time varying from 1-2 to 5-4 hours, and the loss of stimulating effect of nicotine was Wd to be in no way correlated with the negative cbrono. tropic effect of reserpme. In auricles which were quiescent through the action of reserpine, and Mo longer reactive to nicotine, epinephrine, norepinephrine, isopropylarterenol; and histamine, were all able to restore the rhythm and strength of contraction to a value only alightly less than nomtal. When, in the presence of reserpine, the auricles were pretreated with epinephriue and eserirm, nicotine was once again active, which was not the ease following administration of atropine. Pretreatment of isolated guinea-pig atria for 4 hours with 450 pg/ml iproniasid (Uanailid) clearly reduced, and similar treatment with an equimblar concentration of pheniprasine (Catron) completely abolished, the effects of subsequently-added nicotine (Pepeu et al., 1961). In auricles which were quiescent through the actiob of reserpine, and no longer reactive to nicotine, ephedrine and serotonin did not restore the rhythm and contraction to any apl•zeciable degree (Pepeu, 1lannaioni and Giotti, 1959). Soncin and \iaflii (1959) reported that D\fPP produced on guinea-pig auricles an initial decrease in the amplitude and rate of contraction, promptly followed by a positive inotmpic and chronotropic effect; the first phase of DX11'P activity was blocked by nicotine; the second phase, only slightly reduced b; it. Pretreatment with DXIPP abolished the effects o1 ni•etine on isolated auricles. The stimulating acti%it,r of D.NIPP was still present in auricles.in which the effects of nicotine had been abolished by rrserpine. J. J. Reuse and Bourgain (1900) statad that pretreatment with raxrpine appreciably or markedly inhibited, but did not abolish, the nicotine response of guinea-pig atria; the effects of nicotine were also appreciably inhibited in isolated atria from mhbits pretreated with reserpine. 1-. Trendelenburg (1900) studied the action of nicotine alone, and in connection with other drugs, on isolated atria of the guinea pig, rabbit, and cat. On cat atria, 27 µg/ml nicotine rnumd arcelcrstion, preceded by a transicnt dcrel- elation, which was fully abolished by b.67 pg/nil atropine. Hemnethoninm abolished the accelerator tesporux to nicotine without affecting that to histamine ot eerotonin. Choline 2,6-s,vlyl ether bromide (TAt-10), in concentration of 33 pg/mI, abolished the reslxmre to nicotine. Incrcasing amounts (6.7-6s pQ/ml) of morphine sulfate caused increased de- luesrien ci the nicotine response, as did 1; 3-6.7 µg/ntI cocaine hydrochlmidc, but the response wa.c not abolished by these courentra+ii.n.. IncreaQing amounts of the antihistaminies. p~'rlauritw ,unleate (3.3-13 µg/ml) and t.rilwllrnnmine (03.'i- 3.3a N;m)l:, ai.o progrescivel,r decrearrd the nirotine re.lwnse. Dirhb)roit"ptvicrenol (1)CI), in concentration of 0.33 pg/nil, Produced bv 1'",.% Council for Tobacco Research-USA, Inc,

CARDIOVASCULAR SYI3!M1 79 abolished the response to nicotittle. Isolated guinea-pig atria tine; however, on atria from animals pretreated with 15 , ., .t • 7• , ,... . . • , • / t atropine, caused a rise in the rate of beat of rabbit atria. menta have been supplied by Leaders and Long (1962b), Addition of reserpine to the oqgun-bath blocked this action who also reported some additional experiments on isolated of nicotine, as did bretylium (which lylocks the effect of stimu- rabbit-atria. lating post•ganglionic sympathetic fibers, without affecting Lindmar (1962) stated that the positive inot.ropic and the action of norepinephrine). When norepinep'hrine was chronotropic responses of nicotine were abolished on auricles ,CAtw.......n u,. ao I'ly..... v.. e ...... .. .........u..,... - ...O...p e..«............. ....... ...y .... .. . , ............... regular increase in amplitude of contraction and in rate of action of nicotine was observed. Similarly, naserpine added beat. Since changes in amplitude were more conveniently to the bath in concentration o15 jug/ml had a weak inhibiting measured than changes in rate, the former were used for effect on the action of nicotine, but in atria from animals calculation of results; and the pnthgonism of the nicotine pretreated with reserpine (2.5 mg/kg i.v., 2-1 bours pre- efleet by the substances liated above was essentis0y similar viously), there was almost no reaction to nicotine. to that found on cat atria. Rabbit atria responded to nico• Leaders and Long (,1962a) stud:ed the mechanism of the tine similarly to guinea-pig atria; iexcept for some quantitative cardio-stimulant action of nicotine, using cat•atria prepn- diBerences, the results with the kubstances listed (excepting rations with parasympathetic and sympathetic nerves intact. DCI, which was not tested in this species) were essentially Inhibition of the parasympathetic nerves by 20 pg/ml hedni- similar to those on guinea-pig atria. The isolaled atria of cholinium abolished both negative and positive chronotropic cats pretreated with reserpine did tpot respond to the usual phases of the response to 10 jug/ml nicotitre; the inhibition concentration of nicotine, nor to twice this amount; atria was reversible. During the period of inhibition, sympathetic from reserpinized rabbits showed little or no 1+ecpnnse to stimulation praiuctvl , it= rharncterictic responftc. (`hronic nicotine; and pretreatment of the animals with retterpine gangliectomy or reserpinisation abolished the positive chrobo- appreciably or markedly inhibited, but did not abolish the tropic response to nicotine. ;:orepinephrine uptake N•as nicotine response of guinea-p(g attia Asarnoff and )iura observed in atria front reserpinized and sham-operated, but (1961) found that 2.2-4.5 Kgido! nicotine, in the presence of not from gangliectomixed, animals. Details of these e?cpCri• allowed to act on the attia for 20-3m min, and then removed taken from guinea pigs pretreated with reserpine, nor coiild from the bath by repeated changes of the bath-fluid, the the action of nicotine be restored by the presence of norepi- act.ion of nicotine was greatly iatcrea.§ed (the frrat addition of nephrine in the bath-fluid. norepinepbrine often had a mtich sinaller effect on the re- Philippu and SchiSmann (1962) reported that the inotrooic spom;e to nicotine thar did later additions). and chronotropic effects of 1.6 µ.l/ nicotine on guinea-pig H. Iioyal, Chang and Rand (1961) also reported that 40 atria were inhibited by 0.4 p.U guanethidine, 9 pDl chlori- pg/ml bretyliwn completely blocked the increase in rate sondamine, 0.3 µ.wl cocaine, and 0.9 101 tetracaine. and amp'.itude of beat normally produced in rabbit auricles \iattila (19ti3a, b) reported that 0.03-0.05 pA!/mi nicotine by an equal concentration of naeatine. In comparison, in the produced an initial bradycardia, iollowed by acceleration presence of concentrations of plnocatne which produced a of rate and augmentation of amplitude, of guinea-pig atrium. marked effect on the refraetory period and slowed the rate The nicotine effect was antagoniaed to some extent by nornic- of beatthe response of the attid to nicotine was either otine and ethylnornicotine, but the poRitive chronotropic unchanged or very slightly affected. and inotropic effect was not antagonized except in larke, Schoepk:e and Shideman (19!B1) reported that a concen• depressant doses. Thiamine (200-40D Ecg/ml) eliminated the tration (5 pg/ml) of nicotine, which was capable'of blocking chronotropic effect of nicotine and abnost entirely its inotropic the positive inotropic and chrobotaopic rtsponses, of isolated effect. Tettaethylammonium (50 pg/ml) and hearetnethonklm auricles of the cat to tetramethylarnmonium. failed to alter (50-100 µg/mI) blocked the action of 2-fold doses of nicutine. the positive inotropic and chunnotropic responoes of this dtropine (2 µg/ml) blocked the initial btgdycardia due to tissue to 4-methyl-2-aminopyridine, or to its tsomets (g- nicotine. Ephedrine (50 pg/m!) prevented the positive met.hyl-2-tiaainopyr.ttine; 5-tnethyi•2-aminopyridine; 6- chronotropic and inotropic effect of nicotine. Cocaine (1.5 met.M•1-2.tdminopyridine). Also, the negative inotropic pg/, tl) diminished the nicotice resllnter; aeonrentaalion of effects of pytidine, 2•methylpyridine, 3•methylp'yridine, 4- 6 Kg/ntl lowered the rate without any effect on amplitude. methylpyridine, and 2,6-dimet.hylpyridine on cat auricles In atrial preparetions taken from reserpinized guinea pigs, were not blocked by this conventratibn of nicotinr:, although the adrenergic action of nicotine was almost totally inhibited, 5 pg/ml nicotine were suflicieitt to block any response to while the negative iuotropic and chronotropic action was further addition of nicotine (Rchoepke and Shidemen, 1982). prolonged and enhanced. bichlornksoproterenol (2-4 pg/ml) Timsit (1961) studied the antinicotinic actions on guinea- prevented the positive chrunotropir and inotropic actaons of pig auricle, of the following subatances: pheniptasine (phenyl- nicotine. 1-isopropyl-2-hydrasine HCI; Catron®); d-arnphetamine; Sugimoto and co-workers (196a) rcqxtrted that 0.01-1 bydtaaino-2-octanesulfate(D.1514);amino-2octaneterttste jug/ml of the mnnow-us,•osidn-P t\lA(ts inhibitor, pheni• (D. 2524); hydrasino-2•methyl•6-heptaane oxalate (D. 1301); prasine (b•phenylisoprop. ihydraainc HCI; Catrona'), did amino-2-methyl-6-heptane tartrate (J. L. 1340). All of these not affect the contraction ol atria f•run rabbit4 and guinea substances exerted a positive inotropic and chronotropic pigs, but, on atria pretreated with 10 jtg/mi nicotine, 0.1 action, which was not suppressed by paralysant (250 rag/ml) pg/ml phenipraabne cauml utorked >.timulation, even whei concentrations of nicotine. In the presence of atropine, these the nicotine was no longer ptesent; pretreatment with 0.1 substances inhibited the positive inotropic action of 1-2.5 jug/ml phenipranine had little influence on the effects of Ng/mi nicotine, without having an, effect on the positive subsequently-added nicotine. The stimulant action of 10 inotropic effect of epinephtine or nonepinephriue. pg/nil nitrotine was slightly deqrressr,l aftcr a prior addition lr;adaielawa (1962) found that gue,ttethicane added to an of 0.1 Wt/tnl pvro;tallol; thr carly inLibitamy and later stimu• organ•bath containing isolated rabbit atria for 90 nun, and lant actions of nirotinc Nrric strrnRtl,ent- I by prior addition then washed out, did not reduce the stimulant effect of nico• of 100 pg/ml f-methionine; prior addition of 10 jug/ml reser- Produced b1t The Council for Tobacco Resc-arch-USA, Inc, im 0002991

-~----------- - -- ----- ----- - _ - °--~ i I 8D TOBACCO-EJCPERfME\TAL A\D CLINICAL STUDIES. SUPPLEAIB\T I n;oa rwdnna+l ~bn a/Gm~don/ wnlGnn hnf al;nfitl~- o.,mnnnfai the inhibitory effect of nicotine. VVhea 10-pg/ml reserpine were added before 100 pg/ml 1-methionine, the aupmenting effect on nicotine was nullified. S.c. injection of 1 mg/kg rescrpine given 24 hours prior to removal of the atria did not prevent the inhibitory effect of nicotine, but, in most caFes, its stimulant action was "scarcel+• observed"; when I mg/ml 1-methionine was administereo before nicotinr•, inhibition was markedly augmented; and isolated atria from animals pre- treated 24 hours earlier with both reserpine (1 mg/kg) and l•methionine (100 mg/kg) reponded to 10 yg/mi nicotine as well aF did atria from control animals. According to Oldsser (1960), the naturally occurring base, leptodactylina (which baa cholinergic and adrenergic' effects of the nicotine type), had a greater inhibitory effecl than nicotine on guinea-pig auricles; after atropine, high doses of leptodactylina, like nicotine, increased both mte and amplitude of contractions. For a discussion of the effect of nicotine on phosphorylase activity of isolated atrial preparations, see Iluffoni and Giotti (1961), below, 338. 337-C. Ventricular Strips. Wenzel and Ingianna (1964) gave s.c. injections to 3 groups of 8 Sprague-Dawley rats of either sex of, respectively, 2 mg/kg epinephrine in oil, an equimolecular dose of norepinephrine in oil, and 0.5 mg/kg nicotine; the drugs were administered daily for 7 days, and the animals sacrificed on the 8th day, when the isolated hearts were tested a•it.h 178 Ng/mi nicotine. A fourth group wns treated by stressing (by mes.iw of restraint) for 15 hours prior to sacriIIce. As controls, heart ventricular strips from 16 untreated animals were tested with nicotine. Pretreatment with epinephrine or norepinephrine significantly increased the effect of nicotine on contractile force under 4/sec electrical stimulation; but pretreatment, with nicotine, or preliminary stress, did not significantly alter the responses of the ven. tricular strips. 83i-D. Papillan Muscle. According to Forster and Hoefke (1960), 1-100 µg/mi nicotin: acte.d on papillary-muscle preparations from the cat., but not from the rat, exerting a positive inotropic action for the first 2-3 mitt. Nicotine in eoneentration of 100 µg/ml e:errid a negative chronotrupic action on the spontaneousty contracting isoi®ted papillary muscle Pyridine, SmethyIp,•ridine, 8-methylpyridine, 4- methvlpyridine, and 2,6-dimethylpyndine, all produced a transient negative inotropic effect, followed by a much more prolonged positive inotropic tksponse, on isolated papillary muscle of the cat; the Former effect was not abolished by nicotine (Shoepke and Shideman, 1962). 838. Chemical CAangea in Cardiac Tiasues On the premir-es that the stimulating action of catecholam- ines on the heart is developed patallel to an activation of phosphorylase, and that nicotine stimulates the heart through the release of catecholantinea, nicotine should cause an in- crease in the percentage of phosphorylase a, and such an increase should no longer take place in conditions of impov- erishntent in cateeholamines. With these premises in mind, I3utfoni and Giotti (1961) studied the effect of nicotine on isolated atropinised guinea-pig alria and on its phosphorylase activity. In normal atria, the positive inotropic and chrono- tropic effect from 50 pg/mi nicotine bitartrate was accom- panied by a stimulation of pho.nphorvhise nct:vity equal to that obtained with a dose of nurepinephrhre, equiactitr from the inotropic and chrorotropic standpoint. In reserpinized A1r/9 nD whlPh niMtlnP t7ad nn IN/AIIIYa inntmnie dnd ehmnn. tropic effect, nicotine did not cause incrmsec in the percentage of phosphorylar;;e a, while norepinephrine had a nornul stimulsting effect on phosphorylase activity. 1-sukovets (1961) investigated the effect of nicotine on phosphorylafe activity of heart-muscle uAng Langetadorit preparatiops of the rsbbit heart with ganglie intact, in which 0.14.2 mg nicotine was in;ected into the bulbus aortae 32 sec after 0.05 rrtF atropiue; muscle samples were obtained at the,lmal di the mechanieal effect. In 16 control runs, active hhoa. phon°la.re (e.cprc:wd in ternu of total activity) was found to be 10.7 f 2.774 units; and, in 6 tests with nirntinc;, the value rose to 23.0 :1: 3.48 units. In other expetiments on the rMuue prepsiration, an increase in active phosphorylase ot about 23,,i occurred synchronously with the increase in contractility produced by 0.2 mg nirotine (Rukovets, 1962). Kukovetz concluded that increased phosphorylase activity paraltel.d the relea,~e of adrenergic substances and the in- crease in heart-rate and contractility. In rats in which cardiac necrosia was induced by corti- costervidtlectrolite-stress (see above, 333), Ireatment with nicotine increased the incidence and rrverity of the myo- cardirhl leEions, snd myocardial potassium was significantly decreased (Wensel and Stark, /f)65). Nicotine bad no effect on the serotonin content of the hearts of mice, rats, and guinea pigs (Hat>sAon, afasuoka and Clark, 1963); see above, 331. See also above, 331. Cat- erholamine (norepinephrine; epinephrine; DOPA; dopamineJ Content of Heart of Nicotine.Trested Animals/Organs. 339. .tleelaanfarn of :Ictian of tlu 11 frotirte B,Bed on 1leart.Rate is Uammala (145) Yosshikata (196t) studied the effect of nicotine on heart• rute, blood pressure, and the electroeardiogram of dngs, before and after transection of the vagi.q carotid sinus, and spinal cord, and interpreted his fi-,dintts as indicating that the 'brnd~•cardia and suceeeding blood-presnure fall were excited partly by cardiac reflex via the vagus, while the tachycardia and blood•prescure rise were attributable to the action of nicotine per ae (one satqposes, for example, in in. ducin;¢ catrcholadrilne release). Tauberger (1964) pestulated a primary central stimulation by nicotine, which a•as subject to rnnrked tach;•hhylaxis, and was reduced or eliminated by iucreai:ing the depth of narcosis. This worker gave small (0.017~-O.0i5 mg/kg) femoral-vein injections of nicotine ..: cats under chlomlase.urethane anesthesia, and these doses activated efferent total action-potentials of the cervical portion of the autonomic va(tus, along with inspiratory phrenic-nerve discharges. On the basis that the doses used werc• below the threshold for ret1ex stimulation of autonomic centcrc, and that the same e/feets occurred after bilateral vagotoAny and exclusion of the carotid sinus [all questionable as.rum4ttions or reasons), a"pmnary central •tinmlation" was then assumed. The inereuscs in cardiac frequency and force following nicotine hece been attributed to the nicotinc-induccd release of ehhnephrine from the adrenal glands (Qolti et al., 19GOa; Ring. 1960), or, more often, to catecholamine (norepineph- rine; rympathomimetic-amineR) release at syynpalhetic nervc-smdingx (lih+g,1960; Leaderx and Long, 1962a), and/or chrotmlffin tissues (Leadern and Long, 1962a; Fawas, 1963), aud e-•lrris111• from stores within the heart itself (Ginael and Kotte;,m (=Fottegoda), 1f151; J. H. Burn, 1PH0a; J.J. Reuu• mid liourytain, 1060; Lindmar, 1062). These storee may he inlra- or cxt'aceUular (Lindmar, 1962); their release, Produced bv The Council for Tobacco Reseaicll-USAf Inc, 0002992 1

CAItD1OVASCI'LAIi SYSTFM Sl QIRY't ul ///W/tla4 poW,ble mechanism, the released catecholamines may then activate myocatdial phosphorylhse (13ing, 1960). •if•he more detailed t.houghts, and discussion, of certain of these writers are worth notibg: J. H. Burn (19BOa) has bumntarized the action of nicotine on the heart by first necallinR that the drug stimulates auto- nomic ganglia, and causes inhibntion of the isolated perfused organ, as might be expected from the presence of intracardiac ganglia on the course of the vagus nerve and the release of acetylchoUne from its postgangliouic terminations. Sur- prisingly, however, this inhibitory effect is followed by a stimulant action (best studied M•hen the inhibitol effect is esefuded by atropinel. There is evidence that the increase in rate and amplitude caused by nicotine may be due to release of an epiaephrine.like substance; and, in fact, norepi. nephrine, plus amall quantities of epinephrine, are present in the heart (see abo.'e, 331); and, following pretreatment of the animal with reserpine, norepinephrine can no longer be extracted from the heart, and nicotine no longer shows its usual stimulant action, on either rate or cardiac force, on isolated atria from such reserpinised animals. Thus, it ap. I~rs that the arceleration of the rate and incretue in the force of the beats which nicotine causes in atria from normal animals is due to the discharge of norepinephrine from the store in the atria. Discussing the relationship between biochemict,l activity and mechanical function of the heart, Bing (1960) asked whether activation of these stored catceholaminea influence cardiac activity. In the isolated, heart, suprathreshpld stimu- lation and nicotine have a short pharroacologieal action similar to that following release of cr.rdiac catecholatnines; but, with respect to nicotine, the situation is quite different in the heart in situ. Here, nicotine causes an increase in catecholamine concentrat:on. Apparently, stimulation of the adrenaf gland and sytnpathetic nerve.endings elevates cat- echo)aminc levels in blood, with subsequent stot8ge in the heart [sre K; Iisko, Chrysohou and Bing (1961), above, 331). 1Yhile, therefore, the importance of eatechodamines in blood for cardiac action is itidisputed, the pbarmaeologic importance of the sto" catecholamines Is as yet uncertain. It 6 Iwssible, lv,arever; that their activatioo triggers the rapid fornuition of phosphon•ldrr 6 to the active form. The activation of atrial phmphoryla.r due to nicotine, and the disappearance of its activatin)( effect simultaneously with the disappearanre of nicotine inottopie and chronotropic stimulating effects after treatment with reserpiner was con- sidered by Buffoni tuid Giotti (1961) as further proof sup- porting the interpretation that the stimulating effect of tltis drug is effected by means of the release of catecholamines. 1{ukovets (1961) also concluded that increased phosphorylase activity following administrati,o» of nicotine paralleled the release of adrenergic substances and the increa'sed heart- rate and contractility (see above, SM). It ha.s been postulated that nicotine stinculates patasyTr pathMic ganglia in the atria, which resuita in the negative chronotropic response; and, according to Leaders and Long (196Ya), the poFitive chronotropic response to nicotine also im•olves initially the parasytyipathetic nervcs, apparently resulting in trtesse of catecholamittes from sympathetic nen•e-endings or chramaf6n-tiaeue stores. Alter a more detailni acccmnt of these e\periments, Leadera and Ln (1962b1 concluded that invoh•ement of the parasympathetic nPr\'ou8 AyRtem in both the dregative and positave phases oi the atrial response to nicotine obviated the necessity of m4ivlnrinv Rvrotnathetic aanalia in the heart to explain the positive chronotropic response to nicotine. 'l he increase in coronary vascular resistance observed in dogs receiving niem tine may or may not be related to the positive inotropic effects of nicotine; Leaders and Long (1962r) have discussed possible mechanisms of the former effect (see below, 394). 340. aLoon PaCSSVBE (148) 511. Su6-rnornmaJian Fonns (148) In chickens under wdium.phenobarbital anesthesia, I.v. injection of 5 mg/kg mecamylamine blocked the pressor effects of i.v. nicotine tartmte (liufiag and 11'alaFaek, 1901). 342. 1larnnHI/s (148) 393. Dog (199). Variations of vascular reactivity, as meas• ured by arterial blood-pressure change, were studied by 1. H. Page and Taylor (1949) in 722 dogs; the dose of nicotine used was 0.15 ml of 1:1,000, i.v. Blood pressure was found to change during the same experiment and on different days, variability being particularly great with nicotine, leading to the conclusion that, unless an average response is obtained and confirmed over a reasonable tirne, individual determina. tions may be grossly misleading. There was no direct rela- tionship between the response to one drug compared to another, although often they paralleled one another. Nor was it a function of a height of the blood pressure, weight, tem- perament, length oi kennel residence, depth of anesthesia, body temperature, voluntary muscle rela.ration, or intra- arteriall-v administered salt solution. As compared to dogs under pentobarbital anesthesia, nicotine responses were greatly increased, and unusually variable, when only local proraine was used to prevent pain during camwlation of the artery for blood-pressure recording. l.v. infusion in anest.retised dogs of 20 pg/kg/min nicotine resulted in an increase in arterial pressure (Bellet et aL, 1962; R. K. Larson and Murray, 1963; Larson, Ftitkuda and Murray, 1965). In 14 tests in 9 normal, unanesthetized dogs, the pressor activity of 15 pg/kg nicotine [by infusion] ranged from 20 to 65 mm Hg (E. tfaas and Goldblatt, 1959). Larger (45 pg mg/kg) doses of nicotirr- induced blood-pressure rises of 125-165 mm Hg. Intra-arterial infusion of nicotine into the renal artery of dogs alw•ays caused a strong pre_csor effect, without notable change in heart.rate; sbnilar infusion into the artery vtesen- terica cra»alil; also always cau~erl a pressor effect (Dremer and Felix, 1960b). Following injections of nicotine into the ascending aorta of anesthetized, open-chest dogs under ar:i- ficial respiration, avstolir and diastolic blood pressures rose, the average peak rise beine 857c and 655c. respectively, of Initial values (K. Braun and Stern,1964). Following injections into the abdominal aorta, the iwrssur response was markedl,v dintinished or ahwent-, follon9ng injections into the femoral vein or pufmonar.• artery, the pn'csor reslwnse was also d'unin- ished. In dogs anesthetized with a nature of pentobarbital and barbital i.v., with vaFcal.s.vmpathetic trunks cut bi- laterally in the neck, and under artificial respiration, i.v. injection of 5 pg/kg nicntine raised the mean blood pressure 19 t 5 mm Hg; after intm-aortie injection, blood pressure inctea ed 32 f 10 mm Hr (Gntttounis and Aycock, 1963). Intra•aortic adntiinistmtion uf 10 p)t/kg nicotine in anesthe- tised, open•che=t. hilatet-i"h• vatnitomir.ed dogs. produced arterial binod-pt•--orP re}- -vise: al+nroximately equivalent to 2 pg/kg epinc phrine i.% (p:. F. Woods and Richardson, 1955). Produccd bv Tth i rouncil for 0002993 Tobacco (~aseai i;,'j-USA, Inc.

82 TOBACCO-EJtPE1tI\fEKTAL', AND CLINICAL STUUIES. BUPPLEUENT I hieotine was not absoi^bed from the dural catial rtrpidly enough to produce characteristic vascular responses (13echt, 1920). Inhalation of cigarette-emoke had a prtssor effect in dogs (de ltoum, 1959; SfcOiH, 1963), but inhalation of smoke from a plant other than to6cco was without effect (de Moura, 1959). The pressor action of cigarette-smoke was therefore ascribed to the presence of nicotine. 34-t. Cat (151). In,18 experiments on spinal cats, i.v. injec• tion of 0.1-0.3 mg/kg nicotine elicited a mean blood-pressure rise of 114 f I1 mni Hg (A. Jones, Gomez Alonso de la Sierta and Trendelenburg, 1963). No effects on blood pressure of chloralosed cats were observrrl folloning injection of 0.01-1.0 mg nicotine bitartrate into the submasillahy duct (Emi»elht, lfuren and StrSmblad,1954). Al. F. , C•,thbert (1964) reported relative potencies to nicotine tor tyramine methiodide, dopamifne methobromide, and norepinephrine methochlor•ide on blood pressure of the eat. One puff of smoke from an unfiltered small-diameter cigarette (which had been smoked for about one-half its length) produced effects roughly equivalent to an i.v. injection of 5 pg/kg nicotine in raising blood premure in lightly atcesthe• tized cats (M. S. G. Clark and Rand, 1904). Smoke from filtered cigaretles aas less effective, and smoke from a ditmtny cigarette made of tissue-paper produced no definite iise in blood pressure. 345. Ratr,bit (152): Although not strictly relevant, we may note here that, following i.v. injection of tobacco mosaic virus, even in non-sensitized rabbits, the blood pteaslire fell (Chopra, ~). (For an account of the im^: .'..t, <.; t;,bacco mosaic virus, see below, a156.) 346. Other Afamanafs (152). Lv. Injection of 0.1 ong/kg nicotine on rots resulted in a fa)1 in blood pressur^,, aUtile larger dokes induced only a rise (Grewal, Lu and Alhnark, 1962). ln sheep, at doses of nicotine higher than 0.1 mg/kg, carc)iae slowing was not manifest during the hypertensive phase (Brurmud and aavarro, 1954). 347. F.,$ect of r?eprotEd Doses of Nfeotfne (TarhJphJtnzis) (152) A second dose of nicotine rulministened to cats had less effect on blood pressure than the initial orde (A. Jones. 1982). Lv. injection of 0.1-0.3 mg/kg nicotine in spinal cats elicited a mean blood-pressure rise of 114 * 11 mm Hg, and a second dose given ZO-30 min later showed little kariation in pre:isor response (Jones, Gomez Alonzo de la Sierra and Trendelen- burg,1963). 348. Blood Presaurc Foll..aing Chroriu Nicotine or To6aceo•.Sneoke Adutini.strotiorz (153) In rabbits given nicotine In their drinking-water over a period of 24 weeks, with or aiti,,,ut the addition of cholesterol to the diet, systolic blood pre.•ntre was found to be signifi- cantly increased (11'entxl, Itnmel and Turner, 1960) lfor erpetimeintal details, see (333.h) tutd bPlow. b96-a1. 1\'enzel and his oswociates (1961) emphasited the ptvgt•esaive nature of such rises in blood pressttro in rabbits sintilarly treated; in these experiments, an additive effect of :.icotine and the hyperchalesterolemic diet also ol•peared to have occurird, with or without i.v. injection of et11t•onovine maleate at 4-week intervals. Groups of rabbits receiving 1.14 mg/kg/day of nicotine In their drinking-water, or nicotine + 5 rtt)eiLg caffeine i:v. w-eekly, or nicotine + the hyperchole`terolamic diet, or nicotine + weekly i.v. injections ot 0.025 mg m:a. bain + tjte hypercholeste,rolemic diet, all showed significant increases in blood pressure, compared to untreated aninml- (1CenEel. XlacCarthy and Rutledge, 1964). Wenzel, \1•attanapongsiri and Vedral (1964) studied nirn. tine and renal hypertension in Sprague-Dawley female rats given an average daily dose of 1.14 mg nicotine (2.28 mg/kk) in their cainking•.cater, which w•as calculated to be equivalc•nt to a two-pack-a-day dose in the smoker; 10 other rats served as contro . Eighteen of the animalw were madc hvpeMenave by partial constriction of the left renal arter}•, and 10 of thehr were simul+aneously treated with nicotine, aa above. thc otlrets serving as controls to these. The 10 untreated controls showed no significant deviations in systolic pressure (tail-cuB teehnique) over the 55-week study-period. In contrast, the rats receiti ing nicotine, but tinoperated on, showed a ptngres• sive rise in pressure from a control level of about 145 mm Hg to a peak of about 200 mm Hg 15-20 weeks later, followed by a progressive decline to control levels att about 30 w•eeks, with further progressive drop to about 115 mm Hg at 55 weeks. Rats rendered hypertensive, but not given nicotine, showed a, progressive rise in prewsurc from about 145 ntm HR to about s00 mm Hg at 20 weeks, and a plateau at this level for the rentainder of the 55-week I>eriod. In contrast, the mt- rendered dtypertensive in addition to receiving nicotine show-ed a progressive rise to about 215 ntm Hg at 20 weeks, followed by a progressive fall to control levels at about 35 w•ee)w-, with a further progressive drop to about 120 rnm Hg at 55 weeks. The authors noted that, o•hile the human re'slwrise to smoking may not entirely resemble that of the rat to nicotine, their data could explain the lack of a significant relationship be• tm een smoking and h)pertension (see also beloa, 444-A). Blood-h•ressure determinations taken at regt:lar 2-month intervat.shohed no diHereucec between white rats eax,sed to cigareRte,smoke every half hour, 14 times daih• for their entire lite-span, and mimali kept as controls in the animal• room, and still others treated more or less similarly to the experimental ones, eecrpt (or actual exposure to ciFarette• smoke (Haag, Larson and Weatherby, 1960) (153b). 349. Pressor Responses Fofionrng )6eme Scrtion (153) Injection of 0.1-0.3 mgA;g nicotine, i.v. or into the right ventricle of cats and dogs, reintlted in a fall in blood pres`ure, .ollow•ed by a secondary rise (Takasaki, Yokoo and lagasnki, 1959). Bilateral vagotomy just below the hilus of the lung did, not affect these changes; after bilateral vagotomy ebore the hilus, the fall in blood pre4sure was slightly lers. Scr ah Yoshikat'a (1964), below, 375. 330. Effect of 1firatinr on Pressm and Deprewmr Re,(Ihrea {155) According to Anikina (1961), Chemiaovskii (Affcrent,rc Si.tenty Unutrennykh Orge+not•. Kirov., 1943) was the hra to establish the diver.re effect of diKturlwnce of the metabol6m of chemoreceptors caused by the action of monoiodoacetir acid. Under these conditions, raipirhing carbon diosidc to the receptors resulted In none of the changes in blood pressure or respirmtion characteristic of the receptors in a normal state: a•hfle refk•.rs caused by nicotine and arntyleholinc were maintained. From this, the author hypethcsiaed that acida (as well ata oxygen lack and c)•anide compounds) do not nct directly on the chemoreceptors, but cause essential chattge. P~®duced b~f P~^ ~fniP~l ~~ Tobacco Gc~ca~cfi USA, INt 0002994

CARDIO\'ASCULAR SYSTEM in the cell tr6etabolism, which stimulate the chemoreceptors; while chemical stimulants ol the second group twhten mciucqes nicotinel act directly on the receptors, and possibly cause no essential metabolism changes in the cell tissue. Also according to Anikina (1961), V. A. Lebedova (Byull. Ekaper. Biol. BBfl1): 19, ?9fr1) confirmed this finding, and noted that the excitabi'ity di the receptors in response to chemical stimulants of the second group [e.g., nicotitie] increased. On this baqis, this author developed a theory that the disturbance in meta• bolie procesees is reflected in the general functional state of all receptors{ and is manifested in their elevated excitability. The disappearance of pressor reactions to carbon dioxide under these i•ottdit,iona, and an actual transition from preseor to depressor reactions, was treated under the theory that attt apparatus in a state of constant and sufficient ly strong activity should more easily fall into a statie of inhibition than a quim cent and inactive apparatus. Thus, this writer ceased to aecrrbe any importance to the difference in the reactive arts- tems receiving the different grotRps of stimulants, and gave more attentibn to the change in excitability of the receptors. 350-A. Carotid-Sinus Reflex. In his discussion of the evolu- tionary aspect of chemoreception of the glomus caroticuin, Chemigovaky (1902) stated that Kulaiev and Beller had observed cessation of respiration, followed by cardiac arrest, on applying nicotine directly to the vascular lab}rintb in frogs. This dnd other observatiohs (see below, 508) led him to conclude that the chemosensitive properties of branchial vessels were immediate precursors of the chemoreception of the carotid glomus, and that there was a clear-cut morptio• genetic relationship between the btattchiwi arteries and the chemoreceptive apparatus of the glomus caroticum in main- L-l9I6. In chloralosed cats, local application on the carotid-sinus region of cottor-wool pads soaked in I% nicotine acete,te caused the normal circulaton reflexes from the carotid sinus to disappear, demonstrating the paralyeing action of high eoncentrations of nicotine on the baroceptor mechanism (vbn Euler, Liljeatrand and Zotterman; 1941a). Electtoneurogtams of the nerve, of Hering taken at this point showed that the small "chemical" impulse' -egttlarly disappeared, whereas the large impulses were leA rapidly influenced. As a result of prolonged action of nicotine, the large spikes too became greatly reduced in number, uou•ever, even in casea whetr the general blooi pressure showed nd significant danges. Shehegoleva (1931) found that the chemoreceptots of the carotid sinus in old animals were niore sensitive to the action of chemical stimuli than thnse in young animals (cited by Frol'kis, 1962). In old eats, nicotine caused changes in tlhe blood pressWe and tespiratiou in an average dose of 32 d: 0.72 µg/kg, compared to 0.8 * 1.3 µg/ktr In young a»hnaEs. This higher sensitivity in old animala was obterved both after Lv. injection and after injectdon directly into the isolated carotid sinus: '1'he pressor effect df nicotine was abolinhcd by dettervation of the sinus. In dogs subjected by Taka.caki. Yokoo and Iragasaki (1989) to the perfused-head technique of Heymans, nith both carotid-sinus nerves denervated in the recipient animal, injection of 0.1-0.2 mg/kg nicotine into the recipient dbg produced a fall in blood ptessttre and heart-rate, as well as reflex alutea, u•hich were followed by a secondary rise in blood presssure and acceleration in the depth and rate of respiration. After vagotoiny, the circulatory depreacor response to nicotine no lontvr apl mamd, although the secondary rise in blood pres- sure re,nnineiL See al.w 1'oahikal& (19frt), below, 875. 83 In isolated perfused carotid-sinus preparations from the cst, tne mcreaaeo nroig trutu acuvr prrmur rreeptuia reaui6jag from 0.5-1 µg/ml oratronger solutiors of nic•otine was blocked in the presence of 0.1-10 pg/ml tubocurarine or hexamethar- nium (Diamond, 1955). The electrical activity in the carotid. sinus nerve induced in cats by injection of nicotine into the lingual artery ures diminished by the non-barbiturate am- esthetic, diethylamide of 2-methoxy-4-allyl-phenoxyacetic acid IG-29505j ()3rialing, Shapiro and Sigg, 19g2). For carotid-sinun-reflex effects on heart-rate, see above, 313, and on respiration, below, 508. 330-B. Aortic Reflex. Emplo~ing the inQenious technique devised by Comror and 1lortimer (1963,19frt) to separate the effects of stimulation of aortic from those of carotid-body chemoreceptors (described above, 313), these workers found that aortic-body stimulation by nicotine led to hypertension in all dogs tested, while carotid-body stimulation by the drug resulted in hypotension in the majority of animals (Comtoe and \fort9mer, 1963). In a typical result for nicotine in a chloralosed dog breathing spontaneously, injection of 4-10 pglkg nicotine bitartrate into the ascending aorta resulted, within 2 sec, in hypertension, tachycardia, and hyperpnea (aortic-body response); 00 sec after the injection, greater hyperpttea occurred, accompanied by bradycatdia and hylw tension (carotid-body reafwnse). A few instances of non- tvpical carotid.body (increase in blood pressure and heart rate) and aortic-body (decrease in blood pressure and heart rate) responses occurred. In general, the cardiovascular re• sponsess a ere similar whether the dogs breathed spontaneously or were ventilated artificially, although initial tachycardia occurred tess frequently following stimulation of the aorta chemoreceptots during constant artificial ventilation. S. Stern, Ferguson and Rapaport (1964) also studied the beao- dyytamic effects of stimulation of the aortic- and carotid-body chemoreceptors; these workers injected b-2U µg/kg nicotine bitartrate into the base of the ascending aorta of anosthetited, artificially-ventilated, open-chest dogs. Beginning 1.b-3.0 seo after the injection, there was a significant reflex rise in pul- monary vascular resistance, the sensory receptors for the reflex being aortic chemoreceptors, and the efferent paths being sympathetic fibers; similar effects were not elicited by stimulating the earotid-body alone (for f,trther detaiLe of this study, see below, 305). Following denervation of the aortic and carotid chemotrceptors in anesthetized, open-chest dogs under artificial reFpiration, injection of nicotine markedly diminished the rise in systemic and puknonary pressttreR, and the Iatent period was prolonged (1:. Braun and Stern, 1969). 350-C. Perl- and Epicardial Cbemonrrptora. I n dogs an- esthetizcd with urethane and morphine, i.v. injection ot 0.1- 0.2 ma/kg nicotine resulted in a fall in blood pressure and heart•rate, followvd by hypertew;ion; and the circulatory ttflcxes were blocked by tetraethylnmmonium, hexamethu- nimn, and procaiue (Tskaziaki et sI.,195tta). Procainc instilled into the 1>nricardial sac blocked the circulatory reflexes nor- mally caused by nn•ntine Cl'alau.aki et al., 1959b). ln order to anah•ee reflex rhnun~- nrrurring in the cardiovascular system during the action of nirotine on ]mricardial chemotecrptors, Tmrbenko (1962) cuudurtcd arute experiments on cats under umthane anesthesia, recordinpt frequency and strength of cantinr ront ractiun- an.l vascular rre iaance in various organs. In tb-• •Irl•rPswor tvi• rnm rw•eurrinu in restmnse to l+erirardial ad i-•tnltion - I •..... ~mn•, a de,•elcmtioa of cardiac rhythm ah.a~- oreurred. ui•• i• ~ tlm t;trength of cardiac contractions Produced bv Thi Council for Tobacco hQs~axh-USA, Inc. 0002995

84 TOBACCO-EXPERII1fE\TAL AND CLINIC.AL STUDIE;.S. SUPPLENtE\ 1P I either diminished, increased, or remained unchanged. Reduced ...e, uias ' u,uwuce w•curlui uui) iu t6re 4tAlC/D uJ wt: elueeu, intestinr, and kidneys, bbt such resistance changes were not always distinct, and a•ere even absent in a number of cases. Rrvluced vascular resistance did not occur in the posterior eatremitim orgwans of thh lower pela•iia, lungs, neek, or head. Experilments with a temtwrary exclusion of various vascular areuu+ frdrpl the general rqrculation showed that the develop- rnent of ahe depressor reaction consequent upon the nicotine art inrl nll prricardial rere~'~tors a•as connected with changes in the pulttlanary vesRels. Tprere was diniinishtd circulation-rate in the veuanrx vrxrelspreceding the arterial-presstrre reduction; a>• to the brteriea, the circulation-rate iremained unchanged or drr-nara•U after the anerial ehifts. The efferent route of this reNer ardw ruid to paR+ In the vagus. KnlaPv (111ti2. 1P0.7) reported that alferent libens from the epicardial receptors senwitivo to nheotine were fouud in all cardiac nerves, pat- ticuhtrly In the branches of Ihe vagus and depressor nerves of rabbits and cats. Impu1sa1 tmveling along fine, unmyelinated, slue•lytmidurtbtK fibers of the C type (slow impulses) were said to piay a major roM in effecting reflex changes in the systemic blood pressure (n mspouse to the application of 0.005 pg-10 tnptJmi nicotine to the epiranlium (chiefl}• the ventral surface of fhe ventriclea). Control experiments, in which a dye was substituted for the tlinstinc MolulionP, showed that the stimulus trached only the a•plcnrdium bordered by the margins of the (cloatetl) htcisinn In the thoraa. Slow impulses from the heart were found to be capable of cansing both pressor and deprewr arterial•p.mrlaurc rractionN; the former developed a•hen a relatively small nusdrer of alft-retlt fibers u'ar activated by weak stimulation, aml the latter develnpnd after stmnger stimulation exciting a larger nrmber of the same fibers. No types of aAferent impulseP svlwrific to pmsMOr or depressor reac. tion were rhoted in the ex>kriments. 350-D. Cerebral i7remoa+eceptors. Taylor sind Page (1951) studied the; effect of nicotine on cerebral chemorirrptors using the following experimental set-up: Donor and recipient dogs under pentobarbital anesthesia were arranged so that the circulation of the head of the recipient animal was serarnted from the body, and its carotid sihuKes n•cre drtlere•aterl and vagi cut; the cardiac ends of a jugular vein and carotid artery of the dont,r were connected to similar vessels leading to the brain of the receipient for perfusion of the isolated central nervous system. Injection of 0.15 ntu nicotinc into the arterial side of the perfusion circuit reduced the peripheral blood prea• sure of the tecipient animal bv 15-98 mm Hg (intense rexpira- tory stimulation lasting 2-4 lnin also resulted). Thus, it ap- peared that these effects resulted from stimulation of cerebral chemortMeptors. (ln this set-up, drugs which normally caused a rise in blood pressure in intact animals produced a fall, mtd ones which normally caused a fall, resulted in a rise. The authors thorad'ore concluded that cerebral ehemorereptors seemed to dct as a buffer meehanism.) 350-E. Pulmonary Chemorrceptors. 'Nicotine injected in- tra-arterielly into the l'nnited puhnotuzry circulation of open- cheat dogs under morphine plus pentobarbital anesthesia re- dured systemic arterial prcssttre snd decreased l.part-rate (1'akasaki,1962). These effects did nott occur after lef t cervical vat,rotomy, from which it was concluded that they were medi. ated through vagal chrmorer•eptors in 1he lung (for further details of these exreriment., see below, 393). Bevan and Murray (x963) reported that nicotine and lobeline stimulated the pulmonary-arlery ref3exogenic sonc in man, leading in half of the subjectA to bradvcardia and eeuniuu. 'oe3au, :uwyirri aou ttnruY ttmrrr rna•esthgated. in anesthetized cats, some of the structural requiremrn». ~ drugs which stimulated this zone, utilizing the reflNa liyta, tensia-e response following tight-atrial administration of d. drug. The relative activity o1 nicotine, Q-nieot}rine, o•dil.l. dronicotine, fi.dihydronicotise, myosmine, and a nuailrer d other compounds is given ky theue workers, and stnrrtwsl requirements discussed. 350-F. 1lntestinlal Chentomccptqrs. In an ulce.•tit;utiui n1 the action of nicotine and acids on ehemort e•epton of t1w inle.. tine, At»kina (1961) used cats under urethane ane,thr.-ia in which a sect ion of i ntertine, isulated except for it. ncr,•onsa.u. nectiona. lva.a perfrrred 1•iait+ve wlk. while hlnui.•rw.ia.- recorded in the carotid artery. Continuous perfusion of thr intestine with low (0.0005 M) concentration of morloiodoaretlr acid initially intensified the pressor reapome to injertions of 0.05 mg nicotine, but continued perfusion nith the acid re• sulted iu a decrease in the reflex response to nicotinr. 3her rinsing with Ringer•Locke solution, the reaction to nicotn,r was restored. When the intestine o•®s Imrfused with 0.5 nll ot 0.5 % lactic acid or n•ith 0.03% hydrochloric arid in the perfusing fluid (which contained 0.0005 ,ll monoioduacetir acid), the pressor response was also initially intcnsified, but continued perfusion with monoiodoacetic acid resulted in a decrease and disappearance of the prraror reglronM• to arid; and inversion of the acid re9e.r-a occurred only after they had first disappeared; and, foiloaing rinsing with Ringer.l.u•{,e solution, the acid reactiuu became diminctly delarsrive. Front these and other experiments +hou•ing that the iueswn response to acid was not restore<I following riruiug, while that to nicotine w•as. Anikitm agreed with the earlier theary of Chemigovsltii (see above, So0), and argred for the exi.tcnre of separate receptive reactive scxttmts, each with a differrnt metabolism, which is easily disturbed by monoiodoaertir arid. Levtov (1963, 11964) also studied the effect of nicrotine on intestinal chemornceptom using cats under urethanc aa>• thezia with the superior-mesenteric artery and vein isuland along with the nerve-trunks; the intestine n•ar herfuacd either with Ringer's solution or by cross-circulation with blood 4um a donor animal. The author himsrlf remarked that dew•nptinn of these experiments with nk•otitre presented certain difli• culties, inasmuch as the reactions of the 1>Prfusiun pre~,mrr were rather quiekly extinguisherl in the eourr•v of an exlMri• ment, and perhaps the reader Iwd better cm4Qult dirrK•tly thc English translation (Levtoe, 191if), rather than relying eunt• pletely on our su:runary..apparently, injev-lion of I pp nhvNine in Ringer's solution produced taaorlilatation or a bipho-ir reslx,nse (pressor-depressor tenclion); 2 µg nicotine ptuluea.I abil-hasicreslww.e, thepnxwwphase uf which te•asdiunini-hol or absent following a setrond dorc of the drug. 11iQs blmd perfusion, 0.5 pg nirotine produced local caronlotor reartionri but tart prcr.ncn• n•flrx efferts un sN:•tt•nric lduu"i pre~ nn•. M higher nieotine doses, systemir reflcxex were observed with both RinKer's solution and blood as the perfucate. I),sw.- nf 0.1 and 0.2 ppt nicotine were, generally, subthreahnld; in onc experimrnt, 0.2 pg nieotine pnrluced small depmawr ehwics- in syKlemic blood premun-, but, whcn more com•eutmted nirotine solutions were used, the reflex acquired a premnr chararter. 3.'i0-C, Chemorerrptor Rr11ra frorrm the Ear. .llirtkif-u>ki (1650) relxn•Ied a drpmwnr m8rN invnla•ing rhrm~trvrptnr• of the rabbll ear. 11'Ith the rabbit ear IMdateJ fnxn the auimal I Produced hi Th-a Cn;mcil for uoo299u Tobacco fzescalch-USA, Inc.

CAItAIOVASCUI.AR S1-STE.'ul 65 I i except for a nervous connection, and the vessels perfused, . . , .u, ~ .~ . , .. . _ tors to cause a sudden Uin artet•• sl blood pressure. In the perfused rabbit•ear, conrtected with the body only through the N. suricularis magnuc, stimulation of these afferent fibers elicited a reflex fall in blood pressure and rise in respiration; 0.01-0.04 mg nicotine had a stimulating action, which, on repeated injection, decreased and finally disappeared (Lem• beck,1957). $30-H. Qremoreeeptor,Reffex from Hind •Limbs. Bremer and Felix (1960b) have demonstrat.ed that nicotine elicits a depressor reflex via afferents in the hind-limb (dog); for de- tails, see below, 392. 351. 1'resaor Effect of Nicotine in CondUions CLarocferised by Lorn B1God Pressure (156) j\o new data.) 352. Preasvr B,Q'ect of Nicotine in Conditfone Choraeterised Lg High Blood Pressure (158) The effect of nicotine ory blood pressure in rats made h,vper- tensive by renal-artery constriction has been studied by Wenzel, t1'attanapongsiri hnd Vedral (1964); for a description of their findings, see above, 34fi. 353. B'Baetof BpinephrineSeo•efion vd .4drenalertomy (157) In comparison with 5-10 pg/kg nicotine infused i.v. in cats, eafuipressor doses of epinephrine, vasoprer,cin, and diruethyl- phenyl piperasinium iodide (D11PP) were said to be, respec- tively, 2 gg/kg, 50 mPU/kg, and 3-5 pg/kg (K. Ytunamoto and Domino,19g4). Following adrenal ligatpon in cats, nicotine had no, or a murh reduced, preseor effect (Ling, 1959; A. Jones, 1962). In dogx, adrenalectomy largely or completely abolished the prcssor efferts of nicotine (E. F. Woods and Richardson, 1955; Gstgounis and Ayc•0ck, 1963) and of cigarette•smoke inhalation (deAloure,1959; AfcGitl,1963). However, nicotine hrjertetl directly into the aorta of decerebrate, adrenalec- tomized cats strongly elevated the blood pressure at doses a•hich, if given i.v., would have had only a slight effect (h:rone• berg, I9bs). Quantitatively, lipation ol the adrettals of spinal catr re- dured the ptewr response o nicotine to 32.8 3 6.6% of control injec.tions (A. Jones, Gomes A)ont:o de In Sierra and Treendr)enburg, 1963). In 14 dogs under pentobarbital an• exthrsia, injection of 0.1 tno</kg nicotine caused a pressor rise of 128.1 * 9.0 tnm Hg (from a control pressure of 129.4 t 7.9 mm Hg) prior to ndrenalectomy, e.nd a rise of 45.7 :t 6.9 mm Hg (from a control pressure of 70.4 ck 6.8 mm Hg) after adrvimlcrtom) ; end the reduction in the nicotine pressor teRlvrnsc was significant, despite the difference in control pns..ur" (co•varianee analysix, p e<0.001) (Gardier et al•, 1960). Although, in the experiments of Gardier and co-.vorkerc (1960), just deacribed; the nicotine ptrmr response was sir:oificantly tttluced after adrenalectomr, the ganglionic pmwor eBert In adrermlectomized do)te was su/l'irient to allow a yuantitath•e study of the gangHonle effects of hexantetho- uium and I•2S6 ~V,A"diisopropyl•~1"•Isoam)1•1"diethyl• aminoethylurca). Each of thcr•e agentH n•aa tested in equimolar dosxge for ability to block the pmxsur effect in 7 adtrnalec- tumir•M animals; both significantly reduced the prexwr r+`I-Lv to nirotine, the nrore potent bcing hexsmethoniwn. In npnul estx, cocaine did not sitmificantly affect the pnK.w+r response to nicotine after adrenal ligation, and this was also Y/u~.:v. uu.~.LWVUtl +A. JVYrA, ~JVUrKil HrWaW UC al blernr anU Trendelenburg, 1963). Choline +^.•6•xyl}-1 ether bromide (Tit,i-I0) reduced the nicotine response after adrenalectomy abotrt one-thud. In bilaterally adrhnalectomized cats during thedepolarisingphaseof nicotine block, the pressor response to ang~otensin wea said to have been reduced (BeneUi, Della Bell's and Gandini, 1964). I.v. injection of 0.1-0.6 mg/kg nicotine in anesthetized adrenalectomiied dop produced marked pressor res)wnxcx+, which were associated with several-fold increases of plasma catecholaminea, predominantly norepiaephrine (Weiner, BuriStck and Draskocey,1961). A subsequent report by 11'einer, Dra+skoczy and Burack (1962) presented a somewhat different pfm-0rrn. hicotime in do-ee of 0.5 mt; wae here mid to produce usually a marked, brief preasor response, but this rise in blood pressure was not associated with a detectable rise in placcnra cateeholamit>es. With doses larger than 0.5 mg• the pressure rise was more sustained, and was accompanied by a definite inert~•~e in plasma concentration of norepinephrine; an increase in plssma-epinephrine concentration did not occur consistently, nor was the rise as marked as that of norepincph- rhie. In adrenslectomized animals pretreated with pheno-ry- benznmine (Dibensyline), no blood-pressure rrsponsc to nicotine was obtained; and doses of 0.5 mg nicotine again lailed to produce an elevation of plasma catecholamines, although larger amounts were effective in increasing plasnur- norepinephrine concentrations in most instane•es. According to some writers (e.g., Ling, 1959; Gardier et al., 1960), nicotine hypertension is primarily dt.e to epinephrine or catecholsmine release from the adrenal glands isec• also 373, below, and (172b; 173a)J. 354. Ir}puenae of Other Drugs on the Preasor Action of .\'icotine p59) 355. Central Nervous System Depressanls/Stimulanls (159). E. F. Woods and Richardson (1955) found that it required about 4 times that dore of nicotine effective In the conscious state to produce comparable precsor and other cardiovascular effects in anesthetized dogs. Lt the cat, 10 mg/kg hydroxydione sodium succinnte [a ba."l anesthetic agentj hardly altered the pressor effect of nicotine, although the time-course was slightly modified (Lermat. and l'aton, 1960). With 40 mg/kg, however, the lne.sor a.•tion of I ntg nicotlne was abolished• Hazard and his associates (1954) have fnrnished data on the antinirotinic action of diethylaminoethylphenobnrbital (1), its iodomethylate (Ia), meth•rk•amphosulfonate (Ih), and brombhenz.latc (Ic), and of diethylaminocthylbutobarhital (II), its iodomctM•latc (11a), mc•h;-kamphosulfonate (Ilb), and bromobcnzylate (Ilc). Tested on dogs under chloralose anestheis, substances I and 11 in dosage of 5 mg/kg sup• ixc.-,NI t!m ichia) va4.t) (h.•potensive) phase ol 0•05 mg/kg nirntinc i r. ,i, blood prc; ure; their quaternary ammonium derivdtivet. were morv active, producing the same effect in dose: var)inu from 0.5 to 2.5 mg/kg. To reduce the sym• pathrtic (prr.wr) phase of nicotine action, dores of the order of 7.5 tug/ka of 1 and 11 arre required, while hle Mrresponding brom, licntylaa•s nere inhibitory in doses of 0.3 (Ic) and 2 (11c) mg/ku. The other quaternary derivatives arre only r=liuhth• m-+m )ootent than the baw., and required doses of 4 ho 5 nue ku. t.• ••d I mg/Icir• morphine and methadone did not to',•rt -,••••••r nspunso of spinal cate to 0.03-0•3 mg/kg Procfuccd hv Thl Council for TOb;L,,J kc-icaldh-USA, Ince 0002997

TOBACCO-FJiPERIAIE1TAj. ASD CLIAICAL STUDIES. BUPPLF-ME\T I i _ r .~ ~ .4: 1- _f. ..I ..A...N ... ...«...... : ... ... ...... .. . . . tias also'; noted following adrenal ligation, but the effect of this procedume was d.ot tried in the case of morphine (Jones. t^,omes Alonzo de Is Sierra nnd Trendelenburg, 1903). Tested on rats, cats, and doga, ~pethidine (meperidine) nnd its two analogues, benJ.ethudine (ethyl-l-(2-betuyloxyethyl).4-phPrr.•I-piperidiae- 4.carbhrnylate] and furethidine (ethyi-4-phenyl•/-(2-tetrabc- drofurJurvloxyethyl)-piperidine4bc•arboxybate], had no effect on the rsponses of the vascular systein to nicotine (Lister, 1960). lnjection into cats of 0.3 mg/kg Raubasin ]-y-yohimbin. ajrrtali~eiin; an 4lkaloid of Rauwolfia) inhibited the lrressor effect of, n,icotine to a greater degree than that of norepneph- rine in the majority of experiments; with 1-2 mg/kg doses of Itad,basin; difletentiation between anti-adrenergic and anti- rdicotirhe effects was not possible (Kroneberg, 1958). Rats pre- treated tvith reserpine showed a markedly reduced pressor rzspnnse; to nicdtine (A. M. J. X. Blair, 1962). Following ad- tltinistfiation of oeserpine to rabbits 24 hours previously, J. J. Reuse (1960) obtained two types of results: with moderate doses of reserpine, the cardiovascular response to nicotine was chardcterized by a fall in pressure with strong brad.•- <ar•diaR without a hypertensive component; if atropine had lieeth pruviottsly given, nicotine caused only a week pressor effect. One month after reserpine, the responses to mcotine had rei,urned to normal. After strong doses of reserpineo blood pressure fell stnbngly from cardiac arrest, and, although the heart recovered after a time, blood pressure remsined very low. (F'o'r detailsof drugadministration, see tbis writer, above, 180-C.) , Increments in arterial blood pressure produced by adrenal stimulation, normally proportionate to catecholemine levels, were not sigrµi6eantly altered following prior adminis- tration of reserpine to dogs (Iiabnvell, Woods and Richardson, 196U). In jiogs anesthetized with pentobarbital, i.v. injection of l mg/kg reserpine enhanced and prolonged the pressor effect of subseqttently-administered nicotine; the potentiating ef{ect appeared in about 10 min, and persisted for several hours (\V;alaszek and Burford, 1963). Reserpine prevented the hypertensive response to cigarette•smoke inhalation in anesthetiaed dogs (~IcGt~7, 1963). The risC of blaod,pressure following nicotine injection into the 'spinal cat was keduced by chlorpromazine (Kolwra and At•rnitage, 1954). Lv. injection of I mg/kg dimethylamiao- p'ropyl-71'-'chloroNltcnothiasine HCI 14560 RP) marked)y decreased the pressoreffect in aneethetizcd dogs of 0.05 mg/I:g nicotine i;v. (Pocidalo et at., 1952). (See Courvoisier et al. (1953) (98a).] I.v. injection of 10 mg/kg (s.piperidyl.A'- eihyl) :V-eth,vlaminoacetyl-l0-phenothinaine 1II.101 in the dog abolished the hyqroteusive action of 0.02 mg/kg nicotine, and slightly diminkshed its M•pertensive effect (Frommel rt al., 1'8616). 9'he tronquiliaing agent, haloperidol, reduced the pressor response to nicotine in dogs and cats, prcbabh• by an adreuoh•tac action (Henri Schmitt and Hi•l~nc 5chtnitt,1.'tfi2). As judge<I by auto-assay using blood-pressure increments as criteria, the pressor response to nicotine was signHicnnth blocked b~' the prior administration of selected doses of several monoamine oaidase (\I aO) inhibitors, while hrior administro- tion of the non-NIAO inhiuitor, isoniazid, was without effect (Gntgounis and Aycock, 1983); see below, 762. Gntgounir, Richardson and Clayton (1964) determined, in anesthetized, vagotontited, open-chcstt dogs, the relationship between changes in heart force, blood prrrcurr, and adreunl-venouc. plaNna caterholamine levels productsl M' nicoline injected before and after the NIA(1 inhibitor, pheniprazinc (.1. 11. 516). Thc mean changes in blood pressure to nicotine were 02 * 14 ...... }7.. h,.L,... qnd dR .a 7 mm Hv wftpr. hiiectibn of fi mv/kr pheniprazine; deart force and catecholamine levels were also reduced. The ani6de of 3•(1'-pyrrolidino)-n-butyric acid IAVS 1016 rlassified as a psychopharmacologic agent with neuroleptic and thy'pioleptic properties (Frommel et al., It3$1a). In dogs under chlorelose anesthesia, i.v. injection of 20 mg/kg of this compouitd abolished the hy}wtensire phsse, and marke.0) reduced the hypertensive phase, from 0.2 mg/kg nicotine i.r. Oral adpninistration of 60 mF/kfl, followed in B hour by 25 mg nicotine s.c., afforded about a 20!`, protection. In dogs, the pressor response induced by nicotine aw partially; antagonized by y-aminobutyric acid ]GAIIA] (Stan- ton, 1960). 1'rior i.v. injection of small (e.g., 0.1 mgikgi do+,a of sero tonin (5.bydroxytryptamine) in dogs anesthetized with chloralose enhanced the pressor effect of i.v ~injecled nicotine, whereas large (e.g., 5 mg/kg) doses depressed the pre.,Vr effect of nicotine (Jacob and Miclwud, 1902); see also below, 371. Bulbo'capnine is also classeli as a psychomimgtic drug; for its effect on the pressor action of nicotine, see 1Talaszek and Chapman (1962), below, 359. 356. Paa•asympathomimetic Drugs (159). In She dog under chloralose anesthesia, the bipl:asic effect of nicotine on blood pressure was potentiated by physostigmine; on previuvs atropiniaation, only a potentiated pressor response resulted (Buchel, LAvy and Alichel-13er, 1956). When, in chloralosed dogs, refractoriness to the presor effects of repeated injections of tetraethylpyrophosphate (TI:PI') had developed, i.v. injection of 0.01-0.03 mg/kg nicotine still elevated blood pressure, although this action waa slightly diminished (Paulet, 1954). 357. Parasympatbetic Blocking Agents (15r9). Atropinc reduced or abolished the fall in blood pressure produced b,r i.v. injection of nicotine in rats (Grewal, Lu and Ailmark, 1962). Seeal.co Akineton (Bipcriden) (H. 14isasand l;let•ehn (1955), below, 363]. 356. Sympathumimctic Drugs <I60). In aUopioised clits under chloralore anesthesia, the pressor reshotsc to nicotine was not modified by prior i.v. administration of 0.5-2 mg/kg isoprennline lisoproterenol] (Buttct•worth, 1963). In cats, the pressor effect of nicotine diminished with increasing development of tachrphylaxis to amphctamhte; on complete tachy-Irhylaxis, the pressor effect of nicotine was absent, or -%n$• slight (Reinert,1957-5R). Thc hyfxrtension produced 'ai the dog by methy•I-i-d- chlorethyl-5-tbiasole iodomethylate was found by Lechat and co•tivorkere (1963) to be dae to an excitant effect on sym- patiretic ganglie; thi:, .timulunt eRe.•1 aua raid Io V•eur '4lh- out secondary inhibition, since the action of airntine was nnt suppresred. 3"a9. Adrenergie Blocking Agents (S,rmpathol?tir brnpts) (160). In crmm-cirrulatimt exioerimrnta relxrrterf b)• Solti and ac.-aciates (1960a), the injcction of dibt•naminr blorked the h,vlxrtensive action of nicotine (for details, r•er abovc, 321). In dogs anesthetized a•ith cyclopropane, thr blucking effect of the dib•naminc t.-pes, Sl'-2.ti IA'-(2•bromo• eth.•h-\'.l.naphthnlcne methylnmine Hlir] and SY-21• ~.~•f'~•ritlomcth~l)-\'•cth.1•9-fluomn.lartine H('1) on nier• tinc.induced cardiac arrhythmias wa.~ generally &&-ociateJ Produced by The Council for Tobacco fiesearch-USA, 1110, 0002998 ! s

CARDIOVASCULAR SYSTEM 814 with a reversal of blood-pressure response (Drill and Hays, effect on the blood-pressure rise produced by i.v. Injection of .n-.• .. ,.. ~ •.n•.• r,~ n \•.+ ..q....• , •• •' l•.N. v. \l. 4,1a16 tlllu L171//u, INI}I) • tne curare-Uke butylamine HCl) prevented arrhythmias following nicotine, subst,.nres, diiodomethylates of a-phent•i-a-tertio•arninoace• but, with this drug, an increase in blood pressure w'as always tates of Q-tertioamisoethoxyethyl, were without eJ(e!t on the obtained. pressor response to nicotine of cbloralosed dogs (Che)ntol et Thetisein blood pressureenduced in rsts by doses of nicotine al., 19601. greater than 0.1 mg/kg i.v. was blocked by phenoxybensamine Following administration of tetraethylanunonium, nicotine (Dibenzyline) (Grewal, Lu and Allmark, 19G2). 1V-Penta- had little pressor effect in the cat (Nfattila and Vartiaiuen, methylene•¢-phenoxyisopropy)amine, N-metbj9-R'-eyelo- 1962; Alattila, 1963a, b) or in the dog (Page and Taylor, hexyl-d-phenox) isopropylaimine and its iodomethylate deriva. 1947a, b,1950). tive, and A'-cyelohexyl-0-phenoxyisopropylamine, in doses of Hexarnethonium blocked the pressor effect of nicotine in the 5 mg/kg, all abolished the pressor effect of 0.05 mg nicotine rat (.4. M. J. \. Blair, 1962), cat (KroneberF, 195g; Mattila in ch/omiosed dogs (Polonavski, Schmitt and Pelou, 1953)• andVartiainen, 1962; \lattila, 1963a, b), and dog (C. Tardieu Lv. injection of 18 mg/kg asapetine given in divided injec- et al., 1951; Ludena and Long, 1961; Roszkowski,1961; D. E. tions to dogs under rhloraloac• anesthesia blocked the pressor Knapp au.1 Domino, 1962). AQmiii6tration of he.smetho- effects of 0.05 mg/kg nicotine, but accentuated the depressor nium also prevented the pressor effects of inhaled eigarette- effect (J. Mercier et al.,1956). smoke in anesthetized dogs (de Nloura, 1959). However, after Administration of phentolamine (Regitine) inverted the pretreatment with both hexamethonium and atr+opine, nico- pressor effects of inhaled cipatette-srnoke in dogs (de Uoura, tine still gave a pressor response in the anesthetized cat 1959). In dosage of 0.1 mg/kg, phentolamine prevented the (Mattila and Vartiainen, 1962; Alattila, 1963a, b). Tbe nico- pressor response of anesthetized dogs to inhaled cigarette- tine-produced pressor effect in eats tvas completely abolished smoke, or terminated arr established pressor response, con- by hexamethonium, while that of equivalent norepinephrine comitant)y with restoration of renal blood•flow to control doses was not affected (){roneberg,1958). levels (Mc0iil, 1903). Aiecam•vlamine blocked the pressor effect of nk•otille in In dosage of 5 mglkg i.v. choline 2,6-xylyl ether bromide dogs (D. E. Knapp and Domino, 1962). The pressor response (TbI•i0) did not affect the pressor response of spinal cats to of nicotine in the cat was inhibited by certain aminobieyclo- i.v. injection of 0.03-0.3 ftlkg nicotine (A. Jones, 1962). 12,2, llheptanes (congeners of inecamy txmine) and bie.rlo- After adrenal ligation, however, T.lf-IO reduced the pressor ;3,2,11asaoctanes (bridged congeners of pempidine) (Edge response to nicotine to less than one-third of that iu the ab- et al., 1960). sence of T\I-10 (Jones, Gomez Alonzo de Is Sierra and Tren. In normal, unanesthetised dogs, the pressor effect of 0.015 delenburg, 1963). ntg/kg nicotine aaa abolished by previous infusion of 0.015 In the anesthetized cat, 20 mg/kg guanethidine reversed mg/kg/min pentolir-ium for 10 min (E. Haes and Coldblatt, the pressor response tT nicotine (Mattila, 1963a, b); but 1959). The pressor effect of 0.045 mg/kg nicoth\e was about guanethidine did not prevent the hypertensive response of 70% inhibited by the above dose of pentolinium, and was anesthetized dogs to inhaled cigarette-smoke (\icCifl, 1963). abolished when 0.15 mg/kg/min pentolinium was infused. A dose of 1-2 mg/kg yohimbine reversed the pressor effect With a still higher (0.1 mg/kg) challenging dose of nicotine, of 0.05 mg/kg nicotine in the shloralosed dog (Hazard et al., however, a small residual pressor a^tivity of about 20 rnm Hg 1955). could still be elicited. Acetylcholine in rather large amounas ln non-anesthetized dogs, the pressor effect of 0.02-0.03 infused sirnultaneot:sly with pentoliniurr, slightly enhanced the mg/kg nicotine i.v. was decreased by s.c. administration of ganglionic blockade to nicotine. Expressed in mg/kg/min, 211 mg/kg of the sympatholytic, N-(3'-pheny)propyl'(2))- the equipotent doses of several ganglionic blocking agents 1,1-diphetrylprvpyl-(3)-arnine [Segontin®), 24 hours earlier against the tranplion-stimulating action of nicotSne in the dog (Lindner, 1963). were: tetraethylammonium chloride, 0.50; hexamethonium 1•P'iperidinomethyl-tetralan-2 (Na•86)--an aminoketone bromide, 0.07; pentolinium tartrate, 0.15a chlorisondamine with adrenolytic properties-antagonized the pressor effect of chloride, 0.04; and mecamylamine hydrochloride, 0.10 (Haw nicotine in eats (Pbrsedsz et al., 1957b). and Goldblatt, 1960). (For details of the test of equipotency, In cats under pentobarbital anesthesia, bulbocapniue•- see aiwve,187-A.) termed here an adrenergic blocking agent abolished the The gang)ion-blocking agent, trimethadinium bismetho- pressor effect of nicotine; instead, a small depressor response sulfate, abolished nicotine-induced blood•pressure changes in was observed (A'alaFo.ek and Chapman, 1982). the dog (D. E. Knapp and Domino, 1962) and in the cat (K. Yamamoto and Domino, 1964). 360. Ganglionic Agents (!61). l3. Tardieu and associates In cats under chlotalose anesthesia, doses of 1-3 mg/kg of (1951) bave ightly pointed out that abolition of the pressor 2- (p- butox.yphenyl) • 2- (tetrah}-dro-1,9 - oxazinometh,vl) - response to small doses of nicotine by ganglionic blocking dioxolan-(1,3)-methylbromide(Q 160) completely blocked the agents is not an adequate criterion of total blockade of the pressor effect of nicotine for about 10 min (Kadatz and s)nspses of the .MWonatridor system. For example, they Klupp,1956). found that 1 mg/kg pentamethonium blocked the hyperten- Investigating a new series of polypropplrne glycol po)ymers, sive action of 0.025 mg/kg nicotine, and also the carotid-sinus In which terminal hydroxyl groups of polypropylene glt•eol reflex; but the effect of excitation of the splanchnic nerve was had been replaced with a diethylenetriamine moiety, \fcCart y qot abolished, and even very large doses did not block the and Chenoweth (1962) found that I mg/kg of these polymerr, hyperteneive effect of central stimuiation of the vagus or that (which iwss~s curaredike properties) sufTieed to block the of niedullary piqAre. Similar findings were observed with pressor effect In dogs of 0. A mg/kg nicotine. hexamethonium, 2 mg/kg of which will block the action of The pressor action of nicotine in anesthetized cats could not nicotine. be impeded by nomicotine or derivativeR, except in large, ln lightly anesthetized cats, I mg/kg p-erythrvidine had no equiactive doses (Afattila, 1963a, b). (For equiaetkve doses of Produced hv Th~ Council for Tobacco Res :ai•ch-USA, (nc, 0002999

i 88 TOBACCn-EaPERIAfEKTAL AND CLINICAL STUDIEB. SUPPLEMENT I w.ear wu~tnruu~w, aee uei~•., :o:~. xu a:ev euv. ~, :.±:, :... :..- blocking action of nicotine itself on subsequent administration of nicotine.) Following administration of ii00 mg/kg cotinine to dogs, i•v. injection of 0.125 mg/kg oud•otine produced a further fall in blood pressure (Bonelleca, Bowman and \IcKennis, 19n2). In the dog under chloralose anesthesia, Lv. injection of morphnlim•lfonnoylcholine-considered to have a nicotii,e- like act5on--antagonized the pressor effect of nicotine, and vice versa (Hazard et a1.,1955). ICicotine hy)x•rten>:ion nas suproressed in dogs by sparteine (Lu, 1952, Frommel, Vincent and, Fleury, 1959). However, a dose of 2 mg/kg sparteine scarcely affected the hypotension from 0.02 melkg nicotine (Frommel, \'incent and Fleuf v, 1959). Canglionic 'blocking agents prevented both the hypotensove response and the increase in renal vascular resistance resulting from cigarette-smoke inhapation in anesthetized dogs (McGiff, 19631; but, in lightly anesthetined cats, 1 mg/kg fi-erythroi- dine had no effect on the pue-s-sor drspotLre t•1, cigarette-smdke inhalation (M. 8. C. Clark and Rand, 196•1)- 361. iaca1 Alnesthetics (162). In dosage of 1 mF/kg, cocnune did not affect the preseor reapof>`~ of spinal cats to 0.03-0.3 mg/kg nicotine (A. Jones, f962). This lack of effect was shodcn either beiore or after adrenalecto)n,v (Jones, Gomez Alonzo de la Sierra and Trendelenburg, 1963). The prrssor effects of nicotine in unanesthetised dogs were temporarily abolished by i;v. injection of 5 ml of 2;•o procaine (Page and Taylor, 1950). In cats ttnder chloralose anesthesia, Lv. injection of 10-100 ing procaituunide reduced or abolished the pressor response to 0.1-0.5 mg nicotine (Paton and Thompson, ,191'rt). 0rthochlotoprocainamide moderately diminished the initial (vagal% hy)iotension, and very weakly reduced the 'subsequeat hypertension produced by i.v, injec- tion of 0.05 mglkg nicotine in chloralosed dogs (liesanton, 195i, 1958). Hazard and his associates have published a series of reports dealing w•itlr the anu•uicotinic dction of local anesthetics; in these experiments, dogs under chloralose anesthesia were emplo.•ed, and the test dose of nicotine was 0.05 mg/kg i•v. Diisopmpyla6ninoethy1'p-aminol)enzoate (Isocaine) in dose of 5-10 mg completely inhibited tl.e initial depressor and subse- quent pressoi e8ects of nicotine, and the action of the iodo- methyl derivative was even more pronounced (Hazard, Corteggiani and Cornec, 1951a). A dose of .ri0 mg/kg m-diethylnmhro-2,6-dimetM•lacetairilide (Xylocaine; Lidb- caine) sufficed to suppress the parasympathomimetic hyµo• tensive, as well as the syh»ftathmmimetic pressor effect, of nicotinP, and the iodometln•1 dcrivairve proved even more Iwtent in this respect (Hazard, Corteggiani and Cnrnec, 1951b). Assig,rinp procaine an tlnlinicwtinic potency of 100, the comparative values of some o( its derivatives were reported (Hazard et a1,, 1952) to be as folloa•s: correspondinK icdo- methyl derivatives, 6fx); p-deaminated derivative of pro• caine, 60; corresponding iodomethy~l derivntive,100; dimethyl derivative of procaine, 40; conrsponding iodomethyl deriv,a- tive, 600; p-deaminated dimethyI derivative of procaine, 20; corresponding wdomethyI deriva¢ive, 40. Thus, p•deaminetion lowered antinicothtk lotency in all instaa•:es, while iodo- methylation elevated it. An i.v. injection of 10 mg/kg p- ethosy-\'-(:-diethylaminoeth.•1) benzamide HCI (111) totally supprrr-.sed the hypotensive pIw•e• and redured by 7017, tpne hypertensive pF.ase of nirotine; aut, to obtain comparable cBects.vith p-anmino•,V•(2-diethylaeninoethyl) benzamidc HCI • ::,. .n ..a ~• in a:...6../.. ...F..h /....,. ..,:A., . . - ...:.,.. . u ............:........ . HCI (II) required dor•ea o1 the order of 25 mF%kg (Hazard et al., 1953a). An i•v. injection of 10 mg/kg p-amino,l'-(2- diethylaminoprop)•1) benzamide HCI (lY) or N-(2diethy)• aminopropyl) benzamide HCI (V) totally suppressed thc initial hypotensive effect of nicotip:e, while p-ethoxy-5•-(2- diethylaminopropyl) benzamide HCI (VI) reduced it only about 50 % (Hazard et al.,1953b). The hyperhensive phase wss reduced about 151 , ' by 10 mg kg of IV and ib 1~"C; by 20 mF/kg; 50 ~ by 10 mglkg of Y, and T0 ~ by 10 nrg/kt: of Y7. Weak doses of the iodumethyl derivatives of 1. II, and lll scan-eh• affected the presor action of nicotine, and suppre.`ed or abolished the initial drpmssor effect, but reductions of about fin- in thP prr-nr eRw•t of +virntine cnuld Ew obtained u•ith 1.0 mg/kg of IX (the iodomethy1 derivative of 111j, 2.0 mcikg of VII (thc iodomethyl derivative of IJ, and 7.5 mg/kg of \'lll (tlle iodoinethyl derivative of 111, qlemonstrrting that iodo• methy-lation markedly enhanced the antinicqtinic activity of 1, 11, and II l(Hazard et al., 1953e). The WomethFl deriva- tives of IV, Y, and VI in very weak dusage had an inhibitory eftectonly on thedepres.sorphase of nicotine action; in do.e of I mt;/kg, X1 (the iodomethyl derivative of 1'J and XII phe iodomethyl derivative of VII markedly reduced the pressor effect of nicotine; X f iodomethyl derivative o( VI) being some- what less active (Haaard et al„ 1953d). The analogue of procaine, tetrahydro-1,2,5,6-pyridinoeth•vl-p•amnrobenzoate HCI, like procaine in similar dosage, reduced or supprecsed the effects of nicotine on blood preraure (Hazard et al., 195S). 362. 4ntihistaminic Drogs (163). (No new data.) 363. Centrally Acting Skeletal Muscle Relaxants (164). H. Haas and Klavehn (1955) studied a number of antiparkin- soni.nn compounds (for details, see abave, 130-B), and re- ported that blood-pressure experiments in the cat showed that all of the antinicotine substances studied had anti-cholines- terase characteristics. As an e,~ar.tple, Akineton (Iliperiden) depressed the nicotine pressor response. 364. Drugs Acting Directl.• on llfuscle (164). (Ao new data.J 363. Aliscellaneous Drog E/tects <164). Supek, \lilkot•ii• and t'roic (1958) reported that slow i.v. inGusion of ferrous sulfate into ancsthetized dogs, cats, and rabbits had no effect on the pressor response to nicotine, although it lem-~emvl the response to epinephrine, norepinetrhrine, and several other vasoconstrictors. Indoresof 35 µg-5 mg, thiamine hydrochlodide did not alter the hlood•prrcsure response of the anesthetized cat to nirotine (Ilhagat, 1963). In the dog under pentobarbital anestheFia, however, the pressor response to nicotine was inhibited by prrtreatmmnt with 5 or 10 mg/kg thiamine (L Yamamoto, 1963). IThe larger dose of thiarnine did not aftect the pre.,,snr respouse to 1-2 pg/kg epinephr)ne, nor the depresewr resfpnM• to 1-2 µg/l•g acetylcholine,J Not only thiamine mNafnmiznl the pressor response of dolt,,A to nicotine, but also thiazule derivatives containing an amino group at "2" atd/or a phenyl (rroup at other than "2" elicited an augmentation in auta{!o• nistic activity to nicotine (1`amaruoto, 1961). A dose of 10 ma/kg phenetl•-)•IdiKuanidine diminished, aml 22 mc/kg abolished, the hyy>PrtensA•e effect of I ing nicotine i•v. I? dopl (Ashkar, )lurruer and de Peralta Ramos, 195.~). Pk:tnikoff and co-a•orket:s (1963) Rstudied the stimulant effects of tot..~h.•amr:rloltep18yu111011ne derivatives. and Protluccd bY T?-~ Cotmcil for TobaccO he;caich-tiSA, Inc. 0003000

1 CARI)IOCASCUI-AR SYSTEAI 89 I L nf ~n/,.qMF.•In.•.nnl,nvnt,q/P lTCPPI v/;tl nnwt,"-wV effect (Paulci,1954). liow•ever, when nicotine administration was continued to the point of respiratory paral}sis, TEl'P no longer modified blood pressure. 368., Sympathomiruetic Drugs (166/. The pressor action of epinephrine was potentiated in aicotinized dogs (Fujimori. 1955). 1n dogs under sodiuko-anmobarbital anesthesia, i.v. injection of 0.3 mg/kg nicotine also potentiated the blood-pressure response tosubsequentl,v-injected norepinephrine, presumably through reduction of sympathetic blood-vessel tone by the nicotine (FLjimnri, 1958). In rats under urethane anesthesia giveh repeated ix. injections of nicotine until the drtig no longi'r produced a pt+e.~or effect, the pressor respon.i;e to norepinephrine was potentiated (Gokhale, Gulati and Joshi, 1964). Prior ix. injection of t-5 mg/kg nicotine in dogs diminished the h-vpotensire action of subsequently-injected -nethyl diethvIphosphonate; this compound was said to have a stimvlant action on sympathetic ganglia (Salic, 1951). Following doses of nicotine sufficient to paralyze sympa- thetic ganglia in dogs under chloralose anesthesia, d-phenoxy- isopropylamine, A'-meth.•1-0-phenoxyisopropylamine, and A',A"-dimeth)•l-/3-pheno..•,visoprop.rlamine no longer produced pres4or effects, and the first two even caused a fall in pressure (Polonovski, Schmitt n.nd Pelou, 1953). Previous administration of nicotine to dogs in dosages M•hich elicited little or no change in arterial pressure, followed by i.t•. or local arterial injection of small :unounts of various waso- consirictors drugs in dosages n hich had no significant effect on arterial pressure by thetnselves, resulted in arterial-preMare incressex of 50-180 mm Hg (\IcCubbin, Kaneko and Page, 1981). This reaponse to vasoconstrictor drugs injected into a lumlio-adrenal artery, was prevented by simuitaneous ad- ministration of a vasodilator drug. (For the relationship of these ea-periments to adrenal secretion, see below, 762.) 369. Ganglion-Stimluleting/Blocking Agents (167). Aior- pholimlformoylcholinc- -said tc have a nicotinedilx action- antagonized the pressqr effect of nicotine in chloralosed dogs, and vice versa (Hasarcp et al., 1955). Following repeated doses of spartcine (averaging 40 mg/kg) to dogs anesthetized with chloralose and u%der artificial respiration, a slight pressor effect, accompanied by brady- cardig, developed, instead of the usual depressor one (Lu, 1952). Complete nicotiprization railed to prevent this. General ganglionic blockade by injection of large amounts of nicotine in spinal cats blocked the ptt»csor response to dimethylphen}•Ipipcruzinium iodide 1ll\ll'Pj (.4. Joncs, 1962). For an acrount of the effect of ttanglionic blocking agents of the muscarinic type (Yde\-a-343; AHR-602), see above, 367 (A. Jbncs, 1902; Jones et al., 1963; Franko ct al., 196c1; Gok- hale et al.,1961). 370. Drugs Acting Directly on Muscle (187). I.v. doces of 0.5-1'A mg/kg nicotine ht spinal cats (a dose smaller than that requiied for complete ganglionic blockade) increased the secondary rise of blood pre.~sure of subsequently-injected histamine (L'. Trendrlenburp, 1961a). Following doses of nicottne (10-20 mg/kR) producing full ganglionic bloek, a subseiauent iniection of histamine caused no pressor response, or only a verv small mne, An identical series of nicotine in- jections was Fften 30 min later, and it w•as noted that the respoolse of the blood lireseure to aicotine was much reduced, ..~.t .1.-• i.. .1., .....In n.nl.n.l.:.n1 n......h...•. .inrnv : ~ ~. . of 1 or 9 mg/kg ix. potentiatcd in various degree the pressor response to0.025-0.1 mg/kg nicotiue. In the cat, the pressor effects of nicotine were potentiated by low do.qes of these agents, and blocked by high doses. In anesthetized dogs, nicotiue administered subsequently to 5 mg/kg or more of Osthol produced its usual cardiovascular effects (Jamnal. Anand, and Chopra, 1962). Osthol is a nsturally-oecurring coumarin isolated from the roots of Prangos pabularia Lindl, a tall perennial herb found in Kash- mir. Scaeli sibiricum Benth. is a small perennial herb found in the Western Himalayan region. Iu the anesthetized dog, ix. injection of 0 2 mg/kg of the volatile oil ianlated from the aerial parts of the herb did not alter the pressor effect of 0.02 n,F/kg nicotine (Jamwal. Sethi and Chopra, 1963). 366. Pressor Ejlects of Other Drugs in tbe A'irotini*(d Aninml (165) 367. Parasympatbomirnetic Drugs (165). In atropinized doKs under pentobarbital anextheam, a dose of 8 mg nicotine completely antagonized the pressor responses to benzoyl- choline (1{oppanyi, Karesmar and Sheatz,1953). Nicotine injected into spinel cats in repeated doses sufficient to produce depolarization of ganglia blocked the pressor response to pilocarpine; but, during the later, non-depolarizing phase of nicotine ganglion block, when nicotine still failed to cause a pressor response, the pressor respouse to piiocarpine was restored (U. Trendelenburg, 1961a; A. Jones, Gomez Alorzo de Ia Sierra and Trendelenburg, 1963:. (.See also Trenot:mburg (1961a), below, 370.1 Several workers have investigated the pressor response in nicotinir:ed animals to the mttscarinic compounds, \ich-.a• 343 j4-(nt-chlorophenylcarbamoyloxy)-2-but)royl-trimethyl- arnmonium CI) and AHR-W2 I11'-benzy)-3-pyrrolidyl acetate methobromideJ. ln dogs under phenobarbital anesthesia, ix. injection of 3.5 mg/kg nicotine eliminated the pressor response to AHR-81V (Fl•anko, Ward and Alphin, 1963). General ganglionic blockade by injections of large amounts of nicotine a-343 in spinal cats first blocked the pressor response to %IcN3 and AHR-802 (phase of depolarization), but, during prolonged blockade (non-depolarizing phase), the response to these agents was restored (A. Jonea, 1962; Jones, Gomez Alonto de la Sierra and Trendelenburg, 1963). In dosage of 10-20 mg/kg, hexamethonium (which, without previous injections of nico- tine, failed to block the action of AHR-802 and llc\ :4.343) reduced the pressor response to both agents after large amounts of nicotine (Jones, Gomez Alonzo de Is Sierra and Trendelenburg, 1963). In rats under urethane anesthesia, increasing doses of nicotine (0.2, 0.4, 0.0, and 1 mg/kg; total dose, 1.2-2.2 mg/kg) injected i.v. In rapid succession, until the drug nc lon;ter produced a pressor effect, totally blocked the preaeor responses of sutsequentl}--inje,ted AHR 602, 11IcN•A-3l3, and physostigmine (Gokhale, Galati and Joshi, 1964). In the atropinized cat, neostigrnine caused a rise in blood pressure, which could be blocked by 25 mtt/kg nicotine (Fromhera and Pellmont, 1053). In dogs, ix. injection of ncostigmine meftlsulfate produced on marked alteration in blood pressure; but, following pretreatment tvith 8 mg/kg nicotine bitarttate s.c., it• praluced a pressor response (J. P. Long and Eckstein, 1961). After ix. injection of nicotine into chloralosed dngs until its effect on blood pres.cure had just disappeared, an injection Produced bv Th^ Councilfor Tobacco Rcscaicil-USA, l 0003001

I f 90 TOBACCO-EXPERIAfEICTAL AND CLINICAL STUDIES. i3UPPLEkfENT I 1nWCalWg lIW4 We uID~ ecNna w uyquws .. its ganglion.blocking effect. The subsequent injection of histamine always caused a pressor response, which exceeded the control response. A third series of injections of twice the amount of nicotine had no effect on the blood pressure, but histamine then caused an even larger response. Hexametho. nium reduced the pressor respomw to histamine to about that of the controls. Experiments with pilocari,tine gave results similar to those obtained with histamine. The mechanism of this "sensitization" was said to remain obscure. 871. AlisceUaneous Drugs (167). Low doses of nicotine depressed the pressor esponse of chloralo.eed dogs to trypta- mine, while higher doses enhanced it (Jacob and blichaud, 1962). The pressor msponse to 5.hydroCytryptantine (-ccro- tonin) consisted of an initial spike, following by a second, more sustained "dome" effect. Nicotine (0.01-2 mg/kg) progressively inhibited the spike; lower doses depressed the "dome", but hipher doses enhanced it. There was said to be no cross-tachyphplaxis between serotonin and nicotine, but there appeared to be some common pathways, for example, in the case of the 5-hydroxytryptamine spike. (Also see above, 355.) In dogsanesthetized w itl chloralose, the efiect of nicotine on the pressor response to i.v: injected acetylaldehyde varied with time, the pressor response being enhanced when the acetylaldehyde was injected immediately following subsidence of the pressor response to nicotine, but being depressed if injected somewhat later (Jacob and Michaud, 1962). During the depolarizing phase of nicotine block in both nonnal and spinal cats (nicotine injected in increasing amounts, until there was no further response of the blood prrssure), the preswor reaponses to angiotensin were signifi- cantly redttced; but, during the phase of non-depolarizing ganglion block by nicotine, a subsequent addition of angio- tensin always elicited enhanced pressor mponses (Benelll, Della Bella and Gandiui, 1964). Following ganglionic blockade with nicotine in the dog, i.v. injectitm of a mixture of the total alkaloids of Vinca minor L. [periwinkle or myrtle) produced a hypntension (QuBvauviller, Lemen and Janot, 1!)61). [For an account of the effect of vincamine (an alkaloid isolated from Virua urinor) and nico- tine on isolated intestine, see Alachovg and Mokr~ (1964), below, 615-1i and 616-E.) 372. Hornwnaf /nJluerters on Nieotbts Hyprrtension (168) J\o new data.) 373. Wer Inftue/,ces on the Pressor .-lction oJ Nero/{ne (168) An average dose of 3.2 * 0.72 µg/kg was required to produce changes in the b.bod pressure and respiration in old cats, compared to 6.5 * l.3 pg/kg in young animals (8hche- goleva, 1961, cited by Frol'kis,1962). A rise in blood pressure and increase in heart-rate were produced by 1 mg/kg nicotine i.v. in young dogs, as well as in adult ones, but bradirardia and blood-pressure fall in the former were slight (Saito, 1964). Nephreetomized rats were found to be relatively less sensi- tive to the pressor action of nicotine than were intact animals (A. A1. J. N. Blair, 1t162). 374. Comparnttive Prrssor Ejlect of Nirotfne and Cerfatin DeritvlFars (168) Nicotine, nornicotine, ethylnornicotine, allylnornirntinc, benzoylnomicotine, and carbaminoylnornicotine, all caused a s..~ thP qnPC/t1PtY7PIl /"Ar whi1P srrh•1. notnicotine pror!uced a fall (Mattila and Vartiainen, 1962). Taking the relative pressor response of nicotine as 1, the com- paqative activities of the derivatives were: ethylnornicotinc, 1/10; nornirotine,1/15; allyl. and benzoylnomicotine, 1/206- 1/100 (;,lattila, 1963a', b). Again taking the potenr)• of nicotine as 1, the comparative activities for ,\'-ethylnomiro- tine and ~'-propylnoniicotine on cat blood-pressure xere, respectively, 0.06 and 0.006 (Erdtntan et al:, 1963). Compared to nicotine bitartrate on the blood pressure of rabbits and cats, the nirotinr•like action was retaiiurl in nicotine monomethiodide, which showed a marked derrcasc in ihe adrenergic effects, while both nicotine iaomethiodide and nicotine dimethiodide showed no nicotine-lilte action (Shims- mdto et al., 1y5b). In dogs, incidentally, nivotine nnom» methiodide produced a blood-pressure rise withuut accom- pamying electroencephslographic activation (D. E. hnalry) and Domino, 1962) ibut, being 100 1~; im»zed, this comlound would not be likely to pass the bloud-brain barrier). 375. .Vechani.smm of tke Pressor atttioa of Nicotine (Mamnlafs) (170) It seems apparent that invesctigators in the field must conr sider the mechanisms of the presror action of nicotine to be now adequately underst'ood, at least with respect to the organ. tissue level on which t4tost experiments continue to be per- fotmed, and beyond which there is still small venturing to. wards cellular or subeellular pharmacology. The quite detaikd acaount of these mechanisms in Tobxeo (170b-173b) has not bei!n materially affected by the relatively fe.v subseq,lrntlyd published researches or speculatiun; and it is, only possible in this Supplement to document vith a few additional references some of the mechanisms described therein. As summarized in T+mbaeco, the blood pressure ol animals treated with nicotine may be elevated through: (1) central vasomotor stimulation, either direct (Solti et al. (1460a); butsee H. Schmitt and Hrl@ne Schmitt (1963), belowi or, via chemoteceptor reflexes (see partictjlarly 350-A and 35i)-B, above); (2) sympathetic-ganglion stimula6on (Page and AlcCuh. bin, 1953; Kroneberg,, 19513), possibly together with some parasympathetic ganglionic depression (see J. I'. Long and Hlghgenboten,1969, below, 390); (3) stimulation of )wst-gal>glionic adrenelrgic 6hen=, n:w'1- ing in local release of catecholamhmr in the vascular wall (Gross and Bohr, 1964; and below, 387 in particular); (4) a direct effect of the drug on the vset ular musculature itself (see below, 3&i); (5) epinephrine release from the adrettal glands (Palm and MeCubbin, 1953; Solti et al., 1960a; Gatgounis and A)rork. 1961, 1963; and see 353, above); (6) peripheral vasocollstnctlotl, independent of central action (consult Slmcial Citrulatians, below, 3f36-403); and. possibly, (7) vasopressin release (210a); but thiR mechanism was ssid to have been ruled out (Solti et al., 1960a). According to H. Schmitt and Whie Schmitt (1903), In. jection of nicotine into the dieneepbalon, mecencephalon, a• medulla oblongata of anesthetised cats had Oo effect on binnd pressure, although inhibition of central aulonomic reactions evoked by electrical stimulation of ther.e structuren did maull. I.v. injection of nicotine also Inhibited the effects of elertriral stimuli applied to the above stntrtnrec. (For details of Ih(-..e exieriments, see above, 154.) The maximum blood-pressure response of dogs folbwing Produv•f ro: 1n6l for Tobacco I;, s~u1c;]-USA, Inc. G00 3U:.'G

CARDIO\'ASCULAR SYSTE.t\1 91 uiLUuuc txi,uwir,uauuu uim a Miua ituigrui uwee pa~aiiriaw the mavimum bEood.epinephrirte level (1\'atts,1960); and the sympat#tomimetie properties of nicotine have been explained solely oht the basis of the amount of norepinephrine it releases (Faa-az; 1903). There appears to have been little recent mention of the hypote>osive phase of nicotine action. Yoshikata (1964) observed changes in blood pressure, heart-rate, and electro- cardiogram in dogs given i.v. injections ot 0.1 mg/kg nicotine, before and after transection of the vagus, carotid sinus, and spinal cord; and he interpreted his findings to indicate that the bradycs.rdia and succeeding btood-pressure fall were excited partly by cardiac reflex through the vagus, while the blood• pressure rise and tachycardia were attributed to the action fit nicotine per as. 376. Effert of N&oti.ne oin Venous Pressure (170) Becht (1920) measured venous pressure in dogs in the torcula and in the stump of the a-dllary vein (where the latter joins the jugulara) in dogs before, during, and after the ad- ministration of nicotine. Arterial pressure showed the charac- teristic rise, but the general venous pressure nas essentially unu'uinged in 2 e.eperiments, although torcular venous pres- sure rose from 30 to 33 and from 20 to 28 mtn of sodium- carbonate solution of op. gr. 1.088, respectively, during "nicotine action". In dogs under ehloralose-morphine anesthesia, injection of small amounts of acetylcholine into the portal vein increased portal-vein pressure, and this effect was not modified by previous administration of nicotine (Bennati, Ghiggino and Zexbino, 1955). In cross-circulation experiments (see above, 324), injection of nicotine in the acceptor dog had practically no effect on venous pressure in either the ac.ceptor or the donor animal (Solt.i et al., 1960a). 877. EtFEC'f ON BL09D %'ESSELS AND Oti Ct&taL/1TION TNBOllaN 6pEClAt. nCOt01•S (173) 378. $lood 1 "ersels (173) 379. Invertebrate (173). (\o nea• data.) 380. Fish (173). (\o new data.) 38). Frog (173). Nicotine in 'roncentrationa of 1:300,000- 1:10,000 caused strong %twwl dilatation in isolated, perfu.a•d frog-lungs JR. csrnicnta) (Fedotriu•, 1934). 382. Toad (175). (\o new data.) 3113. Turtle (175). (No new data.) 3&1. 'Mamnwl (175). Against euuUuvtions of surtir-chain preparations of the rabbit induced by 25 pg/mi nicotine sul- fate, E.I).,o concentrations (in JrF/ml) to produce relaxation for various smooth-muscle rela?ant- were found to be: atro• pine sulfate, b; lwpnverine HC1, -10; hexamethoniam, 0.25; pil)evidinoethyl-3•rnethyl9avrnie.8-carboaylate HCI, 4.7 (Setnikar, Itavan and UaRe, 1960). The average cotmentra- t ion in pg/ml for seve-al other muscle relasauts a•ere reported to be: morpholinoetM•1-3•meth,vlflavone-8-carbo\ylale HCI, 42; flarone•7-eth,vlowaretatc /II2ecotdilj, 40; 2,0-bis(dictha. nolamiuo)-1,R•dipi}mridinop~riryiido {5,4 d)-pcrimidine, 22; 2-ethcl-3•(3',5'•diiodo•4'•h•vdro.,%I,vbenzoyl•ctmwrour, -1.5; ami. uvNu.W ui,#e, iaJV, /Sio cyv js i uiuiua.C, U.wu; praWrr~li,ru) i~ tetranitrate, 0.004 (Setnikar, Aturmann and Ravasi, 1961). Macmillan, Smith and Jacobson (1962) studied the response to nicotine of normal, denervated, and reserpine.treated iso• lated carotid-artery segments of dogs. On nonnal arteries, single injections of nicotine in amounts up to I mg Idoses of nicotine ea.preased as the dihydrogen tartrate salt) had no eftect. n'hen 4 pg/mi nicotine were perfused through the arteries, the most frequent response was vasodilatation, and this was observed in isolated arteries from normal and reserpine-treated animals and in denervated vessels (denen•a- tion a•as accomplished by peri-arterial stripping or by re- anastomosik,g of segments removed and reversed, 12-17 days being allowed to elapse prior to testing). The vasodilatation involved both the aartulial ..all yer ar 8110 Ilo-.ara vaeorum. Only occasionally was a constrictor response observed, or t•asoronetria•tion followed by vasndilatation. Neither I pg/ml atropine nor 10 Mg/ml hesamethoniurn had an effect on nicotine vai3odilatation. In contrast to the findings on duRr, nicotine had only a4 constrictor effect on arteries from normal or from rer-erpine-treated domestic swine. The action of nit•otine on isolated vascular smooth muscle was also studied by Gross and I1ohr (1964). Nicotine in con- centrations above 0.01 m.11 caused contraction of iwlated helical strips of rabbit-aorta, which was blocked by I µg/ml dibenamine. 1\icotine contraction did not appear in strips taken from rabbits treated with 5 mR/hg rcacrpine 24 hours previously. Nicotine (0.5 mJf) caused relaxati,m of u+dated helical gtrips of dog-coronary arterioles (-f00-500 p), a% did 0.01 µg/mI norepinephrine or epinephrine. itolh nirotinv and catecholamine relaxation were blocked by I t,u/mi nethalide. In the presence of 0.01 m.11 nicotine, epinephrine crontractiuns of aortic strips showed no depressed fast comlwnent, a4, the authors stated, might have been expected if nicotine loarened bound calcium I\ayler (1963), 300, above(. Nicotine in con- centrations subthreshold to that required for contraction (0.001-0.005 m.V) did not hasten the lo~s of epinrphrine contraction in calcium-free physiological saline. Itoth linec of evidence indicated to these arorkcrs that the mechanism of action of nicotine on vascular smooth n.uRClc duea not involve calcium mobilization; and they concluded that the vascular effects of nicotine on i.wolatcd anrtic anil coronan• smooth muscle are mediated entirely throuah the release of notepi- nephrine. In concentration of 100 pg/ml, ni,rotine failed in cause con• traction of isolated filYSral strips from the ductus arteriosus of fetal lambs (l:ovaleff:, 1963). J.1'. Long and HiFhgenboteu (19tr1) studied the mechanism of nicotine-induced vascular resistancc in tF.e superior mesen• teric artery of the eat; see below, 390. i'sing a standardized suction procedure for studying capil. lary fragility in the sl:in of the white mt, I:ozam (1952) noted that local injection of I or 10!; nicotine caused no hemor. rhagic manifestations. 383. Spcrief Cirridations (176) 386. Skin (176). I.v. infusion of 0.02-0.05 mg/l:g/min nico- tine bitartrate in cats decrea:xrl bhwdJloe• through nkin (as measured by flow through the saphenous artery), but the effect subsided +vithin a few minutes after stopping the infusion, and to rehroducc it the second time required approxinmately 10duld higher do. (ltt•cmcr, 1962). Wenzel and his evolleaguci (1901) measured peripheral eirculation in rabbits through the use of the thermal circula- Produced by The Council for 0003003 Tobacco Research-USA, Inc.

t TOBACtY)-F.XPiiR1AWTAL AND CLINICAL STUDIES. St1PPLEStE.1T I 92 tion index: skin temperature - room temiierature/rectal tem• Perature - satn tem)rcrature; uie a.iiuwie ..... . ~:... 2: weeks with either 1.14 mg/kg/day of nicotine in the drinking• water; 1% cholesterol and 5 % cottoaseed oil in the diet; or a combination of t!.e two, with or without the injection or 0.05 ung/kg ergonovine maleate at A-week ihtervals. Peripheral circulation became progressively depressed during the 24. week period in the groups receiving nicotane or nicotine plus cho• Desterol or nicotine plus cholesterol plus ergonovine. The authors pointed out that reductions in skin temperature finduced by smoking or by the administrotion of nicotine are well-known 1(218) and 4371, but that such studies, heretofore, have related only to the acute eff'ects of nicotine administra- tion. 387. Ear (170).10icotine in dosage of 0.62 mg tsiuatid atrung constriction of vessels of the perfused ear of the rabbit (Lem- beck, 1957; I. Yamamoto, 1963). From their several tntperi- ments (Ginzel and Kottegoda (1953) (177a)], Ginaef and Kottega (-Kottegoda] (1954) concluded that the stimulating actions of nicotine (and acetylcboline) on both normal and denervated preparations of the rabbit-ear were not mediated tbrough axon•reflexes in the corresponding post-ganglionic sympathetic 5ixts; instead, they advanced the h,vpothesis that ganglion cells, or ganglia with adrenergic neurone;, or chromaf6n structures, are present in the manunalian heart and along the vessels of the rabbit-ear, and that it is on tht+ce that the actions of nicotine (and acetylcholine) are to be attributed. When the sympathetic 6beps had degenerated, nicotine constriction of the vessels of the perfused mbbit-ear was greatly reduced (J. H. Bum et a1.,1959). Petfusion of the ear of the reserpinised rabbit with norepinephrine restored the vasoconstrtctor action of nicotine (Burn and P,end, 19ti0a.). In this preparation, a large dose of nicotine wres able to abolish the effect of further doses. Summing up these observations and conclusions, J. H. Burn (19W wrote that the isolated rabbit-ear preparation does not contain sympathetic ganglia, and that the constric- tion produced by nicotine must be due to an action on struc- tures very near to the blood ves.cels themselves. Since the coo- strictor action of nicotine was reversed to vaF.odilatation under circumstances in which the constrictor action of epinephrine was also reversed, it appears that ihe constrictor action of nicotine might be due to a release of an epinephrine- likesubstance. Now, norepinephrine cnn be ectrat twl front the w a1ls, of ttrteries and veins, and also from the skin of the rabbit- ear; furthermore, in rabbits prelrcated with trserpine, norepi- nephtine is no longer present in the thoracic aorta nor in the skin of the ear. Nicotine no longer has a constrictor action on the perfused ear of the reserpinieed rabbit Iliurn and Rand (195Sd)). It follows from this that the constrictor action of nicotine is due to the release of norepinephrir.e, either from the vessels (Burn and Rand (195Sd)] or from structures in the skin nearby. Cells containing chromafftn tissuc (which is tissue containing granuLes in which epinephrine or norepi- nephrine is prerent) have been described in ht.man skin, and such cells tue ak.o found in the skin of the rabbit's ear. When rabbits are treated with rtserpinc, the t•bronwffin cells iu the skin of the ear can no longer be seen. Thus, Burn con• cluded, it is possible that the vasoconstrictiuu causQd by nieo- tine itr the vessels of the skin of the rxbbit's ear is due to the release of norepinephtine from chromaf6rt cells. On the perfused rabbit-car, when the reaponse to errnpa• thetic•nert•e stimulation had been blocked by hetnicholinium (which inhibits the syytthesis of acetyIcholine), the re-pon4vs to nicotine and norepinephrine a•ere not only still lueNnt, ti..• . .. ,e4orMi nl t'hnnv and Rand. 1980). On this same test-organ; the polymethylene.bis•phosphonium com- potmd IlCt1Js)i=P•; (CHs)~-1^=(Cel7e)rJr' 21ir- (Q..), blocked nicotine vasoconstriction (Ginsel, Klupp et al.,1951), In dosage of 0.2 mg; thiamine had np effect on the coutrar. tion induced in the perfused, e.cured rebbit-ear by 0.02 tnK nicotine, but an increase in thiamina dosage caused irthibi• tion, and 2 mg conlpletely antagon4zed the nicotine eBa•1 (I. Yamamoto, 1903). Mattila and Vartiainen (11kaa) tested the var•oeonstrictor action of nonticdtinc derit•atices on the perfused rabhit-ear; allylnornicotine and carbaminoyhtomicotine did not rause vasaron.triction; nornicotine, acetti•Inonticotine, benzoyl. nornicotine, ethylnornirotine, and ni~otine did constrict, the last two being the urost active in this respect. I asodiiator resporrses were observed in sonie preparations, particularly from the allyl and acetyl derivatives, but also after nicotine and ethylnornicotine. The vasuconatriMor effects of all of t.hese compounds were Inhibited, and converted into a vaso- dilatation, by tolaxreline. Tetmethylsmmonium antagonittrl the vasoconstrictor effects of the notnirotinr derivatives, as did thiamine also. Re,Prpinitation of the rabbit cautrlleil or weakened vasoconstrirtor responses to nomirotine deriva• taves. 388. Kidney (177). Page and McCubbin (1953) made an extensive study of n'nal vascular and systemic arterial lrn's- sure responses to nervous and chemical (nicotine and other drur,rn) stimulation of the kidney, using dogs under pentu- barbital anesthesia trith innervated kidneys perfused by a constant-output pump with blood from donor dogs. Small m,12 :rO .Ire;es of ttiirotine h:i „ trti into the perfusing blood caused strong t•asoronstrirtor response in the kidney, and, surprisingly, also produced a rise in arterial pressure in the body of the reeipient animal. injection of nicotine into tlre recipient's general circulation caused fharp, very briei rises ia resirtance in the renal circulation, followed by a fall in pressure. &allyl-0.~ dihcdro5•/! dilmnz(r,e) azcpitre phos• phate (RO 2-32•i8; azalxiine phtuphatc; Ilidarl] blurkwl t)re response to nicotine injected into the kidney circulation. Blockade of epinephrine arrd norepinephrltte trywrr~rs in the recipient's body by piperoan (lienodaine) or tolarnline HCI (Priscoiine) blocked nr reverttvl the blood•pm.sure rise elicited by nicotine injected into the tvnal circuits, without affecting vasoeonstrictor ms),bnse ht the renal vascular bed. In prepara- tSons in which the syritutl tord had been cut under sterile conditions at C-6 one day before the experiment, nicotine injected into the rrttal circulati.ut cauEed powerful vaxarntt• striction, with Rood reflrs rise in pmxsure in the recipient's body. This sy:;temit• rr.ryionrr was generally larger x•hen the spinal cord was intact, and sectiun of the vagus nerves Mu- larly increased the response still further. Nicotine pnulured the stmP sharp, brief rise in thc )mrfurt•d kidney when giren Into the body of dogs after section of the conl. Itotlt responses to nicotine injected htto the renal ri:culation were blocked when tetraethylanuAim»um (TEA) was given into thr kidney, but when TE-A tvo.Q aiven into the recipient's bndy, the syH- temir rrslumse was nugrnented. On the other hand, adreurqtic blocking agents in the recipient's body blocked nut the sys- temic rrsponse; given into the renal circuit, they blocked Ixdh retral and systemic response. t'rtravertt•bml lumbar nynpa• thcrtomy (L•I to 8.3), or sertinn of tne renal nerves, had no effect on the ability of nirotinr to elicit rrspnnsM+ in the recipient's lv,d.• when the drug waa {tiven into thr renal artery. Produced hv T~^ Council for .Tobacco Inc.

CAItUlU1•ASCCLAR SYSTE.\1 :.y..::.... „1 ~ :o r•:.;,i.:..: :,... ........ gland and the perfused kidney failed to block the response in the recipient's body, as did also painting the entire area with 5% cocaine.l3ut when a single ligature was passed around the band of tissue connecting adrenal and kidney, and tied tightly, the reRpomre in the recipient's body to nicotine injected into the renal perfusion system entirely disappeared; release of the ligature usually restored response in the recipient's body. From the above, and studies with other drugs, 1'a'ge and 1fcCubbin concluded that nicotine administered into the arterial supply of a,perfused kidney stimulated ganglia in both kidney and adrenal, gland, respectively eliciting renal vasoconstriction and discharge of pressor amines. (Adrenal ganglia were stamulaQed by passage of small amounts of the drug through that part of the normal blood supply of the adrenal gland aristng from the renal artery.) Post-ganglionic nerves mediating these responses were adrenergic in both kidney and adrenal. Emplo)•ing a constant•flo.v perfusion technique, '6rom, Crafoord and Samelius-llrbberg (1904) studied changes in the renal vascular resistance of cats and doge; they reported that i.v. injection of 10-20 mg/kg hexatnethonium blocked the vasoconstrictor effect of close intra-artetial injection of nico• tine. In the atropiniced chlodslosed dog, following i.v. injection of nicotine in increeasung doses of 0.05-2 mg to the point when its effect on blood pressure and kidney volume had just disap- peared, an injection of tetraethylpyrophosphate (TEI'P) still produced a pressor effect (Paulet, 1954). Regional blood-lfow response to cigarette-smoke inhalation in anestheti.,ed dogs was dexatnined for humoral and nervous influences by AlcGiff' (1903). Sintultaneous and continuous measurements (by rotameters) of femoral and renal blood• floa•s revealed a's-30-foid increase in renal vascular t'esistance, associated with a marked pressor response. Femoral vascuk+r resistance was only moderately increased. The response of the renst bed was examined in detail by measuring flows from both kidneys, one of which had been denervsted; denervation did not preclude the increased renal vascular resistance. Adrenergic blocking agent: prevettted the pressor response, or terminated an established prewor re.cponse, concomitantly with restoration of renal flow to control levelc. Ganglion- blocking 9„-rents, teserpine; and adrenalectomy prevented both the h)•pet•tenpit-e trstxmse and the increase in renal vs.ccular res6tance, while guanethjdine did not. The incrrased renal vascular re.spor.se elicited by cigarette-smoke was -aid to be as great a+, or to surpass, that induced by asph..ia and increased intracranial pressure. Aleasurrs designed to interrupt release of eaterholamines, or to bpixrse their action, prerented this regiwnse. 389. Spleen (179). In dop anesthetized with Dial and pento- barbiwl, in.vhich the spleen was freed from nervoin, cotmir- tions, placed in a plethystnogtaph, and perfused by anastamos- btg the splenic artery to a femoral artert (outflow from the splenic vein pansing into a femoral vein), 15-2D µg nicotine injected into the splenir artery caused contraction of the spleen; this contraction was abolished by he\remethonium and by dibenayline, but not by atropine (Del3urgh, Daley and Scott, 1981). In 3 at.ropitiieed spleens, which had been dener- vated et a previous operation, nicotine in doses up to 80 µg had no effect on spk•nic volume. Ticoeitte elicited a response on the perfused dog-spleen similar to that of acetylcholbte (Lesdetx, 1904a). Hemicholitumn (50 pg/ml) inhibited the tresponse to nicotine, but not that to acetylcholine; and during 93 this neriod of inhibition, nerve stimulation produced its char- acteristic response. Chronic denen'atton atwusaea tne re- sponse to both nicotine and acctjkholfne. (These data r-ug- gested to Lmdera the prem-nce of separate cholinergic and adrenergic components in the inned•vation of the spleen, rather than a cholinergic junction in the,adrenergic fibers.) As hr 8'obacco, it is convenient in this place to mention histopathologic and hiatochemical changes in the spleen fol• lowing nicotine administration or tobacro-smoke exposure. No significant changes were noted by \icol and Cordingley (1964) in the weight of the spleen of mice given nicotine by stotnach•tube or a.c. injection; the animals appeared to be unaffected by the drug, indicating sub-toxic dosage. h.omesyn- aki (1958) reported finding hemosiderosis and phagocytes loaded aith black granules (cottsiiderid to repreYent rngidfel cigarette-smoke components) in the spleens of white mire chronically esposed to eigarette-smoke. Histopithologic changes in the spleen of white rats ehroni- cally injected with nicotine have been described by Sasaki (1959). Lupu and Velican (1961b, 1962) found, in the spleen of of guinea pigs subjected to chronic cigarette-smoke exposure, certain very marked modifications in the histological and histochemical picture (for details, see below, 109f1). In mice sacrificed 60 min following i.p. injection of 0.1 or 0.5 mg/kg nicotine, or after 0.1 mg/kg every hour for 4 houts, no definite eBect was found on the total catechoiamine levels in the splaen (lians`rott, \lasuoka and Clark, 1964; Hansson, 19B5). l.p; injection of 0.1 mg/kg nicotine I and 3 hours before sacrifice did not significantly change the norepinephriue con- tent of heart or spleen of mice from that of control values (Sharman, Vanov and Vogt, 1962). In the rat, however, i.p. injection of 0.5 and 1.0 mg/kg nicotine resulted in an initial decrease in norepiuephrine content of heart and spleen (West• fall, 1965a,b); for experimental details, aee above, 331. I.v. or i.p. injection of nicotine alkabid into guitea pigs, as well as mice and rats, had no e6ec' on the catechoktmine and serotonin content of tho spleen (Hansson, Mssuoka and Clark, 1963). According to J. 11. Little, Radfotd et al. (1964), the spleen of 3 smokers and 2 non-smokers had ao average polonium-210 content of 0.02 pa/gm of tissue (range, 0.001-0.0f1Y): no difference in "Ol'o levels between smokers and non-smokers was obseryed 390. Intestine (181). lntra-arterial infusion of nicotine into the superior mesetUeric artery of dogs always caused a pres- sor effect, the local vascular effect consisting of a vasoron- striction, frequently followed by a longer-lasting vasodilata- tion (lireitter and 1'elix, 196pb). The vasomotor reactions to nicotine during perfusion of the stnali intestine of cuta, retrorted by Levtot• (1983,1961), have been described above (350•F), J. P. l.ong and Highgenhoten (1964) have also investigated the mechanism of nicotine- induced vascular tesistance in t he pedu.ced superior mesenterfe artery of the cat, in 55 animal, anesthetized with pentobarbi- tal. thrr a dosatqe•range of 10f,}-400 pg nicotine bitartrate intra-arterially, the responses were directly related to duse• Doses repeated more often than 15 min resulted in a decrease in rasponse. As indicated by the perfusbn pressure, nicotine usually produced a biphasic rexponse, the first being an in- crea.md perfusion pres.wre (indicating ineteasc in tesistat,ce), and this was followed, in 35ro of ea•periments, by vasodilata- tion. In ahout 105;, only a vasodilator reglw»sr occurred. II: 30 e\IMrilnents, nicotine produced a mean inctrase in per. 0003005 Produced bv Th~ Courcil for Tobacco Ptcs~aiclrUSA, Inc.

I 94 TOBACCO-1•:XPEIilA1E\TAL A%1) CLItiICAL t3TUUlE4. BUPPLEME-tiT I ,.._... ~ . r non_ 14 o.».., Rr.. A r,... avctnmir nil- ministration or 1.0 mg/kg atropine sulfate, nicotine produced a significantly lesser rise in perfusion pressure (18.1 f 11.7; 24 observations). To eliminate any possible influence of changes in inteetinal tone on the recorded vascular chanRes, the above dose of atropine was administered prior to the fol- lowing drugs: Guanethidine, bretylium, and reserpine in- hibited the vasaronstrictor response of nicotine and of sympa- thetic•nerve etimulation. Hexamethonium bromide did not antagonize the response to nerve stimulation or to epineph- rioe, but readily blocked the vasoconstrictor response of nicotine. Hemichofiniurn had no effect on the response to sympathetic-nerve stimulation or to nicotine, if the vagel nen•eR were not stimulatedc how•ever, with vap,al-nen•e stinw- lation, hemicholinium readily inhibited the vaaaconstrictor action of nicotine. Sympathetic-nerve degeneration also inhibited the response to nicotine. The authors postulated that the vasoconstrictor response to nicotinc is indirect, and that the site of action for nicotine is the vagal nen•ea, and that this represents an example of paraky-mpathetic activity modifying sympathetic transmission. 391. Liver (182). Injection of small amounts of acetykhoGne into the Iwrtal vein of anesthetized dogs increased portal-vein pressure; this effect was not modified by previous administra- tion of nicotine (liennati, Ghiggino and Zerbino, 1955). 392. Hind-Limb (182). The t•ssoconstrictor eftect of nico- tine in the perfused hind-limb preparstion of the ral was abolished by hexamethnnium (Schmitt, 1956). In hind-limb perfusion studies in the rat reported by Mattila and 1'artisinen (1964a), nicotine, nornicotine, acetyhtornicotine, all.•Inomiro- tine, benzoyluornicotine, carbaminoylnomicotine, and ethyl- nornicotine, all caused vasoeonstriction in varrinF degrees, and this effect was antagonized by 30D µg/m1 thiamine. As a note of possible interest, the fall in blood pressure, ob- served in even non-sensitized robbeta following i.v. injection of tobacco mosaic virus (T\iV), was always accompanied by a reduction in carotid and femoral blood-flows (Chopra,1964). (For an account of the immunology of T.Ni V, see below•,1156. ) An analysis of the circulatory effect of nicotine was under- taken by IAemer and Felix (1959, 1960a) using caGr under chloraloseanesthesia, in which U.02-0.2clo solutions of nicotine bitartrate were infused intra-arterially (femoral or deep femoral artery). Total circulation in the extremity, and also muscle blood-flow and skin blood-flow, and blood luecsure and heart-rate were recorded. The circulation of the skin was always decreased by nicotine, but the effect on blood-flox in skeletal muscle was complex: small doses usually dilated the muncle vesssels, while medium doses produced a biphasic reaction (primary dilatation and secondary constriction). The constriction began during the course of the infusion, and stolqx•cl with the infusiGn; this was also truc with thc purr vasodilatation observed with low concentrations cf nicotine (Bremer and Felix, 1959). if the infusion lasted longer than 1-2 min, there often followed a second increase in blood-flow•. With still longer infusion, the local Imripheral effect of nico• tine wrea overshadowed through changes in heart-rate, blood pressure, and vasomotor innervation; the alkaloid now passed more and mote into the venous return. The reaction picture varied mar:edly from attimal to animal with the same dose, and even within the same animal. This would appear to be due to the reflex effect of nicotine on vasornotor tone. Afterartion of the sciatic nerve, the tendency to vasoconstriction was as clearw evident as before. In a number of experiments with hnra-arterial nicotine infusion, a significant fall in blood pren- sure and heart-rate was obsen•ed; tms coutU nut iwve oxn+unoi frorn an -intravenous" nicotine efkTt, since it sppParwl simul. taneously w ith the peripheral local e8ect,. An analysis of tht+r actions wres undertaken by a croa=-cireulation study in a•hich an extremity of a recipient w-as iwlated except for its bmen•a- tion, and perfused by a donor animal, nicotine being infuwrl into the donor's blood flowing through the femoral artery of the recipient. The blood-preamure fall and bradycardia ap. I>eared unchanged in the rec•ipient, although nicotine had uo accem to its general circulation. 'I'he heart and blood-yireavure effects were absent, however, after deep narcosis or se',ction of the connecting nerves. Further data svcre furnished by exlMri- ments with blocking drugs (13remer and Felix, 1900d,b). liy prctrcating the vascular region ..itb hrwtnetlwrimu or tiriah high doses of nicotine itself, the effects of subsequent nirotinc infusions were prevented (13renmr and Felix, 1960a,b).1'hen- tolamiae HCI (Regitine) inhibited the constrictive e'liects of nicotine, while atropine inhibited the dilating effects (Hremer and Felix, I9(i0a). Intra-arterial administration of epineph- ruie, norepinephnne, histamine, or acetykholine did not prw duce these vacedlar eBects, nor did it weaken the effect of nicotine (lfremer, and Felix, 1960b). These workers concluded that nicotine influences circulation indirectly, that is, reflexh•, by stimulation of nerve structures, and that this probabi.• involves pos4gangUonic sympathetic nerve-fibers (adredergric one•c with a constrictive eBert), as well as cholineriic ones (with a dilating effect). The threshold dose which just pn,- duced the local-vecsel reactions was higher than that n•hich, on i.v. infusion, produced changes in the blood pres`ure snd heart-rate (liremer and Felix, 1959,1960a). Thus, local vavu- iar effects of nicotine in the skin and skeletal muscle probably play only a slight role in circulatory clranges in the case of low i.v. doses of nicotine or in the case of inhalation of cigarette- smoke. I3ee also liremer (1962), below, 393.) "Complete nicotinization" (re+ulting from 10-30 mk/klt nicotine i.v. in divided dosee.) of dogs under chloralose anex• thesis, in w•hich, peripheral vasodilatation was recorded by means of a leg oncometer, kreatly reduced the vasodilatation produced by quinidine, although mar:•ed vasodilatation still occurred to an i.y. injection of 0.01 tnl/kg of spirit 9f glyceryl trinitrate (Lu, 1951). This defnre of mcotiniration prevented the peripheral vasodilatation produeed by sparteme (Lu, 1952). Intra-arterial injection of 0.02-0.2 mg nicotine in ane,the• tir.ed dogs caused a prornpt inc.rase in blood-flow In one or both hind-limbs; constrictor responrrs were not observed (Winbury, 1959). Hexamethonium blocked the dilator re- sponse to nicotine; the effect of atropine was variable, but, in the majority of experiments, had little effect on the nico- tine dilator action, although in no ease was it eliminated. The dilator response to nicotine was not enhanced by neo- Atigmine; it aas dtlncked by cor•ainc and also by type Aor t)•l.e C botulinum to-'in. Prettratnent with remrpine did not alter the dilator tespoitse to nicotine, and, as in the non-trserpiniecd animal, hexamethonium still efterteil the block. In the chtvni• cally denervated limb, nicotine had a markedly reduced reslwnse, and, in several animals, there was practicallyv no response. As judgeti by perfusion experimento periomted 11 days after the section of the sciatic nerve in the dog, nirotine caused a dilatation of the blood veccels; the same result was obtained in an animal asich had received daily injections of rescrpine (Ling, 1959). Hind-limh croxs-I+erfusion pr,•t,etatim~w w•ere rnade by Leaders (1961b, 1965) using fenwle dogs in which Ixrfusion- Produced b-f Th'., Council for Tobacco hcscaicll-USA, Inc. 0003006

CAltl)lO1'ASCGLAIi til'FTj:hd ro1o xnc mninfoinat hr manna nf nmmna qnr) dntpm were In• jected intra-arterially into both animals vin cannulns from the pumps. Nicotine (0.1 mQ/kg) and also acetylcholine resulted in a decrease in perfusion pressure (vasodilatation). Hemicholinium bromide (I mg/kg) administered to the donor animal, followed by syTnpathetic-nerve stimulation (post• gauglionic fibers from the last lumbar ganglia on each side) for 30-sec intervals every 2 to 3 min did not significantlv alter the perfusion-pressure reslanse to nerve stimulation (as be- fore, an increase in perfusion pressure), or to nicotine or acetylcholine. Additionnl animals were prepated as above, except that no donor animals were used, a single pump being inserted into the external iliac artery. Reserpine (2 mg/kg) given i.v. 1 hour earlier appears not to have affected the re• slanse to nicotine, but did enhance the response to acetyl• choline and reduced that to nerve stimulation. SN-t:en gttaneth- idine (1 mg/kg) was given I hour after reserpine, subsequent administration of nicotine or acetylcholinestill lowered peMu- sion pns~ssure, and the response to nerve stimulation w-es re,versed, a fall resulting (Leaders, INS). These effects were not changed by subsequent administration of hesamethonium or gallamine; hemicholinium also did not alter the response to nicotine and acetylcholine, but both were inhibited by atropine. Findings with nicotine were felt to be consisterlt with the hypothesis that this drug acts post-ganglionically through the cholinergic system. The author suggested the possibility that nicotine could act through release of endoge• nous stores of acetylcholine, and postulated a possible role of the neuroeffector junction of the somatic nervous system as a site of these endogenous stores. The over-all data suggested the praaence of separate cholinergic and adrenergic conr Iwnents in the vasculature of the dog's hind•limb, rather than a cholinergic junction in the adrenergic fibers (Leaders, 1964b). In contrast to the results reported by Herbig (1942) (184bp, F. 1C. Brown and Remington (1955) showed that vasocon• striction produced by nicotine in adrenalectolmized dogs was as Rreat, or greater, thatt in control animals; in these experi• ments, blood-flow through the peripb.ral vascular bed served by a femoral artery was measured. lioatos and co-workers (1904) studied the effects of intra- a. teriai injections of large doces of nicotine on reactive hyhter- emia in the hind-limb of anesthetized dogs. In 13 of 21 exYteri• ments, such dosea of nicotine injected into the femor•.1 artery abolished or markedlv diminished the hyperemia in response to short periods of isehemia, and this effwt aaQ accompanied by a decrease in resting oxygen uptake and carbon•dioaide release by the tissues of the hind limb. During reactive hyper- emia, t.here was an increase in femoral venous pCOs, which correlated well with the changes in blood-flow; this rise was absent or diminished in experiments in which nirotine in- hibited teactive hytiieremia, suggesting that the effect of nicotine was related to a decrease in the accumulation of carbon dioadde iu the ischemic tissues. bt anesthetised dogs inhaling cigarette-smoke, continuous meusurement of femoral blood-flow showed that, in contrast to renal vascular resistance (see above, 89R), femoral vascular resistance was only moderately increaced (ZleGiff, 1963). 393. Aiuvcle (184). Ae Bremer and Felix (1959, 1960a) dem- ans4rated (see above, 392), the effect of nicotine on skeletal• muscle blood-flow w•as dose-dependent• with low cottcentra- tions (about 30-50 pR/ml), the reaction was one of fleeting vasalilatation; with higher dosea there was an initial dilata- tirnt, follow-ed by secondary constriction. The reaction of the 9•i muscle vowejs depended not onl).• on the dose. but also on t6e initial condition (litemer and !•'eItx, l9tlUa,b). liy the use of measusrs which changed the ve,s.•tel tone, it was poiksible to show that nrcothm had a predominantly constrictive effect in the c•ase of lbw initial tone, and a predominantly dilating one in the case of a high tone (Iiremcv and Fclis,l9l>Oa). By means of thetmi~toirs, lfremer (1962) riiessured blood-flon• through the femordl artery (muscle) and dhrough the saphenous artery (skin) in 9 cats infused i.v. with 20-50 µ{t/kR/min nicotine bitartrate. These doses had but slight effect on blood ptea• sure, heart-rate, and respiratory rate; but blood-flow through mu,wlee was increased and that through skiu decreased. The effects on bjood-floa• subsided within a few minutes after cessation of Infusion, and, to repnoduce them a szecond time, required approximately l0•told higher doses. Itrcmcr con- cluded from his studies that the increased flow through muscle must resuit from vasodihitation, atsd not I>assively from nieo• tine-inducal blood-pressure changes. In experiments performed by Hirvonen and Sonnensehein (:fnii), nicotine u•as, administered intra•arterially and i.v. in active gitstrocnemits-soleus muscles of the cat; intermittent maximal contraction of the mu.ecles was produced by stimula• tion of their motor nerves. In the active muscle, early in an experimend, when the accompanying hyperemia was at a high level, no further increase in blood-flow occurred; there was only constriction when nicotine was given as single injec• tions at a sufBciently high dose. Later on, when muscle blood- flosc during activity tended to be lower, ahik systemic blood pre,o-ure remained at its initial level, nicotine caused a transi- tory increase in the flow. During continuous arterial infusion, progressive decrease in flow occurred. ll'hen nicotine was injected i.v., h t ransitory rise in arterial pressurr, accompanied by a slight hicrease in flow, was observed, whether the muscle was reFting o: active. In experitnents on cats under chloralose anesthesia, in which volume changes of the fetnoral artery were continuously measured by means of an oncometer, Hilton (1959) found that dilatation of the artery tesulted from injection of 0.1 mg nico- tine peripheral to the oncometer, the threshold dose being about one-fifth of this. A dilatation also occurred in response to contraction of the muscles of the lower leg, end this dilator reslwttse w^as fonttd to travel along the artery wall at avmry slow rate, of the order of 10 cm/ser. The known nerve-6bct systems o•ere shown not to be responsible, and the author suggested that the conducting elements were the smooth- muscle cells of the media. Leaders (1985) arranged to perfuse the gracilis muscle of dogs in situ, and reported that hemicholinium did not sig• ai6cantly alter the teslxlase to nicotine or acetylcholine (that is, the lowering of perfusion pressure caused by these dru)ts; see above, 392), while atropine blocked the responses to both. 394. Coronary (1S5). 1Cakowcrki (1908) had noted that coro- nary blood-ffotv increased during nicotine perfusion of the isolated eat• or rabbibhesrt. Initially, the isolated, perfused heart.c of rabbits made atheroscletotic by cholesterol feeding had a higher coronary flow, lower contractile amplitude, attd slower heart•rate than isolated hearts from normal animals (Travell, I#inaler and l:arp, 1960). Nicotine in dosea of 0.01- 0.1 mg produced graded respontrs on heart•rate (acceleration) and contractile amplitude (int•rease) in both groups. Coronary 8ow decrtasetl Initially in every heart; following this, a sec- ondary rise; in flow occurred in every normal, but in no athero• sclerotic he.arl. Notepinephrhte showed a similar diHerence in Produced bv Thn. ''b11r16l Nr Tobacco RcscaiciWS'Al, IIio,

96 TOBACCO-EXPERIMENTAL A1D CLINICAL STUI)1E6. SUPPd.ESfENT I its effects on cononary flow for normal and atherosclerotic h. Hashimoto and co-w-orkers (1964) used innervated Langen- dorff dog-heart preparations in ventricular fibrillation, with completely separated crosa.circulations to the head and the heart, to study emotid-chemoreceptor reflex effects on coro- nary flow. Nicotine in dosage of 0.02-0.1 mg (rapidly injected into the blood perfueing the head) caused temporary arrest of the atrial beat, which soon reappeared, and tended to over- shoot the control rate. Coronary 8oa• began to increase during atrial arrest, and became maximal when the atrial rate was returning to notrital or above. IN'hen the carotid area was completely denervated, there was no change in coronary flow or auricular rate. After administration of I mg atropine to the ianlated heart, or after cutting both vagi, the increase of coronary flow, and initial decrease of atrial rate, disappeared, and a decrease of coronary fiow and increase of atrial rate took place. When a large (0.5 mg) dose of nicotine was given, the atrial acceleration lasted longer, and the decrease of coronary 6ow was reversed to a long.lasting increase. In experiments on intact animals, Papp and Solti (1959) measured bkood.flbw through the left coronary artery in dogs under chloralose anesthesia and artificial respiration. Follow- ing i.v. injectaon of 0.5-1 mg nicotine tartrate, coronary re- sistance diminished or remained unchanged in most instances. No direct relationship between the severity of the electro- cardiographicchanges observed and the changes of coronary resistance was evident. Neither cigarette-smoke nor nicotine administration resulted in constriction of the coronary arteries (Kien and Sherrod, 1960). In normal but anesthetized dogs, femoral-vein infusion of 20 v6/kglmin nicotine led to a rapid increase of coronary blood-flow between the first and 5th minutes, the increase being as great as 9-fold, after which the flow decreased, and leveled off to a value higher than the control (Bellet et al., 1960). The mean iwntrol coronary blood-flow was moderately decreased in dc- with coronary-artery ligation, and markedly decreesed in animmls with narrowing of the two main branches of tne left coronary artery (liellet et al., 1962). (Gradual coronary-artery harrowing was accomplished by placing totsein rings around the circumflex and/or the anterior de. scending branch of the left coronary artery; the casein swells, and within a period of 2-3 weeks, the arterial lumen is consid. crably narrowed.) Nicotine produced an increase in coronary blood-flow which, in normal dogs, was observed to be as high as 125% on an aoerage over the control values; the increase was considerably less (82.57r) in animals with ligation of one coronary-artery b,ranch, and in dogs with narrowing of two main branches (83.3%). The degree of coronary narrowing and/or occlusion was directly related to the response of the coronary flon•; the greater the coronary impairment, the smaller was the increment in coronary blood-flow, and the latter frequently dropped below the control value in the post- infusion period. The response of the coronary blood-flow to nicotine resembled that of anoxemia in the presence of coro- nary insufficiency. In normal dogs, the cardiac output and work increased significantly; the coronary AV difference de• creased; and the oxygen consumption of the myrocardium showed au increase (liellet et al., 1960). In animals with cotronary-artery ligation, nicotine caused only a slight and slow increase of coronary blood-flow between the 3rd and Oth minute; cardiac output, work, and oxygen consumption of the rnyocardium utereased; the coronary AV difference de. creased. The results in dog.c with coronary insufficiency pro- duced by carein vinµ: were simiiar. GeneralU , in animals n ith impaired coronary flow produced by either eoivnary.-ahtery lovatiam or 1» esreln rinrta, the pattern of respon5e to nicotine vvas significantly different from that observed in normad dogs: as compared to normal animals, the coronarti• blood-flow showed only a slight increase, and this was associated with a smaller change in cardiac output, oxygen consumption, and coronary AV difference. Rather shnilar experimenta by Forte and co-workers (1960) on ianest6etized dogs, in which nicotine was infused at a dosage rate of 15 pg/kg/min, and its effect compared to control saline infusions, showed increased h'eart- rate, , increased respiratory minute-volume with a resulting greater arterial pH, elevated arterial and coronarv-sinus blood-oxygen content, and reduced arterial enrbo"otddc content. Coronary arterial venous oxygen difference was un. chanRed, without any greater coronary.oa~-gen-e.xtraction or any change in coronary-sinus venous-blood oxygcn saturhtion or tension during the nicotine infusion. LQft ventkrcular mechanical efficiency stayed the same. Although, the coronary flow land cardiac oxygen,consumption in individual eklperi- aienta appeared to increese proportionately with the aug• mentation of pressure-work by nicotine, the mean changes of the group were not considered significant, probably due to the wide coefficient of variation of coronary blood-flow (t80 %) in thpse experiments. Tbese experiments were interpreted by Forte and associates as confirming the obsercat6ons of West, Gusman and ifellet (1958) (185b) that intta-arterial nicotine does not "cause constriction of coronary arteries•'. The former e.uthors concluded that their project had extended the fimdings of N'est. Guanaui and Beliet in a more inclusive study of myocardial work and meti<bolism, to show that the increased myocardial vigor asociahed with the action of nicotine is accomplished without any change in a•oronary s•i•nous oxygen saturation or tension, which implies that the organism is able to increase the oxygen supply sufficiently to meet the greate- myocardial oxygen demand without any unfavorable tffect on nrechanical efficiency, if the coronary arteries are healthy. l.eaders and Long (1962c) demonstrated that, contrai•y to some previous reiwrts, the ultimate effect of intra-arcerial administration of nicotine is an increase in rorona,ry resistance, which would be expected to result in a dec:ea-ce in coronary blood-flow. These worker; used dogs under thiol>pntal-barbital anesthesia, and found that intra.arterial rnjection of 32 and 64 pg/kg nicotine increased perfusion pressure in the left de. scending coronary artery. Hemicholiniurn, atrophse, stellate gangiionectom,v, or clamping or removal of the adrenals~ had no effert on the response to nicotine; neostigmine inereasivl the reslwnse; heaamethonium, 1„entolinium, reserpine, pan. ethidine, and dichloroisoprot.crenol inhibited the response to nicotine. The authors concluded that the increased perfusion pressure observed was apparently due to increased coronary vascular resistance, in which the sympathetic netwous system or related chromafhn tissue was involved. The release of s,vmpathomimetic amines may have been due to stimulation of pnglion-like structures awMiated w•ith r;•mpathetic titsuer containing catecholamine, or, secondarily, to paresyittpa- thetic-Kanltlia imroleement.'Phis increase in coronary vascular m.Wance may or may not be related to the positive inottopic effects of nicotine. 1n 13 experimentson aneathetiaed cats reported hy Kareva (19&3a, b), i.v. injection of 0.05 nrg/kg nicotine caused a4arir htcrea.te in the tone of the coronary ves.aels, averaging 46 * 6.3 ~j. (The coronary vessels were perfused with the animal's own bl.xid, and meesurec were taken to eliminate the h-tno- dynomi,• effect of changes in blood prescure; resistance hi the system ot the circumflex branch of the left coronary artery Produced btf Th~ eA4anpll PF 0003008 . Jobacca , , i , \a e

CARDIOM'ASCULAR SYSTEUf was recorded.) The increase in coronary vascular tone was _„ .~... .t.~ ..~.r.~ ...o r.,...L...l r+la./.rwnr.linvronlrir changes; these developed immediately after injection, and indicated disturbance of coronary circulation (see above, 325). In some caes, arrhythmia was observed. The prophylactic effect of coPonary-diiator drugs was tested by injecting th'em 2 min prior to nicotine. Po9owing morphine, chloracizin, or papaverine, the constrictor action of nicotine averaged, respectively, 2.6 * 1.6, 7 s 2.9, and 7* 3.4%. Both nitro. glycerol and tetamon prevented nicotine spasm. In a second series dfexperiments, the effect of tfilator drugs given during spasm of the coronary vessels was studied; results were said to have been generally shpilar to the first series. Grewal, Lu and Alimark (1960) found that i.p. injection of 10 mgi'kg nicotine in rate released 3-4 mU of posterior pitui- tary hormone per ml of jrlood, and tlrat, in an isolated per- fused rat-heart, an injecti'on of 4 mU of this hormone reduced the coronary Bow by 25%. In another series of experiments by these eame workers, injection of 2.5 mg/kg nicotine led to a concentration of 4.27 * 0.264 mU of the hormone per ml of blood; and coronary 11ow in the ratrheart was reduced by 25% following an injection of 4 mU of postsrior pituitary hormone (Grewal, Lht and Allmark,1982). In a review of the pharmacol- ogy of the coronary circulation, 1Vt<gria (1951) included the statement, based on his own unpublished data, that, in the whole animal, nicotine increases blood pressure and coronary flow, its effect being very similar to that of epinephrhne. J. W. West, f3eDet and co-workers (1902) classified nicotine rhs a coronary vasodilator, which at the same time produces a stimulating effect upon the heart and increases cardiac work. 395. Lung (186). In open-chest dogs under morphine•pento- barbital anesthesia, with the pulmonary venous outflow of the left lung diverted into an external bypass so that flow-rate could be measured and blood pret•ented from returnirrg,im• mediately to the general circulation, intra-arterial injection of 0.3-0.5 ang/kg nicotine into the limited pulmonary circula- tion reduced systemic arterial pressure by 7-28 mm HR and decrra,red heart-rate 13 be8ts per rnin on the average (Taka• saki, 190"). These effect$ began about 5 see after injection and lasted about 20 r.ec. After left cervical vagotomy, nicoLine failed to produce these effects, which were thought to be mediated through vagal chemoreceptors. Pulmonary arterial pressure was not significantly changed by nitrotire; however, pulmonarJ^-vein outflow decreased 27%, and pulmonary vascular resistance increased ::S % in comparison with the contral. Afoer left cervical vagotonq•, nicotine still did not significantly affect pulmonary pressure, but pulmonary tloa• was decreased about 14°h, and pulmonary vas-cular resistance increased about 38%a fioubrie and co-workers (1903) studied the action of nicotine on the puhnotuu•y circulation of dogs under pentobarbital anesthesia, and such changes as w•ere found were believed to have been secondary to nicotine effects on the systernic circu• lation. The animals received single i.v, injections of 2.5-15 pg/kg nicotine or 10-min infusimix of 40 µg/min/kg, and pubnonary-artery/left-auricle pressure gradient and cardiac output were measured to calrulate the total pulmonary re- dstaru•e. In 23 experiments on 11 anesthetized dogs reported by R. K. Larson and Murray (1963), in which nicotine was infused i.v. at a rate of 20 pg/kg/min, there were statistically signiFcant Increases found in cardiac output, arterial pressure, systemic resistance, and also in pulmonary-arterv/left-atrial pressure gradient (p < 0.01). There was no change in ititra- pleural presanre. Pulmonary vascular resistance increased in 97 15 experunents, and fell in 8; the mean increase of 9.1% was not statisticaUv acQnificant. In 5 experiments, however, the increase in pulmonary vascular resistance was greater tnan a times the standard deviation of 13 pafred controls, while in only I eicperimentt was there a fall of this magnitude. The authors eoncluded that the preponderant effect of nicotine is to reduce the sise of the pulmonary vessels. This study was later extended to a total of 26 experfinents on 13 dogs, with similar findings reported (Lar>;on, Fukuda and Murray, 1965). K. Braun and Stern (1904) investigated the pulmonary and systemic biood•prpasure response to chemoreceptor-stimulat- ing dnrga, including nicotine, in anesthetized, open-chert dogs under artificial respiration. According to their preliminary report, following injections of either nicotine 15-2f) pg/kg as the bitartrate), lobeline, or c.yrenide into the arcending sorta, systolic tdnd diastolic systemic pressures ruse, and pulmonary- artery pressure rose simultaneously with the systemic pres• sure; in two-thirds of the experiments, the pulmonary systolic and dia4dolic pressures rose concomitantly, while in one-third, no changes of diastolic pressure was noted. Following injec- tions into the abdominal aorta, there was no, or a markedly diminished, response in the pulmonary and systemic blood pressures; and following injections in the femoral vein and into the pulmonary artery, the presror respome in both the , pulmonary and systemic arteries was dimuiiahed. Following denervation of the aortic and carotid chemoreceptors, the rise in systemic and pulmonary pressures was markedly diminished, and the latent period was prolonged, from which the authors concluded that chemoreceptor stimulation by these dnrgs had ea.used pulmonary hyperteneion. Hoxrver, since in one-third of the experiments only s}sto1M pulntonary pressure rose, while the diastolic pressure remained mr, changed, the rise in syetolic pressure might have been due to an increase of the pulmonary blood-flow caused by atirnula. tion of the myocutrdium. To study further the hemodyrranuc effects of stimulation of the aortic and carotid-body chemo• receptors, S. Stern, Ferguson and Rapaport (19Fr1) injected 5-20 pg/kg nicotine bitartrate into the base of the ascending aorta of open-chest dogs under pentobarbital anesthesia and artifcially ventilated. Pwa<urrs in the pulmorrary artery, left atriam, and femoral aaeries, and tbt• stroke-volume of the right ver.tricle, were tnea.ured simultaneoualy; and frum these, puhnonary and systemic vasentur m=i-tanm (1'1'R, ul;t) were +Wce'ated. Changes began 1.5-3.0 Fec after the injection, which was followed by a significant reflex rise in PVR, thesenrory receptors for the reflex being aortic che!no• receptors and the efferent paths being symlwthetic fibers. Similar effects a•er+e not elicited by stimulating the carotid body aloae. Rronchcxo»strirtion, changes in bronchial blood- 8ow, shifts ot blood volume between the vascular beds, and liberation of catecholantine were excluded as factors in the I'VR increase. I3radycardia, increased Fi'R, and decreased flow alro occurred after combined stimulation of the aonic and carotid chemoreceplors; however, when bradycardia was prevented by anupiniaation, no significant change in (pulmonarg bluod-jBow occurred. Lundseth and associateu (1903) investigated the effect of cigarette•smoke Inhalation upon the nrterlo-venwys (.a-V) blond•8ow in the rat•lung using the following technique: Graded radioactive microspheree (20p), either glass ("Na ap. gr. 2) or ceramic (1tc, sp. ftr. 3.4), wrre injcctcd Into the femoral veins of adult rat, anesthetized with pentobarbital sodium; the animels were killnl with saturated KGl I min after the injection, the lobes of both lungs were weighed and \ Produced hv Th^ Council for 0003009 Tobacco Research•USA, 1041

I I 98 TOBACCO-EXPERIMENTAL ANl) CLINICAL STCI)IE.-4. t+CPPLhw%lE1 f I radioassa}•ed fn an '•.tmrac" Y-scintillation detector, and thr remnininp rRrrnka trRC n,rlinn-mvvrf In a aimllRr InRnnPr. The fraction of radioactivity of the aliquot found in the car- ca.,x provided the intornmtiUn necessary En calculate the pro- portion of the injected spheres larger than 20µ which pa.sed through the A-V shunts; this fraction represented the .)<-N blood-flow through the lungs in i>er-cent of the total flow, with the previously-riemortistrated fact that the spheres were distributed at artcrial bifurcatium; in the same proi.or- tion as in dte blood. Trac•hcatomized rats were placed hy a constant-volume (9.5•ml)-piec~ure (10 mm Hg) nspirator supplied with 40,1/-, ox)•gen, and cigarette-smoke was de- livered via an electronically controlled smokiug-r•hamher which could be shunted into the breathing circuit for everr 9th respiratory cycle. A KteUdy state was obtained by stop- ing the respirator in inffation position, after which the sphe.res were injected in 2-3 sec. The reported series edn- sisted of 7 control rats, 9 animals which had been subjected to smoke for 5 roiu, and 6 given 0.05 mg/kg succim•leholine (Anestine®) i.v. The A•V shunt flow in the control group was 8%, in the "smoked" group, 9.2 %, and in the succinvl- choline-treated group, 16.5%. Thus, cigarette-smoke un- halation had a small, fnsi;,rniflcant effect upon the A-V flow in the rat~lung. 396. Brain (187). Solti and co-workers (1960a) isolated the circulation to the head of an acceptor dog from the systemic circulation, and lierfused by cross-aruustomosix with the circulation from a dot:or dog. Thus, nicotine injected into the arterial branch leading to the acceptor animal's head reached a high concentration in the brain, but, even 210, cerebral blood-flow ure.m ptsctically ntraHected, as it a•a.• in the donor animal as well. 397. 8pinal Cord. Skoglund (1961) measured blood-flc.c in the spinal cord of decerebrate or spinal cats by inserting thermocouples and at the same time measuring systeniic blood pressure. Systemic blood-pressure changes produced by sttuiller doses of nicotine were rather transient, and the local dectrases of blood-flow observed iu the spinal cord considerably outlasted the systemic effects. 398. Retina; Eye (187). Felloas and Bettman (1960) called attenticn to some of the difficulties in the exy.erimental study of the intraocular blood-volume; discussed their own experiments 1Bettman and Fellows (1956) (Mb)); and re- marked that the effrK•t of cigarette-smoking on the intraocu- lar blood-volume of huntans is not known. However, for what is known about the effect of nicotine and cigarette. smoking on retinal circulation in man, see below, •180-A. 399. Oral and Nasal Dlucosa (187). No new data on ani- mals; but for the effect of cigarette-smoking on the blood- flou• of nasal mucosa in ntan, see brettner (1965), below, 480•E. 400. 1(eproductive Organs (188). In the discussion of their study of the in-vivo effect of smoking and the in-vitro effect of nicotine on human uterine contractility, Kumar and Zour- las (1903) regarded it as questionable whether the degree of increment in uterine activit)• observed was capable of re- during the placental blood-flow (see below, 799). 401. Endocrine Glands (188). [No new data., 402. Exocrine Glands (188). (No new data.] 403. 111iscellaneous (188). N•enzel, ingianna and Grunde• mnn f1iNia1 auld'ted vascular interaction of entoriovine with nicotine or epinephrine in the rat-tail; for an account of their result,, vee abuve, 330. 401. EJkrf of A'irofine on llis(opaUrofopg hf the Blood i•essrla (188) 40'a. General (188). Hi:qopathololric changes in the blood va:,rls of white rats with chronir nirotine poisoning have been desrribed by 5a+aki (1959); but Thienes (1900) ub• served no gross or histological changes in the aortir arch, abdominal aorta, popliteal, artery, or vessrls of the heart, liver, and kidney in mts htjected twice daily with minimum corn•ul4ive doms of nicotine fur 2-(i months (189a); and liersh- baum, ltrllet and KhotwWiau (19ar) f..wnl tdw fu.thulogirr changes in the aorta or the coronary or femorzrl veasels of dow injected daily with nicotine over a period of 6 weeks. In guinea pigs exposed to the rvnoke of one-helf cigarette daily for 180 day-~, Lupu and Velican (1961b) found an accumulation of mucopolysaccharides and of birefringent proteins in the w•alls of the blood veNrla, an alteration in the capillary pemteability, and a stimulation of the jtroliferation of the vascular endothelial elements; these modifications were accentuated in the aortir arch. These woH:ers (Lupu and Celican,1962) also reported finding marked h6stochemical changes appearittg in the protein•mucopolysacetoaride mm- plexes of the connective tissue vascular interstitial spaces ol the aorta of guinea irigs expo-A to 12 periods of inhalation of tobacco-smoke. For further details of these studies, see below, 1090. We may mention again here that Koaam (1952) noted that local injection of I or 10 % nicotine caused no hemor- rha>.dc manifestations in the skin of the albino rat (sre above, 384). •f06.Inflammation. Jancs6, JaucsG-G6bor and Tak6tc (1961) studied pain and infHanimation induced by nicotine, and their prevention by desensitizing agents (for the ob- servations on pain, see abqve, 110). To visualize lucal in- eamnuttor}• reaction, 1.0 ml/100 gin body-a•eigh( of a 12% colloidal silver solution was injected into the tail-vein of the rat, inmiediatel)• after application of the phioRogenir sub• stance to the eye. The conjunctivae, together aroth the cye• lids, were excised, fired in alcohol, deh,rdrated in absolute akohol, and, after clearing, with xylol, mounted on slidev. Following microscopic examination, the silver content of the tissue was determined by the dithisone method. By meam of this same silver method, the authors also demonsrtrated that applicatiot. of nicotine to the mucous membranes of the rec- tum or -•agina elicited transient inflamtnator;y redwneee, which could be specifically inhibited by ganglion-blocking agents. They concluded that the transient inflammatory rraction, asswiated with h) preremia and the developmeat of edema, resulting front local application of nicotinir etimu• lants, was purely neurogcnic in nature, since it could be prevented by ganglion-blocking and uicotinulytir drurs. 407. Experimentai Allteroma In Rabbits (189). No lesions were found kv Csochta-L.•sat.otrica, Gorski and Ked (1059, 1960), in the aorts.a of rabbits given daily i.v, injec tions of 1.5 mQ nicotine for 100 days. Coronary vessels alao showed no lesions (Ctrochra-Lysanoa•ira, Gor><ki and hedra, 1959). Simultaneous daily injection of 20 mR caffeine did not change these findings; but animals injected with nicotine Produced bv Th~ Council for Tobacco Resoarch-USA, Inc. 0003010

1 , CAHllIOt'ASCh1.AN Sl'hMf 99 ..a ..4...........~« r...r ..l...o»........~ ..L........i n.K.. t..o:...,c which were said to be more considerable than those evident after cholesteroi feeding alone l8ntensity ooe(6cients of 2.5 and 3.4, respectively (Csochra-Lysanowics, Gorski and Kedm, 1960)1. Rabbits given cholesterol plus caffeine, on the other hand, apparently showed somewhat less marked aortic lesions; and the aVithcrs deduced from their experiments that, in rabbits fed with cholesterol, nicotine increased the intensificatibn of the aortic lesions, but caffeine restrained their development. 408. Sxperiimental, Production of Peripheral Gangrene (192). Pertinent to this subject, is the study by A. li. Lewis and Moen (1953) of the effect of nicotine on the extent of gnngrene following experimeiatal local cold-injury. In these experiments, a kind leg of mafe albino rabbits was depilated, and exposed to a cold alcohioi-bath at -15° and -16' C For 30 min for determination of skin necrosis and at -12° C for 30 min for muncUe gangrene,(at -12° C between one-third and one-fourth of the animals also developed some skin gangrene). Nicotine was administered s.c. to 71 frost•bitten animals in 3 experiments, using a divided daily dose of 0.21 mg, and the tesults compared,to 66 froeen controls. In the first experiment, the more severe cold exposure was used to determine skin necrosis; the le'fiser exposure was used in the second and third experimentsi in which the extnt of both muscle and skin gangrene was measured. In none of the experiments was t:;~e a significantly greater loss of tissue in the nicotine-treated animals. (The nicotine dose was calculated on the basis of a 70 kg man smoking 1.5 packs of cigarettes daily, and absorbing 0.2 mg nicotine per cigarette (a value which appei~ts to be oat the low side).) Peripheral pathologic changes in the hind-leg vewls of rabbits were minimal in animals receiving nicotine in their drinking-water and/or cholesterol in their diet for 24 weeks, with or without i.v. injection of ergonovine maleate at 4-week intervals (Wenzel et al., 1961). 409. MAN (192) G. :tl. Roth and Shirk (1960a) 'ave renuirked that the effect of smoking on the cardiovascuiar syst.em of man would seem fairly easy to determine, and they pointed out• that some of the confusitln in the tesults of smoking tests has arisen beca,Nse too little attention has been paid to certain factors concerned with the status of the individual. The most importantt of these factors were said to be the environmental tempa'rat.ure; the position of the individual; the taking of food; the basal qnetabolic rate; and previous medication. The authors noted that Roth's "standard smoking test" (194a; 2048; 21 da; 220 [Table 6-1 1) considered these factors, and required simultaneous observation of blood pressure and pulse-rate, but, as we have formerly written (194a), the Tardiovai;cular effect of smoking in subjects in the basal state may not be as important or relee•antt as the effects of amoking dnder ordih'ary, non-basal conditions of actual living; and, until this fuotdamental question is deridcd, or at least investigated, we should not conclude, with Roth, that the mot+e "basal" the conditions of a"standard smoking test", the more valid m more gigtdficant the results. While it is inarguabt,v true that "control" conditions in clinical investigation are preferable to uncrnntrolled ones, it is not always so shnple a matter to dete.rmine precisely which of the available conditions should be controlled in precisely which way. Thus, we may agree that the environmental temperature, the pottit.ion of the Individual, the taking of fiv..l 9.1/t ?tIP I/lPfAtM1IIP M1iP I1r 4ntNIMIf InNtYMMn P VIAnt/i„p tests should be controlled, but there is no a prioH reaaon to supposp that the room.temperat.ure must be the coiiventional one, that the position of the individual must be sitting or supine, and so forth. We have only to reflect upon the "ani- mal experiments" in the preceding sections of this chapter to realize that, the more rigid the controls, the less the nffiults relate to the normal organism-as-a•n•hole-or to the human smoker. De Takats (1963) considered that all the effects of to'aacco on the circulation could be explained by the outpouring of epinephrine and norepinephrine ftom t.he adrenal gland, from the sympathetic nerves, and from millions of small cells around blood-vessels, which store this energising hor- mone. According to Bellet lin JANi.a !r8 (11): 33-37, 19tF11 also, the cardiovascular effects of smoking are due almost entirely to nicotine-induced release of catecholamines. 410. Ptn.sC-sATI: (192) 411. Paiar -Rafe in Smoll•ers and A'on-smokcrs (192) as often before, smokers have been reported by some observers to have a higher resting pulse-rate dhan non- smokers (H. Blackburn et al., 1966; C. B. Thomas, 1900; Thomas, Ross and Higinbotham, 1964; Zwi and Goldman, 1962; Zwi, Goldman and Levin, 1964), whila still others have found no significant differenres between the two cate- gories (Libow, 1963; t1'estfall aud 11'atts, 196U); and, iu a series of 2,000 pregnant women, no appreciable differenrn was found in pulse-rate between m7nukerx and non-smoketa (Zabriskie, 1963). Again, we must point out that statistical significance -or nonsignificance, for that matter -is no test of the physiological meaningfulness of a single function. Some writers (e.g., Blackburn et sl.) explicitly state that direct causal connections are not assumed for such ascocia- tions aith smoking habit; but others (e.g., Thomias et al.) attribute at least a discriminant value to the increased pular- rate of heavy mnokers. It reems likely that single recordings of pulse-rate, whether in individuals or large populatioruc, are merely in themselves but phenomena, influenced perhaps by smoking habits, but also so mam• other and inter-reacting variableR, that competent re~enrchen; and cliniciaris now seek either more sophisticated criteria, or better coatrolled e.pmri- mental conditions, than what is esyentially an aaisa•er to a "smoking questionnaire". 412. NBed of Smoking on Heart•Bofe (l93) Many reports confirm the observation that cigarette- smoking increases the pulse-rate (7.ipp, 1956; lfiksic, 1961; Neveling and Kruse, 1961; Irving and Yantatrioto, 1963; Dock, 1963a; Simon and lglaucr. 1963; Fwlmuttddwicz, Pifer and Marshall, 1964; I:lensch, 1964; 1). C. Aloses, Powers and Soloff, 1964; Pentecost and Shillingford, 1964a,b; Shilling- ford, 1985; Soloft and Powers, 1964; Valori et al., 1964). If this is not always true in the individual cost^, It h. the effect in the majority of cases. For example, in 29 amoke,e, smoking I cigarette resulted in an ktcreased pulse-rate in 27, a decrease in 1, and no change in I; and, in 20 non. smokers, heart-rate incteased in 12, decreased in 8, and did not change in 3 (Klensch,1983). G} B. Thomas and Afurphy (19t)0b) deteriniin,d, the circu- latso}y r~ponse of 9 white male medical students to smokina a single cigarette following small i.v. injections df he%ame- thonium, mephentermine, or isotonic rodium-chloride snlu- Council for 0003011 Produccd bv D i Tobacco Resaa,ch-USA, InC.

100 TOBACCO-E\PERIAfENTAL AND CLIX ICAL >;TUAIR.. SUPPLEMENT I ri~n ~piuy.uu ...~....:..'. 1:...: ~.:,.... ...., .._. ..... ,.A....rn.1 by either of the dntgs. 413. TimeR.elalions of the Pulse Changes (194) (No new data:j 414. Individ'eral Varintf.on in Response to Smoking (194) See C. B. Thomas aad Murphy (1960a), belor:, 421. 415. Comporalid.e EBect oJ Smoking on the Pulse of Smokers and Non-emokers (194) H. Blackburn, ilro'r,ek, Taylor and Keys (1960) compared cardiovascular and related characteristics in habitual smokers uttd non-amokers, data being collccted among 1,618 men agwi 17-67 in groups of studentb. business- and professional men, firemen, raihroad and rural workers in the United Statee and Finland, and found that smokers had a faster resting heart- rate. Another report by I;lackburn, lirolek and Taylor (1980), based on a total of 1,093 of the above subjects, included the observation that young student groups, with short duration of smoking habits, showed no significant differences between smokers and non-smokcra for the circulatory parameters measured (heart-rate, blood pressure, cardiac output), and the associations found did not support evidence for large or imlortant differences in circulatory reactivity between groups of habitual smokers and non-smokers. Following the smoking of I cigarette, Klensch (1963) found the significantly stronger reactivity (significantly increased heart-rate) among %mokers over non-smokens, and the rela- tively-longer duration of frequency F-creased, to be unex- pected (see above, 412). Subsequent experiments ty Klensch (196i) on 8 healthy male smokers and 2 non-smokers between the ages of 19 and 50 led hinm to conclude that the strength of the notsdrenerttic reactions to nicotine aepended on smok- ing habits, and cigarette-smokera showed the most prominent increases in pulse frequency and heart load (see below, 421). The cardiovascular effects (heart-rate, systolic and diastolic blood pressure, cardiac output) follotring i.v. iujection of 1 mg nicotine in normal subject.s a)qwared to be greater in non-smokcHs than in smokers (1'alori ct aL, 1964). Lobeline injected i.v. Into 20 normal subject.- caused bradycaniia and hypotcnaion in 19, of whom 7 were smokers; and Itevan and Murray 0963) considered that the increased response to lobeliue seen in smokers probaNt• refiected an additive action of lobeline with its pharmacological analogue, nicotine. 416. Ejlert of Inhalation (195) (No new data; but see below, 1054-C.) 417. DWrc of Smoking (195) (Xo nea data.j 418. I nJlttenoc of Idtosyacrasp and Hypersensitivity to Totareo (195) (.\o new data; but see below, 466.1 419. I rtRttenoe o/ Fasting and Food (190) According to F.dmundowicz, Pifcr and Marshall (1964), increarea in heartaate, as well as in other circulatory effects, following the smoking of 1-2 cigarettes were similar hi the fasting and post-cibal states. Ftnoking 2 cigarettes In 10-15 min incnmsed the heart-rate of habitual smokere, and the inrwnav ana hloeke>a bv prior administration o}t glucose either i.v. or by mouth (D. C. Moses, Powers ana aoton, 1984; Soloff and Poners, 1964). This finding, and jalso glucose block of the smokiug•induced increase in stroke volume and cardiac output, led Boloff and Powers (1964) to nbte that the cardiac response to smoking cannot be fully characterized by studies limited to the post-absorptive state. 420. Egeet oJ Posture (196) (tio new data.( 421. Relation of Pulse-Rate Changes to Other Corditmascular Elfetts (196) C. 11. Thomas and Murphy (19(10a) recorded on healthy young men the effects of smoking 2 cigarettea on rystvlic, diastolic, and pulse pm3sures, heart-rate, stroke volume, cardiac output, and cardiac index (s4e C. B. Thomas et al. (1956) (197a)J. 11'hen the response to the smoking test was compared to the response to the cold-pressor test in 386 young men, it .vag found that age and smoking habits'affected the magnitude of the responses very little. Correlations between circulatory responses to the two tests were relatively low. Thus, independent information is gained from the smoking test and from the cold-pressor test, and both tests contribute to the appraisal of individual circulatory reactivity (in which striking individual differences were found (C. B. Thomas et al., 1956) (197a)l. 1:1ensch (1904) compared circulation parameters in men with healthy hearts before and after cigarette-sinokiutc and betore and afttr 0.25 tng nitroglycerine, and reported that, aith the exception of the heart-rote and stroke-volumc, all chanees occurring in thesc parameters were of opposite sign, yet mostly of the F9me order in per-cent (me bebw•, 431). 422. Ejlect of Ape (197) Age had very little effect on the magnitude o[ circulatory mponses to cigarette-smoking (C. 13. Thomas and dlurphy, 1960a); see above, 421. 423. Elforl oJ IIodU Sisr (197) (\o new data.) 421. EJfeM of Maternal Suwl,tinp on Fetal Ileart-Rott (197) Hellnutn and co-workers (1961) reported the development of a reliable fetal heart-rate meter utilizing a phonocardio- graph, which they used to study the effect of maternal smoking (1 or 2 cigarettes). In 10 experiments, there was a gradually increasing maternal tackyeardia; fetal tachycardia was usually pronounced, with snme flattening of the beat- to-beat variation. Fetal tachycardia occurred much more quickly than did the response to atropine, sometimes pre- ceiiiug the maternal response. In 5 of the 10 patient., there were short bursts of fetal tachyrardia during the time when the mother was being given the cigarette, but prior to its lighting; the authors termed this type of res)wnsc "anticipa- tory". The rapid onset of the fetal response, and the frequent presence of the "anticipatory°" response, led the authors to believe that the fetal tach,vcardia with cigarette-smokinK is a response to some kind of va4omotor change in the pla- cental bed, rather than a response to nicotine pa.ccage across the placenta. An incrrssc in fetal heart-rete occurred in about half of the 17 pregnant women studied bv Kumar and Zourlas (1963) following their smoking of 2--1 dRarettea; and, Producc(t 1vi Ti-,2 CounO for oO?3012 Tobacco Rescarch-~SA, Inc.

CARDIOVASCI'LAR 81"STE.1M in the rest of the patients, there nas either a decrease or no cna,~ge. Au increase ro maternai putse-rate occurred in au but 1 of the 17 women. ro definite relationship was seen between the patients who showed increased uterine activity following smoking and those who did htot (see below, 799). 42.5. In,lluence af Cardiorasculer Dfsease (198) Kaufrjiann, Bensimon and Bergogne, (1959) described a cigarette-smoking test for determininf; subjects with cardio- vascular disturbances: the patient, under basal conditions and having refrained from smoking for 12 hours, has his radial pulse recorded with a pieco-electric apparatus, then smokes 2 cigarettes, and a second recording is made 5 min later. In most normal subjects, tobakuro in customary doses cawvd nu cardiuvasrular clmuge ercept tachycardia, some- times accompanied by a moderate vasodilatation (19 of 20 subjects showed this pattern); but, in patients with cardio- vs:<cular disturbances, the test showed the appearance of a sliRht br>idycardia, accpmpanied by vnabconstriction (Kauf- mann, Biensimon and Bergogne, 19591, Kaufmann and Bensi- mon, 1960). Regan, and associates (1960, 1961), stbdied hemodynamic and myocardial resporises to cigarette-smoking in normal subjects and in patients with coroner)• , disease (see below, 49,$). Fohloa•:ng the smoking of 2 standard cigarettes with inhalation, mean pulse-rate increase in, 6 control subjects was 6 beatr/min, compared to an increase of 13 bests/min in S suttijects with coronary-artery disease (Itegan et al., 1961). afean hearGrate increases in 14 normal subjects, 5 patients with angina, and 14 patients, 6dter myocardial in- farction, following the smoking of a single cigarette (except for 2 in the post-infarction group who, smoked pipes), were, respectively, 11, 7, and 6 beats/min (F'etptecost and Shilling- ford, 1964s,b; Shillingford, 1965). No differences were ob- served by Klensch (1963) in heart-rate between heaithy and coronary subjects of similar age fcilouing the smoking of I agarette, but, in both control and disease groups, smokers showed a significantly stronger reactnity to smoking than did non-sittokera. 426. F$ed tat the Pulse-Rate .rf 1'artous Forms of Tabacco.t'se (198) D. L. s;imon and Iglaue.r (1960) bnk~e summarized the iar'ious circulatory effects of the use of cigarettes, cigars, pipx amoking, and tobacco.chening. So far as pulse-rate changes are concerned, 14 young mhle cigatette-smokera showed stat.isticalU significant increases following this form of tobacco-use. In 15 male habitual cigar-smokers smoking commercial cigars (1.82!"1, nicotine), hihartbrate increased in all, up to 12 beats/min (average, +i3.4); smoking loa•- nicotjue (0.8gro) cigars, the pulse-rate increased 1-15 beats/ mhn in 14 subjects and decreased 10 betzt5/min in the other, and the average inctease for all the subjects u•a. +5.3 beats/min (Simon and Iglauer, 1961, 1983). In 10 habitual pipe-smokers smoking commercial pilie-tobacro (2.15 k nicotine), 9 showed an inrrea$e of 3-20 beats/mn, while I shodrd a decrease of 4 beats/min, and tite, average for all was +7.4 beats/min. On Iow-nirotine (0.72~7) pipe-tobacco, 7 showed increases of 2-12 beats/mkt; 2 showed decreases of 4 and 10 beats/min; in 1, the heart-rate remained unchanged; and the maximum change for all subjeirts was +4.2 beats/ tnin. Maximum changes occurred 2-30 rimivt after the start of smoking. None of the.re cigar- or pipe-smokers inhaled, and sham smoking produced no changes. In 24 );abitual users of 101 chewing-tobacco, tobacc"hea-inQ increased the pulse-rate, but sttam chewing produced no changes. In 50 smokers smokinF, 2 non•fJter cigarettes for 12 min, pulse-rate increased by 20 beats/min (Jliksic, 1961). Smoking 2 non-filter cigarettes for the aame time, but not smoking the lot third of each, resulted in a smaller increase (+14); and smtoking 2 81ter-cigarettes in 12 min caused still smaller changes (+10). 42% EBect of "Deniwtini.ied" or )licofine-Free Toiiarro (198) As tiotai in the preceding section, low-nicotine cigarn and pipes produced slightly less changft than standard cigars and pipes (Simon and Iglauer, 1963). 428. Fject of Amoldnp A'on-Tobmkrro (.'igareUrs and of Peeudo-Smoking (198) Sharb smoking of unlit cigars or pipes resulted in no sig- nificanQ change in pulse-rate (Simon and Iglauer, 1961, 1963). 429. Coniparalise 4117.1 of J1 uotine and Smoking on Heart Rote t19B) In 21 normal subjects receiving 1 mg nicotine i.v., heart- rate increased as it did following inhalation of the smoke or a cigaretde (Valori et al., 1964). In 2 patients given 0.6 mg nicotine i.v.. a sinus tach)'candia occurred (Irving and Yadrtamoto, 1963). In 14 subjects, comprising smokers and tuon-sntokerr, injection of 0.5 mg nicotine into the antecubital vein over 4 min n•aa followed by a slight increase in heart-r'ate (Solti et al., 1963). In 6 sub- jects receiving 4 mg nicotine bitartratc i.v. in 109 ml sa6ne orer a 10-12-min pereod, pulse-rate rose on the average ib beats/min; and all subjects experienced aching in the arm into which the nicotine was infamd (Coffman and Javett, 1963). 430. Cause of tl.,.? Pt.lse-Rute Inerease FoRosing Srnoking (199) . It has already been mentioned (above, 409) that several miters have ascribed all the effects of tobacco on the circu- lation to aicotine-induced release of catecholamines (Itellet; De Takate; F.dmundoK•ics et al.); and the fact that smoking has a lesser effect on subjects previorsly treated with rescr- pine (11'estfall and 11'atts, 1961, 1962b; above, 412) furnishes evidence for this mechanism. Consult also 339. Mechanism of the Xicotine Effect on Iloart-Rate in ,lnimals. 431. Cardiac Outpul; Stre)x-I'olunu (200) Blackburn and associates (1960), in their study of American and Finnish populations, found no difference in resting cardiac output between smokers and non,stnokers. In a group of 14 young male rigarette-smokers without cardiovascular diseam, no significant change in cardiac output .ras, ruund after suwking urdinary eigarettes (Simon, and Iglauer, 1963). However, although no significant change in cardiac output ures observed, the subjects nhomed concurrent statiatically significant increa-ws in pulse•rates and decreases in skin temperature (Simon and Iglauer, 1969). The cirrula- tor}• effects of cigar- and pipe-smoking were said to be ap- proxtimately the same, though not as marked as those usually fomid during cigatettc-smokinF, despite the fact that none of the pipe.smukers or cigar-rmokets inhaled. Alost workers have found that smoking hncreases catdiac output. Irving and Yamamoto (1963) studied cigarette- smoking and cardiac output in 5 normal volunteers and 15 Produecd bv The Council for 0003013 Tobacco Research-USA, Inc.

102 TUIiACCU-EXPEIt1Af>•ISTAL ANI) Cl.l\ICAL h'TUl)I&S. SUPPMtEXT I hospital patients, who had refrahred from smoking for at ieast a nouns and were m a tmeat state Nnor w ane tt5te; cardiac output was mtertsured by the dye-dilution technique. "Sham" smoking an unlit cigarette, or smoking without inhaling, caused only slight changes in the cardiac output. On smoking a cigarette with inhalation, increases in cardiac output ranging up to 20101% over control values were obtained; and, associated with these, were a sinus tachycardia, an increase in s)retolic blood pn~-utre, a widening of the pulse pressure, and an increase in stroke-volume. In 2 patient; given an Lv. injection of 0.6 ml nicotine, similar changes were obtained. 9'he,authors concluded that the increase iri cardiac output was due to the combined effec6 of an increase in the pulse-rate and in stroke-volume. Valori and co-workers (1961) also reported an increase in cardiac output following inhalation of the smoke of a cigarette or i.v. injection of I rnk nicotine; the increase in "circulation output" was said to result initially from an increased heart-rate, but, after thg hesrt.rate had returned to normal, the effect was attributed to increased systolic output. Tbese authors noted that the action of nicotine appeared to be greater in non-smokers. Edmmndowica, Pifer and Marshall (1904) determined cardiac output (indieator-dilution method) and other cirru~ latory patmnet:ers in 8 healthy medical students, aged 22-?4 years, at rest in the supine position, after smoking I and 2 cigarettes at a standard rate and also after sham smoking. After I cigarette, cardiac output increased by 38'-0, heart- rate by 27%, and stroke•volume by 9%; after 2 cigarettes; the corresponding mean inco+eases were 44, 28, and 13;`c; systolic and diastolic arterial blood preccures increased by 6 and 5 mm Hg, reslx+htively. Results were similar in the faat: ing and post-cibal states; and these workers suggested that the effects might have been due to release of epinephrine. Iiellet (dA\ia 189: 33-57, 1964) also suggested such a mech- anism of action. The effect of itigaarette-smokinK on cardiac output and stroke volume, a: well as on heart-rate and blood pressure, was measured in 14 normal subjects, 5 pn- t5ers with angina, and 14 patients after myocardial infarc- t.ion, all of whom were smokers with the exception of I normal subject (Pentecost And Shillingford, lMa,b; Shilliti<dord, 1965). In the test, all subjects smoked a single cigarette (filter-tap,~ed or not, according to desire). except for 2 in the post-infarction group, who smoked pipe.. Patient•a without evidence of caodiovascula: disease all increased their cardiac output while stnoking by an average of 21 io (range, +4 - +50%); those patients in the post-myocardial-infatrtion group had an avera$e incre~ of onU 1% (rattge, -23 - +33%); MI~d~ C1m»ges irt stroke-volume were +10,70 (range, -10 - +35 %) and -8 0 (range, -39 - +27 0). Two of the patients who showed a fall in cardiac output and stroke-volume while smoking had recently reduced their smoking because of dyspner,; and one of them e.yietienced dyspnes during the smoking-test. Patients w ith angina in the absence of infaretion behaved similarly to normal men. Iu view of tbeir findings that certain patients with ischem:c heart.disea_ce responded to cigarette•smoldug in an abnormal manner, Pentecostt and Shillingford (1984b) concluded that it seems prudent to advise patients who have had a myocardial infarction not to smoke. Klenach (1964) studied circulation parameters in 8 smokers and 2 non-smokers, all males between the ages of 19 and 50 with healthy hearts, beforc and 4 min after beginning smok- ing (10 trials) and before and after 0.25 mg nitroglycerin (18 tests). With the exception of the heart-rate and stroke- volume, all changes occurring in the circulation parameters were said to be of opposite sign, yet mostly of the same onter u~ umgou~uuis. a~irwgi)crriu was e~iu w ~unu~c ~iie iuiuawu. e between ox)^Ren oonsutnfHion and oxygen supply durm¢ an acute attack. while nicotine effects ivere exactly the opposite- that is, nicotine incmaFed those indices characterizing tdtc osygen consumption of the myorardium. This worker stated that, by decr!asing the oxygen requircaent, nitroglycerin can check an attack of myocanliaFl ischemia, whik nicotine, on the other hand, can shift a borderline case of oxygen balance in the direction of ischem'its. C. 14. Thomas and Murphy (1960b) measured canliair output and stroke-volume in 9 white male medical studetitu in response to smoking a single cigarette following appro- priate rmall i.v. injections of hexrmethonium, mephenter- mure sulfatc, or placebo (irntouic sodium chloride) roiution. HexamAthouium did not influctlcc the mean response to smoking; although individual diffrrcnctv were noted, it was difficult tu amess their importaniv. N'hen the mephenterntidtc and plArnbo tests were compared; it appeared that cardiac output was significantly increased; the rise in syxtolic pns- sure was significantly less; stroke-volume and heart -rate were not affected. Cigairtte-omoking incressed the heart-rate, cardiac outlnot, and stroke.volume of 8 (asting habitual stnokers (I). C. Moses, Powers and Soloff, 191W; Soloff and Powets, 1964). The increase in stroke•volume and cardiac output produentil by smoking 2 cigarettes within 10-15 mht was significantl.• decreased by prior i.v. injn•tion of 15 gm of a 10 -, solution of glucose (Moses, Powers and Soloff, 19&1) or by 50 fnn gtlat- cose by mouth either 30 min or immediateh• before snrokint; (Soloff iznd Powers, 1964). No xignificant changes in cardiuc output and stroke-volume occurred after control admini.tra- timi of glucose. Sold and eo-workers (1964) investigated the ncute e8'ecis of I mg nicotnre tartrate i.v. on the cireulation and electrb- cardiogmm of 20 clinical patient:t with a normal circulation. Slight increases in s)atolic• blood prewure and minutc+volume were observed, while peripheral vascula: resistance was vir- tualh• tAnchaprged. Pretreatment with dibenamine prevented the cardiova>:pular efiect+ obvrveii (for further detaiL<, sim below, d36). , In commemt on studies showing that tobacco-smoki• in- creases cardiac output, the Brilish tlediea! Jourrwl (i: 142+- 1429, 1963) stated that it might be tempting to regard this increase of output a ith smoking as a good thing, but that, in fact, there are sound rrasotus for believing that tobacco does only harm to the cardiovascular system. Dock (1983b) has pointed out that the main eBect ot nicotine on the heart parallels that of phyaical ~exertion, inereasing both cardiac output and coronars flow, so that, possibly, in a fetv men, a cigarette may substitute for exercisc. The subject of the comparable cardiovascular effects of smoking and exev- cise is discussed below, 433. 432. Yelocity of Hre Pulbe li'me (2U1) Zipp (1958) recorded the arterial pulse at the thigh, above the ankle, and at the big toe, and also simultaneou.41y re- corded pulse frequencies and blood ptrssures, hi '1t) patients before a6td after smoking I cigarette in 7 min with inhalatitni; some of the subjects were nomtal, and the remainder were afflicted with vasomotor disturbancre and a hypertonic circulatory adjustment. Pulse-wave rate wes found to ba increased in 18 and decreased in 2; blood pressure inereat-q-cl in 11, decreased in 7, and did not ehange in 2; pulse frequenrr• increased in 19 and decreased in 1. ln his anaheis of thm, Produced bv The Council for 0003014 Tobacco Nscarch•USA, Inc.

CAItUI(1'CASCC).Ali yl"sTESf 103 re.ults, the author maintained that, since the pulTe.wave rate is proportional to modulu of elasticitv, and Rinr•e the modulus of elasticity is the reriprocal value vf dilatation capacity, the increase of peripheral pulse-wave rate in, the smoking-test signified a decrease of the vasodilatation ability. Absence of a luuallelism in the behavior of the pulse•wave rate and blood pressure contredicts the assumption that the change in dilatation capacity might possibly be explained mainly by a change of the binod-preacure-dependent, internal vascular pressure. Therefore, one has to assume that smoking results, not only in a pressure-pacAve-, but also in an active change of the dilatation ability of the vessel wall, due to the muscular elements o1 the arterial wall. In most of 19 normal subjects tested by Dock (196:ia), the ballistic forces in systole during inspiration and the velocity of rise of the brachial puh•.~aave iucrease.l as a result uf stnuking. He e.frlainetii that it is not latent myocardial disease, but the type of individual predisposed to heart-disease, which is revealed by the decrease in systolic waves during ex7riration (Dock,1963b). 433. Uyocardial Efficienry ot Reat and durinp Ezerciae (201) In a study of 1,093 F'innish and American men, aged 17--67, 131ackb•un, lirotek and Taylor (1960) noted that pulFe-rate during work and recovery was significantly higher in one group of middle-aged men, if pathological cases in the group were considered. However, there was said to be little evidence for deterioration of cardiovas:ular "fitness" in smokers performing work-tests. During performance of a unifonn amount of work on a bicycle ergometer, no significant dif- ference between heavy cigarette-smokers and non-smokers in a group of 892 Finnish busineasmen was found in the exercise heart-rate (I-Iernberg, 1964). In a study on metabolic protection and reconditioning of the heart.muscle through habitual physical exercise made on individuals ranging from hard-working persons to comf•ietely sedentary ones, Raab (1960) found that smoking habits did nst affect the neurovegetative state of the heart in between the temporarily individual acts of cigarette-smoking. The influence of habitual vigorous ea'rcire, and of lack of it, respectively, on the basic cardiac neurovegetative status at rest was inveatagated in 360 healthy American and Austrian men, aged 17-50 yeam ranging from cotnpetitive athletes, mountaineers, and Alpine soldiers, to completely sedentary persons (Raab et al., 1960). HearGrate and dynamic cardiac cycle (chronodynogram) at rest, with special consideration of the isometric period of the left ventricle (total systole minus ejection period), were used as criteria of cholinera..ic, sym- patho-itthibitory, and adrenergic rhronot.ropic and inotropic influences, respectively. Smoking habits-ranging from non- smoking to heavy smoking--seemed to leave the basic cardiac neurovegetative status unaltered, despite the immediate adrenergic cardiac effects of tobacco-smoking. Raab, \larchet and Deming (1900) reported chronodynograms taken under standard conditions in 272 men, consisting of non-smokcrro, light smokers (up to 10 cigarettes or approximate pipe and cigar equivalents per day), and heavier smokers. They too found that the basic cardiac neurovegetative tone was not demonstrably affected by smoking habits, nor did thesc habits seem to interfere with the profound influence whica eaercise habits exert on the lmsic cardiac cholinergic-adrener- gie equilibrium. However, these latter findings do not preclude a potentially ityurious effect of habitually repeated Inote Klensch (1994), below) nuotine.induced cateeholamine bombardmenta of the heart-muscM and the coronary arteries. In teat, on 22 subiects. with chronod`moarams taken before, during, and at termnmtiun of smukiug I cigarette, and 5, 10, and 20 min after•a•ar+ia. it nnrwnneri that. in the nwioritv (82';i) of subjectx, cigarette-smukinF eliciled, in addition to the familiar positive chronotropic effect, aLw, a Imsittve ino- tropic effect (shortening of the iKrmetric period of the left ventricle). In a minority of subjects, the,:e cateeholamine- mediated manifestations were preceded (9~;) or entirely replaced (18540 by cholinerttir and/or sympatho-iphibitory features (cardiac retardation, prolony,~ation of the iNOmetric period), apparently due to the mmultainmus stimulation of sympathetic, parasynrpathetic, and, possiblo•, syrtlpathor inhibitorc mechanu:mr• b}• nicotine. Regdn and co-worker$ (1960, 1961) measured by means of the nitrous•oside desaturation technique the myacardial response to cigarettesmoking in 6 nonnal subjects and 8 lwtirnt. Nith vcounar.-artcis d6ts-e, %bu lusd iucwivd sn acute tiiyocardial infarction at least I year previously (all but I experienced angina pectoris). After a control deterrnination, cigarette•smoking (2 standard cigarette:i smoked in about 25 mio, inhalation at 45-sec intervals) wa bet,•un, and maintained so that the usual systemic•pre.-vre and pu4:e-tste increase would~~ersist throughout the redetermination of myocardial blood• ow. Associated with the pulse and arterial-pressure increases, there wa.c a significant rise in left ventricular work in both groups during utnokinyt. Left ventrirular work (qtg/.Nl/ min/Jl') increase was 1.40 for the control subjects add 2.52 for the coronary patiente. In the uonnal subjects, the v:ontrol myocardial blood-flow aud ox.•Emn com:eunqNirnt were not altered, despite the increase in systemic fienwdynamic IYaram- eler:. Cottsequently, the ratio of m)•ocaniial ox.-get c»nsump. tion to left ventricular work deelined from 1.45 to 1.13 (p < 0.05). The coronary-artery-di.ean• group had a 30 7v left ventricular work increment a.-wriated with the rate and pressure rL,•e. The resting myocardial blood-flow and axygen consumption were lou•er than in the normal subjects; how- P:er, their response to smoking was the same, in thad there u•as no flow or osygren-consumptio n iucnea.Ye. The oxyµen- corsumption-w ork ratio tvas sirnilarly reduced from 1.46 to 1.06(p <0.01). During pertormance of a unifomt amount of work on a Godart bicycle erRometa r, 5}roung male tron-smokers had a peak pulse-rate of 130 * 7.5 beat>/min, compared to 151 t 8.8 beats/min in 7 smokcn+ (Chevalier et al., 1962). A subse. quent report by Chevalier and co-workers (1903) covered a group of young male subjects, cmisistintq of 18 smokers and 14 non-smokets. Mean heatt-rste while inttinR quietly on the bicycle ergometer was 81.6 (t;D, *9.9) for the non-smokers, and 93.3 (SD, t1.22) For the xnmokers, A statistically signifi- cant difference (p < 0.01). Tke smoking group had a tendency towards higher values throughout the 5-min eaercise period on the bicycle; values for the 5th min of ea;ercise arre 138.8 * 13.7 for the non-smokera and 144.0 f 14.2 for the smokers, but this di6erence was not statistically significant. Three min after work, the heart-rate of the non-smokin,t gmup was significantly lower than tlut of the smoking group (p - 0.02). The eaercistinle heart-rate of normal non-smokers was consist- ently lower after inhalation of 0.5% carLon monoxide (CO) for 2.5-3.5 min, which suMVested to Chevalier, INrumhola and Ross (1963a) that differences in urslmnse to exercise exiating betnren smoker-c and non-smokers might be caused, at least in part, by the effects of CO ivhalation. In another study, in which the subjects were 18 house-staff physiciatts, aged 27-37 )rarz,, 9 of whom had smoked at least I pack of cigarettes per day for 5 years or more, and 9 of whom had not smoked at all for at lea--.t 5 yea", a 5-min eNercise-test was Prnduc^rl bv The Council for Tobacco Researc~rl;SA, Inca o 0 03015

104 TOBACCO-EXPERIALE1vTAA. A.\1) CLINICAL STUDIES. SUPPLEItfENT I performed in the same fashion, the work-load being the same IVI C4LY DWJJCx 4, tlJ4U VA)8eY ut/WYC YN;YDYIW IV, W11. 9/Y,.. before, the 5 min of exercise, and the 10 min after euercise, while heartrrate was determined each adra throughout (Krumho)e, Chevahier and Ross, 1!)li4). Mean heart-rates showed no signi6cant difference between the smoking and non-smoi.ting groupa at rest, during exercise, or after exercise. The effect of abatanence from smoking was studied in 10 young chronic cigarette-smokers 3 and 6 weeks b(rom the time of stopping smoking; all the subjects had smoked more than I paek of cigarettes daily for more than b years (Krwmhols, Chevalier and Ross, 1965b). After 3 weeks of no smoking, beart-rate, oxygen debt, and ratio of oxygen debt to total increese in oxygen uptake produced by bicycle exercise were significantly decreased. (For an account of the effects of exer- cise on ox)gen debt and oxygen uptake in these several studies, see below, 718; and for simultaneous pulmonary-function stui:ea, we below, 523-C.) Some remarks by Kalow (1962) are of interest here. This writer asserted that, in spite of the multiplicity of actions of nicotine, the net effect can be described by stating that the circulatory effects of nicotine are comparable to those of exercise. The stimulation produced by smoking I cigarette is not more than that caused by a modest effort. A healthy heart may become easily adapted, even to a continprous state of simulated exercise, as in a chain-smoker; but Kalow wondered what could happPn to a failing or detective heast under such circuhtstances. Accclyding to Dock (1983b) also, the, main effect of nicotine on the heart parallels that of physical exer• t.ion, causing a rise in both cardiac output and coronarn• flou•. Possibly, in a few men, sedentary and nonaotensive, showing no rise in blood pressure on smoking, a cigarette ma$ sub. stitute for exercise, which is so often praised as protection against atherosclerosis. In fact, Dock coined the phrase, the "'packaged exercise' effect" of cigarette-smokinp, but warned that this would probably be outweighed by various other actions of nicotine, especially in men who smoke a pack a day and in those with even minimal coronary disease, while in a few men with coronary di:xian of great severity, the inerease in cardiac work due to smoking is not accompanied by a rise in coronary flow, and real harm is done by cigarettes. Raab (see Raab versus Dock (1964)) took exception to Dock's suggebtion that a cigarette may substitute for exercise, on the iiads that it may give unwarranted comfort and en- couragement to sedentary sm-.)kers. Raalt argued that the state of being physic+ally trained, in contrast to the individual act of acute exertioru, is characterized by a sustained (prob- ably bypothalamus-inediated) lowering of the 1•ardiac s,vm- pathetic tone, to the advantage of myocardial oxygen econ• omy, and that habitual smoking, on the other hand, produces no suth favorable effect on cardiac autonomic nervous regula. tion. In reply, Dock ktated that he agreed with Fiaab-not in the hypothetical and, hypothalamic details, but in feeling that "Hills do more than "baccy canJTo fortify the heart of man.' So far as his original statement was concerned, Dock thought it clear that he had considered the smoking-induced increase in coronary blood-flow an unhealthy and expenwive one. Re• calling the work of lilensch (1964), above, 431, this writer likened the effect of smoking halt a citrarette to an additional load for the left ventricle similar to that resulting from bicycle riding up a slightly inclined road; and he thought that, for young persons with healthy hearts, the sfngle l;emodyuamic load on the heart brought about by smoking is probabh• no more damaging than when a similar load is brought to hcar on the heart through other means. However, he thoustht it possibly quite a different caAe when the additional Ioadh.g uuu..au uicuwue in wuuuuexi ae io trt~r~i~i~awuviwyy 434. Cardiar RkJftres (W3) No new data.J 4S.ri. THE ELECT60CArtMOOaAA1 AND sAi.tas•rocaabtooanm 03) We should call attention here to a panel discussion on the siguiticance of elea!trocardiographic (EKG) and balgsRo• cardiographic (BCG) changes induced by smoking, moderaled by I. Starr (1960), in which both F.11'. Davis, Jr. and Ejrup reviewed much of their own work. Van Proosdij (1960, p. 118), by the way, considered eler trocardiography an appmveal inethnd of study, built up from sound principles; and he found it regrettable that it should not have been used more widely for systematic inquiries into the effect of tobacco or nicotine upon the heart-muscle. With respect to batlistocardiography, this writer noted that recent publications had been criticalof it, circurospect, or laudatory (01). cit., p. 119). 436. Elecborordi.ogrophfe Chohges in SmoAwe (203) Karvonen and his co-workers (1961) made cardiovascular studies on lumberjacks, who are described as being engaged in the physically hegviest occupation known. Although there were significantly more smokWs, and a significantly greater percent,age who smoked more than 20 cigarettes per day, among the lumberjacks, these subjects showed significantly fewer EKG changes indicativeol ,uut coronary infarction and of myoeaMial ischemia than all of the other men in the romtic of North Karelia, East I'1'niand, aged 40-59. According to Benchirnol (see E. G. Dimond and Benchimol, 1964), the effects of smoking prernt an abnormality in the alx'a cardio- gram in one-third of patients; he suggested that this effect is mediated through the venous system. 437. ElecHOCOrdiographic CAanges Following Smoking (203) In 50 smokers smoking 2 non-filter cigarettes for 12 min, a flattening of the T•bave and a fall in the S•T junction was observed by \Iiksie (1961). Von Alm (19t30) glzain described a flattened T.amve and slight depression of the l+-T se{,mtent following smoking. (1'his is a summary of essentially the'saj»e material earlier published by von An (1954a~ 1) (205a-208ti).1 7.anniai, Conti and Negro (19$3) reported having studied normal subjects andl patients with arterial insufriciency be. fore, during, and following the slmoking of I or 2 ciFarette., a pipe, or a cigar; cutaneous and muscle tempetaturvR, arterial pul.ce, and EKG were said to have been measuredc but, in the abstract available, nothing is said of the EISG findings (the other findings are briefly described below, 453,447, and 476). 438. Biertrornrdi.ogrcphic Chdngea Following 11'irotine Injection (20) In the majority of 20 dlnicrel patients with a normal cireula- tion, EKG recording§ showed a dniform leveling or Inversion of the '1'-nave, and moderate lowering of thc S•T interval following i.v. injection of I mg nicotine tartrate (Solti et al., 1984). Ocossional extrasystoles were observed; the duration of the electrical systole, and therefore usually also of the ir.o. nwtric interval, was,shortened; These effects were preventeJ by pretreatment with dibenamhie. The liliG changes eharacterisQic of tohacro angina could Produced by The Council for 0003016 Tobacco Rescarch-USN Inc,

CARI)IOti'ASCULAR SYSTEM tOci I while ernurol experiments with saline i.v. were negative (Ortun and Sowton, 1962, 1963). 439. Elettrooardioprap~k Changes FoUmoing Smoking in Coranarg Diseaae (206) In 3 patients with true tobacco angina, the EKG changes (accompanying anginal pain) resulting from cigarette-smoking could be modified by certain drugs (0ram and Sowton, 1962, 11963); for details of sucb treatment, see below, 1414. In the opinion of G. P. Robb and Marks (1964), based on current and previously recorded results, ischemic S-T segment depression in the post~exercise EKG is due, with rare ezcep- tinns; to coronary insufficiency and myocardial ischemia resulting from moderate to severe coronary athenr.K•lercmis. In this condition, excessive cigarette-smoking was said to be one of a number of faetors tending to make the prognosis worse. 440. Cause of the Etecb aardiogmphu CAanges .f/ter Smoking (207) Grewal, Lu and Alknark (1962) found that concentrations of poster•for-pit.uitary hormone (ADII), roughly equivalent to those released in the blood following nicotine injection in rats, reduced coronary blood-flow and caused a marked elevation of the 8-T segment of the EKG; this latter finding suggested myrocardial anoxia, possibly due to coronary constrictionOn the other hand, cruss-circufation experiments reported by `olti and co-workers (1980a) in dogs led these workers to conclude that released ADH could not have been P. factor in the acute EKG changes observed following nicotine injection (for detail.,,, see above, 324). These observations on animals are included here because it had been suggested by some writers le.g., Lang and i•lokry (1954) (SOia)) that certain of the EKG changes following smoking in man might be due to reflex nicotine-induced ADIi secretion. 41. BQlUMnnardiopropJst:c Changes Fofl,oming Snwhing (207) As a preliminary to the following accounts of the effects of smokirg on the BW in man, we may recall that Gorbaccio and Wrst (1960a), who studied the effect of smoking on the dogdlCG and noted that the results were influenced by blood- pressnre levels, stated that their findings suggeQt revision of the eurient techniques for performing the smoking tests on the 3CG, with constant monitoring of systemic blood presstre as a prerequisite. In clinically healthy subjects, cigarette•smoking was re- ported by some investigators to have had little or no effect on thc BM (Dee, 1960; D. I. Simon and I)rlauer, 1960), or little effect in some subjects and more elfects in others (Gus• man, 1959), while other workers observed more or less xiitnifi- cant. changes in the wave forms following smoking (see below). \o significant differences were found by C. B.1'homas (1960) in the BCG smoking-test between smokers and non-smokers in a gnwp of 657 medical students. Gusman (1959) found small changes in the BC(3 in 12, and significant changes in 5, of 32 healthy men smoking I cigarette. (By significant changes, this writer meant a sharp increase in the amplitude of breathing fluctuations, with a dreream of the 13CG index below Q4; or a distortion of the curve aud its txpamtc complew, or a deterioration of BCG data in people in a•hom there was risked a pathological 13CG before smoitinp.) In subjects smoking I cigarette, BCG trac• indn, were interpreted empirically by de Crinis ar-d aseociates (1980) on the basis of the following qualitative eriteria: Grade 0: normal tracing; au ioi: w.,.t.:... r F. °^^~^•^~~;^^ and identical; Grade I: norntal tracing; minor varfationR in IJK complexes, which were still normal; Grade 11. probably abnonnal; significant variation in individual complexes, especially in the IJK wsve; Grade III: abrtor•mal; marked abnormalities in the individual complexes, some of which are still identifiable; Grade IV: markedly abnormal; tracing chaotic and no 1JK complexes identifiable as such. Only 10 of their 80 healthy subjects showed changes in the BCO on smoking. Dock (19fi3a) recorded BCG responses to smo),cing in 19 male habitual smokers in good health, with normal blood preasure and no evidence of latent heart-disease; 14 subjects were aged 21-28, and were known to have famil;• histories of va,qmdar diseaae, and 8 were markedly overweight; 5 ot the subjects were aged 45-55 yeans. llock distinguibhrtl tl,tv., types or patterns of abnorntal BCG on smoking: Type l is characterized by expiratory decrease in 13 waves pa.c~ing be- yond the normal range etxf becoming grade 1 to 3; Type I I is described as a pattern of complete chaos, with waves varying from best to beat, and no constant relation to the EKG. Type III is produced in normals only with toxic do". of nicotine, and seems to be due to increased nresygtoGc fnmes resulting from overloading of the left ventricle, causing the "early 11" type of HIJ wave pattern. Type I is the most frequently encountered pattern in coronar•y patients rela- tively free of syn:ptoms, and the only pattern evoked by smoking in people free of all evidence of coronary disease; Type 11 is believed to be due to sonwtic tremor type of coarse muscular movements, and may conceal either a normal BCG pat tem or T}7ies I or I11, but it is not to be accepted as evidence of heart-disease; Type 111 is produced by smoking almost exclusively in men with severe corottary disease, and is the only "positive smoking-test" indicat ive of organic heart- disease (this pattern may be prevented by )yeeryl trinitrate jsee Dec (1960), belowl). With res)rect to the 19 subjects tested in this study, 11 exhibited the tyiie-I response on smoking; in these latter individuals, mean pulse-mte rose, mean respi• ratory-ratc decreased, and the "Aratory pattcrn altered (r.ee be:oa , 498). Dock interpreted his sludie_ to mean that the chanuca in voluutary-musrle tone atul in respiration evoked by nicotine led to a greater e%piratory impairment of trans• missian forces in the head-foot plane; and he stated that the teat is not evidence of the effect of nicotine on the heart, but on the mind and voluntary muscle:. Them chnnges. may be related to risl:s of coronary disease; but the ty7ie•-1 response, like the Brown grade 1-3 pattern Pcen in older subjects with no nicotine administered or atxorbed, is not evidence of exist- ing cardiac dysfunction, but of poor trensmissions of forcea from the heart to certain t.pes of recording s;ystenus. The q•pe- I rcspon-ae indicates a relatively sound heart, but one pre• disfwsed to coronary obstruction, while the tyin•-Ill pattern is evidence of rather mrre corouary heart-dispasr or of h,vlxr- tensive hcait-dirc•asc. Dork (1963b) elas lYlncluded that the common t)pe of lwsitive smoking-teat was due to further deterioration in trammission of force as the voluntary muscles relaxed, oml the subject, tense from baving refrained from smoking M•fore the teet, began to "take it easy"; thuF, the teat merely proved that these suhjects needed nicotine to feel at ease. He added that many men with positive BCG smokintt-tests, both nomtal subjects and patients with coronary disease, are in fwor training, with flabby abdominal mu.~cles, aml that the abnormal tt+ryionses are a somatic, not a cerdiac• t.hennm,mnm and he found it even more reassuring to know that thc twx irive smoking-test merely means that the Produced hv The Cotmcil for 0003017 Tobacco k"escarch-USA, Inc,

100 TOBACC'O-EXPERMfEXTAL A\1) CLINICAL STVI)lE.A. RUPPLIWEtiT 1 man ni•rpts to smoke. rsnu :.' 3` -L.A nmmnwrcial nine-tnbacco (2:1M: nicctuir), of rnhi-mt. F. 11'. Davis, Jr. (in 1. Starr, 1960) stated that it made little had normal control 13CG, and of thcse, 3 retuamed rwrmat di8eveorce in the 13CC response whether the patient with tbG throughout the smoking-te><t, 3 YhoNed minor changrs, and 2 conditiou inhaled or did not inhale; furthermore, it was said >=hwrcd marked abnomtality (development of a de6nite to make little difference whether he is a smoker or a non- "carly M" pattem). Of the 2 subjsct+ with abnomtel RC(i smoker. before smokitog, I showed only tninim.al changeT during pipe. In,a control group of 15 healthy subjectsc studied by Dee smoking, and, in the other, the abnermalitie3 became mum (196(t), no essential 13CC changes were found folluwing the markel. Sham smoking of unlit eigons or 16ipes had nd aKnifi. smoking of a cigarette, except for accelerated heart action. c•ant effect on the I3CG. ln 1>3 of 20 patients with myocardial infarrtion, however, Tubacco-cheninF, but not the cheu•int; mf plarnho gum, dist9iict changes occur•red following snoking, the distortion re~ulted in marked IiCC changes (Simon and lglauer,1fltS0) being generally connected with an lJ-amp)itude reduction, Lv. injection of I mg nicotine in a 65•year.old mnn with while, in 3 patients, tbe I3CG showed no chattgc. In 9 patient.% angina was reported to have led to gross decerioration of the cigarette-smoking was followed by complete curve distortion 1iCG complexes (Davis, in 1. Starr, 1f10(/): fl•yfie I V, hccording to 13roan); in these nitroµlyrerin 442. jipert of Smoltting on Coronory Bfooil-flum (209) admmisiered sublingually rosulted in distiurt nomtalizatiou of the 13CC in 0.5-3 min, but, in 2 cases, return to the prc• Regan. HellemE and 13ing (19Ri0) utudi~ed the effect ot smoking curve occurred only after 15 min, presumably in- cigarette-smoking on coronary circulation d.nd eardiiu w•ork delmnde,nt of the drug. W. Schweitzer and co-a-orkcrc (1960) in normal individuals and in patients with coronarj••artery swdied Rhe effect of several other varodilator` on the changes di,ca,v and with angina pec•toris (none of the paticnu a•aa in in tjie )3CG caused by smoking a cigarette, but found no cont.mstivc failure). Data obtained on pwrmal individuals difference iit the action of these sulstances and of placebos indicated that, during cigamtte.s;rooking, there was a sitcnifi- used as controls. The dmgs used were: 1,3-dimethy9•7-(9'-fn- cant rise in coronary blood-flow and beart•rate, and a agnifi• piperididio)-butauon-(2')-,rl(l')].xanthine HCI (100 mg by cant decline in coronary vascular resistance and myocardial mouth); pentaerythritol tetranitrate (30 mg by mouth); extraction of osygen. In patients xuth corohary-arten• diera.K•, azapetine (25 mg by mouth); nicotinic acid amide (10 mg pulxe-mte, systemic arterial pres5ure, cardiac output, and mixed with the tobacco of the cigarette). cardiac work, all increaced; corr.rary b4nod•dow and myn• U'sirg ultra-lon-frequency ballistocardiography (L'LF- eardial oxygen extraction shotved no marked ehangr.s, al- l3CG), 4:lenseh (1963) studied the effect of smoking I cigarette though, in some individuais, there was a smuill decGne in both on &mokeri an:: non-smokerR of almost identical mean age of thm functions. As a re:ult of these chaitge,:, the ratio of (34 o'ea¢'s). In 27 ot 29 smokers, an increased heart frequency work/oxygen consumption increaseci. Procedures and detailed was observed, 1 showed a decrease, and I no change; in 12 of reau)ts are given by Regan and associates (1960. 1961) (also 20 Aooi;•smokers, heart frequency increased; in 5, decreased, see above, 433). Despiteinerea+cesfollowinRciKarette-smoking and 3 showed ao c)tattge. Thear findings were called a weakly in pulsr-rate, arterial pressure, cardiac mdex, and left ventric• significant result. No differences in either cardiac frequency or ular work, neither the group of 6 control subjects nor that of blood pt+essure were observed between healtM and coronary g patientr with corottarv-arter}• disease had a RiRnifrrant subjects of similar age, but the mean prer.sure in hcalthy chan;,. in comaary blood-flow, so that myrorardial oxygen persons, increased lem vigorously under smoking conditions. usage remained virtually identical to the value before smokink. The author described aQ unexpected the significantly strotger However, there was no anginal Rymptomatolog,v nor EKG reactivity among smokers over non-smokers, and the rela• evidence of isrhemia in the subjeets aith curonary disease tr'ely longer duration of frequeruh• inmrease (an elevation of (uvvious m-vocardial infatrtion). Since small, graded doses of 15% fot 10 houts at rest after a 20•rigaretle.daily consump- ,aterholamine induced changea In coronary Bon before in• tion) in both control and disease groups. He considered the crements in rate and pmqure, Repn and tro•a•orkern (1961) aamplitude-frequency product in smoking to be probably a speculated that simultaneous release of vasoprer.sin during vascular, not a cardiac reaction. Studies on 3 patients with smokinr might account for the absence of chat>Fe in coronan, coronary htart-diseam led {1'. HatLson (1952) to the conclusion flow in these studiea Bing, Cohen and Illuemchen (19G5) re• tltat abnormal reactions to smokiltg could be detected by the viewed the subject of tobacco alkaloids and circulation, in UL~.I3CG. which they presented data obtained by use of a new method With tespect to other fornts of tobacco-use the 13CG showed to estimate coronary blood-flow with coincidence counting definite alteration from normal in 657( of cigar-smokers tested, syRtenu and a positron emitter (tuhidium-84) to show that, ancd in 70% of pipe-smokers, in spite of the fact that none of a•hile, in the normal individual, eigarette•smokhtg resulted in these subjects inhaled (D. L. Simon and lglauer, 196t1). De- an increa-se ht coronary blood-flow, in patientc.rith coronary tai6 of, these findings were supplied by Simon an,l lglaurr diK-ase, this effect was absent. And, in tht'fr patients, the (1961) for 15 male habitual cigar-smokers and 10 pipe•smok- ratio of left ventricular work/oxygen ronsumption n•au ers, aR of o hom smoked for 20 min (wider controlled condi- uniformly and significantly increaeed by smoking. tiotis) i1t tlteirbabitual manner; noneiuhaled. Tnelve subject.. In normal subjects, cigarette-smoking increases coronary smoked commercial cigars (1.R2°'r nirotine); in 9, the 13CC blood-flow along with cardiac output, but in some patients was normal before smoking, and remained norntal during with srvere coronary diwnse land, presuntably, with athero• smoking in 3, showed abnormal increased respiratory varia- sclerotic coronary vecseL`j, the increaz;e in cardiac arork due tlon in 3, and greater changes from normal (form of "earl,r to smoking is rtot accompanied by a rise in cororutrr flow, bt'", or late don n-stroke patterns) in 3. In the 3 subjects with mtd real harm is done by ciRatrttes (Dock,1903b). I3}• meant abnormal control BCCi, the abnormality increased in 2 after of selective arteriograph} and cineradiographie techniques smnking, and lessened in 1. The ma.imum changes occurred ISon" technique), Likoff, Kaapatjttn and Lehman (198•t) ob. within 10-20 min after the atart of smoking, and peraisted tained vicualication of the right, and then the left, coronary %30 min longer after eeacation of smoking. Ten subjects arten,• in 10 patie ts ~O u r h kno.rn coronary hearl•diseaFC, he y Cqunc~ e T pr®dttced bv " Tobacco Etc~carch_uSA, lnc, 0003018

CA Rl)101'ASCCLA lt KY13TE.Sf fore and_ immediately after consecutive smoking hf 2 cilra- (e/ttC. ILI: IYl//bIGU tIKMI{aLC•Ml/VbC MIW /VW/u W 4Y" NV eBkct w•hatscoevet-neither dilatation nor ec/nstriction-on then atherosclerotic vessels. dionttinen '(1964) rregarded the basic mechanisms for the effect of smoking on the heart and coronary, blood-flow as xtill obscure; but, according to 13ellet (JAMA 189: 33-37, 1964), the systemic effects of smoking, including increase in coronary blood•flow, are due almost entirely to nicotine-in- duced releaxe of catecholamineR. Smoking-(nicotine•1 induced v&wprassio release has al.v been suggested as playing a role in changes in coronary blood•flow (Regan et al., 1961; De Takats, 1903). 443. BLOOD PaESBUttE (210) We have discussed above (409) the "standard smoking- tescs' of G. M. Roth and Shick (1960a), in, which simul- taneou.s observation is made under certain control conditions of blood pressure and pulse-tate; and we need only add that Hines (1960b) asserted that there is no universal pattern of b"-preffiure response to smoking, as measured by a stand- anl smoking-test. lt may well be, as we suggested above, that we need, not only a new kind of "standard smoking-test", but also a new approach to testing. Tl,el.ancet (1: 383, 1961) has called attention, inan.tnnota- tioiu, to the many sources of error, both in the measurenientof blood pressure and in the statistical analysis of the readingc obtAined, and has pointed out that the indii•idual's blood pressure is not fixed, but varies incessantly about a mean, which may, but often does not, coincide with the casual reading. It would appear to follow, t,hen, that such casual readings, subject to both technical and statistical error, prowide as little-or as much-positive information about the elfekts of tobacco-smoking as of the hypertension which was the subject of The Lancct's Annotation. 444. Plood-Pressure Leoela in Smokers (210) We may recall that Nye (1937) found raised blood pressure eitiaely absent in Australian aborigines aged 40-70 years and older, although the natives incessantly smoked and chewed trade tobacco which is too strong for most white men to smoke (210b-211Ia). I>owenstein (1961) studied the effects of age hnd sex on the blood pressure of two Braailihn tribes, one relatively eivilized by misstonary influence (the Alundurucus), the other pagan and uncultured (the Carajas), and found a rise of pressurk with age, especially in males, only in the cirilired grouµ. Among the other changes cn life-habits brought by "ciiilisatlon" was the use of tobacco. In a group of 25 professional people and 26 w•orkers in I tal,i•, systolic prmure showed an upward t.rend with increased smoking, whereas diastolic pressure did not (P. L. llattioli snd dfancini, 1959). Intenestingly, though, smoking was not escociated with blood pressure in a series of 167 aduwt male factory-workere of ICeafwlitan parentage, but of American birth or upbtingitrg (bamon, 19g1). We had pretitiously noted (210b) that most observers have found no signiftcant dgerence in blood preasure between smokers and non,smokers. Since then, Libow (1963) found no rigni,ficant diffetpnces between 11 non-smokers and 28 chronic ctp,arette-smokers with respect to systolic, diastolic, or mean aneraal blood pressure (for details of this st.ud'•, see below, 1197); and Westfall and Watts (1964s.) foun~ that blood Pressure was not statistically different in a group of 11 non- s1uokers compared to 11 heavy smokers. In their study of the smoking habits of about 3,000 Jewish male voluotteehe, Spain 107 and Nathan (1961) found no positive arsociation between the tlKY/tlla: {/bV/4 tlllu uuD/Vl/\' U/1NN1 ///YLbu/YA over t>a mm Hg. ln a series of 2,000 iuegna:t women, no appreciablc difference was found in blood preizure between amokers and non-amokers (Zabriskie, 1983). We also quoted Kuhn's (1927) conclusion that it might he necessary• to revise the idea of n nicotine hypertett.sion (2)0b); and, it now appearw, indeed, that blood pressure is lower in habitual smokers than in non-smokers (11'HU Chronicle 16: 363-370, 1901), and this has been observed in various popula• tions. Data on 1,723 men in EnFland over 60 years of age revealed that both systolic and diastolic pressures were higher in men who had never smoked or had given up smoking, than in cigarette-smokers; moreover, ttmpn Irressure .vas inversely related in the numtwr of rigmrenea amokal daily (F. EdwarcL, McKeown and 11-hitfield, I939a). An analysis of the blood• pressure findings on 1,093 FStmi,'h and American men aged 17-87 years indicated tlmt. in "normal" populations somewhat preselected for absence of kNvertension, no diflerenees in resting blood pressures were found between smokers and non :miokers; but, in broader studies, involving samples of the working population, smoking was associated with lower systolic and diastolic pressures (Blackburn, Broick and 7hy- lor, 1960; Blackburn et al., 196b). Young student groups, with short duration of the smoking habit, showed no signifi- cant differences between smokeia and non-smokers. in a group of 657 .tmerican medical r`tudents, the smokers, as a group, had higher mean tecumbent values for pulse preccurc, while non-smokers, as a qroup, had higher mean rt:umbent values for diastolic pressure (C. I3. Thomas, 1960). (Diastolic Ircetiaurc was one of the discrimniant values aleoriated with the habit end degree of cigarette-smoking; see C. 13. Thomas, I;oA- and Higinhothom (1964), betoix, 1129•A.J In a)troup of 179 C'ape Coloured and 181 Gurropean men studied in ('alw Town, South Africa, the arteiial pre.•ure.• of heavy r,mokens tended to be lower than thosc of nou•xmoker:c, but the differ- enceN w'e.r not I,igrri6cant (13rontc 5tewart, 1961). A 10-year Io:IRitudinal t•-tudy of blood prceaure in han Fronci.vo Imrg• shoremen indirated that both syslqlic• and diastolic blood prcs- sure.,~ are slightly lower among the htodemte to heavy smokers than among non-+anoker. (liorhani and Hechter, 1964). 444-A. Blood Pressure in Es-Smokere. It is astonishing to discover, in preparing thu Suliplement, that thete is no• w•here in ToGarro any reference to blood-pressure changes on stol>]>ing smoking, or to blood-pressure levels in e.N-smoketa. In a study published barely too late for inclusion, F. t:dwards, McKeown and 11'hitfield (1959a) did report that both systolic and diastolic pressure w•as higher in elderly men who had given up smoking than in cigarette-smohers. Hines (1960b) noted that stopphtg the use of tobacco may re,cult in blood pressure staying within normal limits in patients who were hyperreactors to the cold-presaor test, but he himself wres able to find only 3 such "•cures" in the records of the afnyo Clinic; and he persorally knew of only one incidence wherein the patient actually did atop smoking with a resultant penna- nent (that is, for several vears) normal blood pressure. How- ever, he suspected that other incidences would be discovered if standard smokhng-tests wrre made on a large number of patients with hypertension, and if the excessive reactors could be persuaded to stop smoking. 11'enzel, \CattanaponRsiri and Vedral (1964), who studied nicotine and renal hylxrtension in the mt (see above, 382), remarked that, while the human tftitonre to smoking mny nut. entirely re.cmble that of the rat to nicotine, their absenei Produccd f)Lt The Council for Tobacco C~eseaic1WSA, Inc. 0003019 Il~

108 TOBACCO-EXPERIMENTAL AN1) CLINICAI. STUDIES. SUPPLEMENT I tween smoking and hypet•tension, and could also explain the paradoxical elevatiorn in blood pressure following diecoptinue- tion of the smoking habit. Otut we •ceed much more data on blood•pres.nire levels in populations or nornial ex-smokers and in patients with hypertension who have stopped smoking before building too much upon the two isolated observations above. 445. .EB'ecl of Suwkirig on Blood Pressure (211) Investigators continue to report that cigarette-amoking elevates blood pressure in iiormal subjects to a greater or lesser degree (Zipp, 1958; l). L. Simon and IRlauer; 1960; Haimbock, 19B1;1n•iag and Yamamoto,19B3; i~lensch, 1963; l`.~lmundowiez, Pifer and \lar-hall, 19it1; lialori et al.; 1964; YPentecost and ShillinRiord; 1964a, b; Shillingford, 1965; among others). Following smoking, blood pressure fell ioc some instances to levels lower than the original readings (Hairnb8ck, 1961). Following the smoking of 2-4 cigarettes, systolic blood Nressure increased in more than half of 17, pregnant women (near term, but not in labor), while diastolic pressure rethained unchanged (Kumar and Zourlas, 1963). tio definite relation- ship was observed between those patients who showed in- creased uterine activity on smoking and those who did not (see below, 779). Smoking filter-cigarettes hrsulted in smaller increases in systolic atcd diastolic blood jnessure than smoking noh-filter citrarettes (\liksic, 1961). In 50 smokers, smoking 2 regular csgarettes for 12 min resulted in increases in systolic and dia- stolic blood pressure of +20 and +10 mm Hg, respectively; smoking 2 non-filter cigarettes for the same time, but not smoking the lact third of each, resulted in smaller increases (,+13 and +7, restiec•tiveh•)i and the increases were smaller still after smoking 2 filter ctgarettes (+10 and +5, mspec- tively-). 446. Foclors .InJleceneing die Pr•easor Eflerl of Smoking (213) Hines (1960b) stated that s ubjects with normal blood pres- sure who are hyperreactors to the cold-pre0sor test have an average rise in systolic and dias-tolic blood (rressure following smoking of 2-2.5 times that of non-hylxrmactors to this test. G. M. Roth and Bhick (19fi6a) reiwrted that the blood-ptes- sure rise in a group of normal subjccLc during omokinQ nas 19/7 mm Hg from an average basal blood lvrrxurr•of 105/72 mm Hg; while, in 60 s7tbject~ ~= who were hyiierreactors to the cold-pressor test, the rise was 32/21 mm Hg. I!'atients with hypertension xhmved similar exaggerated pressor responses to the smoking-test (Hines,19(10b; Roth and tihirk,1960a).) How- ever, no significant differences were found by C. li. Thomas (1960) between smokers and non-smokers in the reRlwtses of blood pressure to the cold-pvesrcor test; and smokers among Amerioan and Finnish men showed less diastolic pressure respnnse to the cold-pmscctr test than did non-snokers (Blackburn et al., 1960). As a result of their study referred to above, Roth and Shick (196Iht) concluded that the effect of smoking on the blood pressure of normal subjects, or of patients with inherently hypmracth-e vascular systems (as measured by the cold-presror test) is not due to a non-specific stimulus acting on a hyI)erreactive vascular system, but is the result, at least in part, of the nicotine in the tobacco producing vasoconetriction. Circulatory responses to stimuli of the cold-pre.,sor te:as b. w o~., u. . wy.....+.. . ~.. ......u ...ww nuwn. ... aged 17-67V years, compared to non-smokers (Klackburn, Broiek and Taylor, 1960). Following a study of several common cardiovascular mea.- ureanents made on 1,093 men, aged 17-67 rean, Blackbum. Brdiek and Tay-lor (1960) stated that them was little evidence for deterioration of cardiovascular "fitnes+" in smokers IMr- forming work-tests. No significant differences were found by C. B. Thomas (1960) between smokers and nmc-smoken in the responses of blood prexsure to the double Master exercif-e-le-m. In their epidemiological study ol cardiovascular distasr iu thc communitp of Tecumaeh, 1lirhiFan, F. H. Elslein, Ostrander and a.bciates- (1965) stated that blood-preK un• elevations were associated with coronary disease among men; and they examined ;w- ~ible intcractions between -mnking and blood pressure. The pressor msimnse to smoking by patients with coronary heart-di.aase W been examined by several workers. In 6 control subjects smoking 2 standard cigarettes with inhalation, the mean arterial-prewure increase was 7 mm Hg, compared to 15 mm Hg increase in 8 patients with coronary disease (Regan et al., 1961). Following the smoking of I cigarette, no differences were observed by Itlensch (1963) in blood pressure between healthy and coro- nary subjects of similar age, but the mean prrFCure in healthy persons increased less vigorously under smoking couditioiw. Smoking I cigarette resulted in an increase in mean blood preesurn of 6.2 mm Hg, compared to rises of 1.4 and 9.3 mm Hg, restMC•tively, in patients with angina or p9tients after myrn•ardial infarction (Pentecost and Shillingford, It161a, b; Shillingford, 1965). In patients with hytimr;eusion, the smok- ing of I or 2 cigarettes caused xignificant elevations of s.•stolic, and sometimcs diastolic, blood Icre..sure (Pcrlstein, 19fi4); see below, 13&4. C. Ii. 1 i-brr.a- and Murphy (1Wb) meacured sy:volic, diastolic, and pulse prea um., heart-rate, stroke-volume, and cardiac output in 9 white male medical students in response to smoking a ringle cigarette following appropriale small i.v. injectior;s of hexamethonium, mephente;mine, or i.wtonic sodium-cbloride sol-ition, administered in random order in a series of separate double-blind tests. The rise In systolic pnx- sure was found to be significantly less following mcphPnter- mine, anrl he.eamethonium did not influence the response to smoking. 1t'estfall and Watts (1961, 19G21r) found that the characteristic increase io bloa: pressure, as well a.c the btcreese it:puLce-rate and decrease in finger- and toe-temperature due to smoking, w-as reduced b.- pretrea:ment with rcr•,erpinc. The subjects were given 0.25 rng rr;erpine daily for 14 dayp, lhen given Roth's standard smokiag-tect Isee above, 409 and 4431. The ma3mum change from control values in 10 untreated university students before and after reserpine treatment aes, reslxrtively, systolic blood p"ure, +17 and +8 mm Ilq (p < 0.fID1); diastolic blood pressure, +14 and +7 mm Hg (p < 0.001). 417. Elfcrt on Blood Preseure of N'firotine-Fmc nnd 14on-Tobarcn Cigareltes (214) No new data; but see below, 448, Simon and Iglauer (1961, 1963), for the comparative pn-mr effee6v of low•-nirntinc cigars and pilm-tobacco, compared to commercial cqu,ers and pipe-tobacco. 448. Preeaor Ejects oj Othe•r F'ornca of Tofrne•eod%er f215) aad carbon-dioxide inhalation revealed a small but significant In elderly pilm :mtoket~ ~tudietl by F. fidWards, dlch/rN11 diminished diastolic•pressum rtwponse to cold, and possibly a and 11'hitfield (1959a), mean diastolic preetnre w•a, appmzi- pr®duced hr ~he Council io~ Iri~t Tobacco Research-USA) 00030w0

CARI)10VASCULAR BYSTRt1 109 mately the same as in cigarette-smokers, but mean systolic pre_"aure was an Wp nn Ill n.CLL LU6 bWUa4lR tbtz nvu$ e, weJ. Cigar- and pipe-srooking Increased the blood pressure in a group of habitual cigar- and pipe-smokers; Dow-nicotine cigars and pipes produced slightly less circulatory changes than standard cigars and pipes; and sham smoking produced no changes (Simon and Iglauer, 1961, 1963). The subjecis comprised 15 c6gar-smokers and 10 pipe-smokers, aged 21-89 years (Simon e nd Iglauer, 1961). The 15 subjehts smoking commercial cigars (1.82% nicotine) showed an increase in systolic pressure from 2-12 mm Hg (average, 7.3); the maxi- mum diastolic qreestrre increased 2-18 rnm Hg (atrerage, 8.9). On low-nicotina. (0.88%) cigars, systolic pressure tose in 11 subjects (+1-15 mm Hg); in 3, there ares no change; in 1, it feq; and the average change of all subjects was +4 mm Iig. The diastolic pressure rose in all (+1-16 mm 11g; average, 6.3). In 10 subjects smoking commercial pipe-tobacco (2.15% nicotine), both systolic and diastolic pressure rose in all (range, +1-:8 mm Hg; average, 8.9). On low-nicotine (0.76cIo) pipe-tobacco systolic pressure rose in 5 subjects, decreased in 2, and remsineil unchanged in 3; diastolic pressute increased in 7, decreased in 3. Tobacco-chewing increased blood pressure in habitual tobacco-chewers, but sham chewing of placebo gutn produced no circulatory changes (Simon and Iglauer, 1960, 1963). 449. Rffed of Nicotine on Blood Preaeure (215) In 17 young subjects, i.v. injection of 0.0406 mg/kg nicotine bitartrste was without significant change in mean arterial pressure; and, in the first 30 min after i.v. injection of 0.01-0.015 mg/kg, no significant change in meo.u arterial blood pressure, was observed (\tertz, 1961). A number of other workers has also tested the effect of i.v. injection of nicotine, but without specifying the body-weight of their subjects, so that the mg/kg dose of the drug is unknown. Hapid i.v. infus(on of I mg nicotine bitartrate in riorntal male cigarett.e-mnokeis resulted in a decrease in both volume and rate of digittil blood•flow, associated with a rise in artetial blood pressure (Burch and DePasquale, 1901). In 6 subjects receiving 4 mg 6ticotine bitartrate in 100 ml saline i.v. over a 10-12-min period, average syst:,tic and dinstoBc blood pres- sure rose 12 and 17 mm Hg, :espectEvely (Coffman and Javett, 1A8"a). /.411 subji'cts experienced aching in the ann, into which the nicotine was infused.) In 2 patients given 0.6 ntg nicotine i.v., an inerease in the systolic blood prescure and a widening of the pulse pre'ssare were observed; smoking a cigarette with inhalation produced the same results (lning and Yamamoto, 1963). In 27 normal subjects, i.v. injection of I mg nicotine resulted in au increase in systolic and diastolic pressures, which appeared to be greater in the non-smokers; inhalation of the smoke of a cigarette had a similar tesu)t (Valori et a)., 1964). ln 14 subjects (smokers and non-smokers), injection o! 0.5 mg nicotine into the antecubital vein over 4 mht resulted in a muderate increase in blood pressure (.5olti et al:, 1963). In 20 other subjects, injection of 1.0 mg nicotine tartrate i.v. caused slight increases in systolic pressure, and ptetreatment with dibenamine prevented this result• (Solti et al., 1964). In a patient with spinal cord Eevered at D-1, i.v. injection of 0.5 mR nicotine caused a fall in blood pressure of 18 mm Hg (Page and Taylor, I850). Lobeline injected i.v. into 20 normal subjects caused brady- cardia and h.7otension in 10, of whom 7 were sntokers; and Bevan and \lurroy (1963) considered thntt the increased response to lobeline seen in smokers probably reflected an additive action of lobeline trith its pharmacological analogue, W W YWC. 430. tlerhanism, oJNu Preseor Response to Sniokinp in Man (215) It has often been suggested that the pressor effect of cigarette-smoking might be due to nicotine-induced release of catecholamines (De Takats, 1963; Iiellet, in JA1fA I89: 33-37, 1964; Edmundowfcz, Pifer and Marshall, 1964), particularly via the very sensitive carotid-sinus and aortic ebemumceptor reflexes (see 375, above). 451. PERIPaERAL c1RCUWT[oK (216) Hirvonen and Sonnenschein (1961) have reviewed the acute effects of nicotine and smoking on peripheral circulation. 432. Capi$ory .1/terostopg (216) [No new data; but see 472, below.] 453. PidhysnropraPhy (216) Extending previously-reported experiments (Coffman and Nood (1957); Coffman, Wood and Wilkins (1955) (217s)l, J. E. Wood (1960) reported that smoking resulted in signifi. cant reductions of foot blood-flow in 28 of 31 tests. Abstinence from tobacco did not enhdnce this vasoconstrictor response (for further details, see belpw, 471). Drettner (1965) studied peripheral blood-flow in 21 healthy perxons foUnning tbe smoking of 1 strorg and I extra-mild cigarette. Blood-flow in the hand was reduced in both cases, and was more pronounced in female than in roale subjects, but less so in the non•smoking females, possibly due to a lesser ability to inhale the smoke. Blood flow in the nasal mucosa also decreased (see below, 480-E). Surface temperature and plethystnographically-measured blood-tfow in the lower extremity did not currelate in a group of SO healthy subjects (Oe Crinis et a1.,1960). In other words, vascular responses to tobaceo in the lower extremities may be difierent in skin and muscle; there tnav be a decrease, no change, or an increase in 8ow, in varying combinations (Redisch, Dfeckeler and Steele, 1960). H. L. Kaqinwn, Payne and 1Cinsor (1960) described a plethysmographic technique for studying tl:e i>eripheral circulation, and included data to show that sctt4tivity to tobacco may be determined in this matter. The in.-trumcnt (Vasograph) used for most of the studies contained three electronic limb pnewnoplethysmographs, three electronic digital pneumolaethysaiographs, a pre%ure parrel, and three thermistor thermometere. ln nomtal subjects under basal conditions, thc smoking of I or 2 cigarettes consistently caused a lowering in cutaneous temperature, particularly ot the fingers; rnuscle tennpcrature rarely clranged, and, at most, iu the direetion of a weak lotcer- ing: and the artcrial pulse (mcasurcd photoplethcsmovraph• ically) was reduced iu amplitude, without rhnnge in .vave morphology (7.annini, ('~onti and \egro, 1993). The effects lasted for several minutes after cevaliat of anokinF, and the vessels maintained their,calmcit,v to react to other ctintulanta (e.g., heat). Smoking 2 cigarettes did not cause prolwr- tionately greater effccts, but simply a longer duration; and cigar- and pipe-stuokcs,were compamble in effects to ciRa- rette-staoke. For an account of perij>heral-vascular changes following smoking in arlerial insuffictenry. see the same au- thors, below, 476, and (or effects following syrnpathectonty, tee 475. ProauCea r.~r N councit i~f 0003021 Tobacco f~Oscarch•USAI ~h s

110 TOI3ACC0-EXP>:ItIAfE1TAL A.\D CLINICAL STUDIES. SUPPLEMENT I Rottenstein and ea-aorken: f19601 used four methods to studv the eHect ot i.v -administered nicotine on the blood-flow of resting skeletal muscle and of the digits in normal subjects. Muscle blood-flow was measured by venous-occlusion plethys- mography after the overh'ring skin had been blanched by epinephrine iontophoresis; in some of these e!tperiments, the Hensel needle was used simultaneously. Digital skin- temperature meesurements in e constant-temperature room (20°C) furnished estimates of cutaneous flow; and digital pletbysmography, simultajteou8ly with limb plethysmog- raphy, yielded further data. Nicotine was administered in doses of 1 and 2 mg during 1, min, and in dosage of 3 nig during 4 min of injection. Cutaneous blood-flow decreased promptly, usually after the first ntg. Subst•quentl}•, there was typicaAy a moderate to marked trrinaitmry increase in musele-floR, frequently exceeding 400 %, and occasionall3• 600% of the resting flos. These marked incrkases usuadly subsided within 6 min after the start of t~e injection. Cutaneous vasocon- striction was usually much more prolonged. Burch and De Pasquale (1961) measured digital blood-flow by means of a rheopletbysmograph in 15 normal men, all established ciga- rette smokers who inhaled, following rapid i.v. infusion of I mg nicotine bitartrate; this restilted in a decrease in both volume and rate of digital blood-flow, associated with a rise in arterial pressure. Solti and co-workers (1964) investigated the effects of I mg nicotine tartrate i.v. in 20 clinical patients with a normal circulation, and observed slight increases in systolic blood presaure and minute-~volume, while peripheral vascular resistance was virtually uttc}nangt•d (for EHG changes in these subjects, see above, 438). 454. Sphygnographu ,Ifefhads (217) Usmg the technique deskribed by SchrBder (Z. ges. exp. Med. 130. 513, 10.59), bas?ed on measurements of capillan• pressure, for the estimation of blood-flow• through skin and muscle, Bremer (1962) studied on 15 healthy habitual smokers the effect of the first cigaret~e in the morning. Varying results were obtained: in 5, there was to change in either akin or muscle blood-flow; in 3, slkin-flhn• decreased, while murele- flow did not change; in 9, t'here was a simultaneous decrease in skin-flow and inerea.ce ii2 muscle-floa. In 3 petsons w•ho showed distinct Jfer,ts frotn smoking, nicutine a-as admin- istered i.v. in dosage of I mg/min for 3 min (this dose caused only a slight feel'utg of disiainess), resulting in increases in muscle blood-flow and dec~eares in skin blood-flow lasting only a few minutec )Sirnilar results were reported by ltremer and Felix (1959, 1900a, b) in cats receiving i.v. infusion of nicotine; see above, 392) In 2 experiments, measurement of fentoral-artery blood pressure showed that increased muscle blood-flonr was not accompanied by a rise in blood pressure, from which Bremer corcluded that the increased flow through muscle must result from vasodilatation, and not passively from nicatine-induced blood-preFsure change. 455. Arleriofe-Oprnirtg 1'etorfty (218) 456. Electricaf Phknornena (21S) 1\o new dat.a.] 457. Ckin Temperature (218) As recounted in the preeedhg section, nicotine and eiga- rette-smoke both decreased finger temperature (Itotlenslein et al., 1060; Zannini et ai., 1963); and cigarette-smoking lowered surface temperature of the lower e-iaremity (de Crh»s et al., 1900). Aliksic (1961), reporting on 50 smokerb smoking 2 non•I~rlter cigarettes for 12 mtn, lound decreases in alan temperature in various regions, as follows: fmgers, 2-3°C; toes,1-2.6°; aben•e the ankle, l.8°; middle of lower leg, 0.5°; under the knee, b:5'; above the knee, 0.8°; upper leg,1.3°; groin,1.1 °; navel, l°iC". Cigar- and pipe-smoking decmased skin temperature or the fingers in habitual cigar- and pipe-amokera, reslKrtiveh• (Simon and Itrlauer, 1961, 1963). bow-nicotine cigan and pipes produced slightly less circulatory changes than standard cigani and pipes [for nicotine content of these several tobarlror<, see above, 4481, and sham smoking produced no circulatory changes. The subjects were male habituall smokers, aged 2149 years, who smoked for 20 min in their habitual marDner, though under controlled conditions; nonae inhaled (Simon and Jglnuer, 1961). ln 15 eigar-smokere smoking enmmrn•iq1 cigars, finger temperature fell in 12, remiined unchanged in 1. increased slightly in 2. In I1 subjects smoking low•-nicatine cigars, finger temperature fell in 9, and the changex t.°ere equivocal in 2. Neither toe nor forehead temperatures showed any consistent pattern of change following cigar-smoking. Of 10 pipe-smokers smoking commervial pipe-tobacco, finger temperature decreased in 9, and the changes were equivocal in the remaining subject. Of 9 subjects smoking low•.niecdine pipe-tobacco, finger temperature dropped in 8, and Ihe chant:es were equivocal in the other subject, As with ciErer- smokrng, no consistent changes were fouad in toe or forehead temperature. T1ammal changes in skin temperature occurred 9-50 min after the start of smoking. Tobacco-chew•inF decreased finger temperature in 24 ha• bitual users of chew•ing-tobacco, but shatn chewing of placebo gum produced no circulatory changes (Simon and lalader, 1980,1903). 458. Skin Calorimelry ft) 459. Tkurmot Conductaare t~23) )No new data.) 4b0.1'enoua Orygen Salurtlion (223) Smoking a cigrrette decreased venous oxygen saturation (A•V difference), but sham smoking did not have this effeet (J. Freund and 11'ard,1960). 461. Radioisotope Uethods (223) J. Freund (1957) (22iib) found that smoking a cigarette significantly reduccd mdio.cod'• :n skin rlearartce in his Fub- jects; sham smoking w•as without sta'tistirall)• significant effect. These findings and conclusions were rr-p•.,blished by Freund and Ward (1960), who added that the acute effect of cigarette-smoking in normal subjects tvaa not as lot>F-acting as hitherto defcribed. Marked indiriduul variation w•az nb. served in normal subjects and also in patients with peripheral vascular dismse (digital plethysmography (217b))7 Kolar, fiova and IfudeFinska-Komatkova (1900) empidk-l escal>P-curves of the radioisotope, nNaC4 injected i.m. intn the lateral head of the gastrocnemius muscle to indimate whether or not the smoking of one-third of a cigarette would affect the cahillary field of a working musrle, and fotmd, in 5 healthy subject.c, that the combined load of physical work and smoking did not change the ecrape.curves; however, in 7 patients a•ith Ifuerger's dirritse, the slope of the curve was lowered under identical conditions. Coffman and JsvMtt (1981-6:, 1963) also used rate of dircap- pearancr of a radioisotope from skeletal muscle as a meature o1 its nutritive blood-floav; in 14 of 17 normal subjects, an Produced bv The Council for 0003022 Tobacco hcscarchUSA, Inc.

t CARDIOVASCULAR SYSTL%f incn'mse in muscle blood-fiow ares observed durmg agareue• smoking (2 unfiltered, regular•sized cigarettes), despite small changes in total calf blood-8ow and the usual decreases in skin tempemture. Muscle capillary blood-flow was measured by the disappearance-rate of the redioisotope, Na"/ and plethysmo- graphte blood-tlow changes measured total blood-flon of the calf, including both akin and muscle. Of 6 subjects receiv ing 4 mg nicotine bitartrate in 100 ml saline i.v. over a 10-12• min period, 5 showed an increase in 'ul disappearance-mte, and the average increase for the 0 was statisticaA)• significant (Coffman and Javett, 1963). Plethysmographic calf blood• 6ow inereased in 5 and decreased in I subject, the average increase again being significant. Average systolic and diastolic blood pressure and average pulse-rate also increased. In 5 sub• jecta tested with sham smoking of an unlit cigarette, 11111 diaap• pa.arance-rates did not change. 462. Fadore alodcfyi,r4 tAe s.6ece of Srtalri+>Q on Peripherol Circulalion (223) 463; Individual Variation (223). See J. Freund and 11'ard (1f#g0), above, 461. 464. Age; Sex; Race; Body Size (224). The reduction of btood-9ow in the hand following cigarette-smoking was more pMonounced in female than in male subjects, but less in the notrsmoking females, possibly due to a lesser ability to inhale the smoke (Drettner, 1t165). 465. Tobaceo-Use (2?A). [IQo new data.J 466, Idiosyncrasy and Hypersensitivity to Tobacco (nA). H. L. Karpman, Payne and Winsor (1f160) re-described 11'in. sor'a (1954) (225a) test for determining seusitivity to tobacco, based on akin-temperature changes following smoking. Vaso• dilat$tion was first produced by applying a wool blanket to the bodj and giving sufficient whisky (usually 30 ml) to bring the dtgital skin-temperature to 36C. The subject smoked a ciga- rettk in 3 min, and the change in skin temperature in fingers or t®es was noted. In the hypermactor to tobacco, the de• cieese in temperature exceeds 2C, and the return to n..rmal is prolonged; in addition, the digital blood-flow (as recorded by eleclronic digital pneumoplethysmograph) after smoking is diminished, and remains low for longer than 10 min. Fontana and w-workers (1959) (225a) tested 80 bealchy adults for cutaneous rmctions to tobacco-extracts and to changes in skin tetnperature and blood-i)ow (large-limb vpnous•occlusion plethysmography). The findings on these same 80 subjects have been republished by these workers (Fontana, 1960; de Crinis, Rediscb, Fontana et al., 1960). Although the actual findings are presumably the same, they are expressed somewhat differently in each of the publications, so that what is easily enough comprehendible in any one of thetli becomes less so in all three together. In the summary by d'o Crinis and eo-workers (1960), 32% of the 80 subjects under investigation were said to have showed a decrease in plethys- rriographirsll.y measured extremity blood-flow in response to smoking; and comparison with skin-testing revealed one sig- naficsnt correlation: 90% of those who did not react to skin- testing with tobacco-eatract had no decrease in peripheral blomd-flow in response to smoking. According to Fontana (1*), peripheral vascular studies indicated that 23:0 of the smokers with positive skin•tests to tobacco had changes in peripheral circulation, Indicated by skin temperature after tnnoking; only 4~'0 of the smokers a•ith negative tests had chahges in skin temperature after smoking a cigarette. How- 111 gr'aphically measured blood-flow in the lower ¢xlremit.v did not correlate in this group of 80 healthy w:hjc,:ta (de Ctinis et al., 1960). Redisch, Nfeckeler at,d Steele (10g0) reported that vascular responses to tobacco in the tower extremities may be different in skin and muscle; there may be a decreatx, no change, or an uncr+ease in flow in varying combinations. Almost every subject tested showing pasitive reactions to tobaceo•extracts showed a decrease in blood•ffow (skin, and, in most casrs, also in muscle) after smoking tobacco to which hk was sensitive; while most subjects whose skin was not sensitive to tobacco-extracts showed either no change or an increase in skin blood-flow as a response to smoking. Redisch (1962a) reiwrted a significant correlation betweeu srau senaitivit;• to tobacco-extracts and ef't'trts of tobacco. smoking on skin blood-ilow in 9 alletgic patients. 0f S patients without obliterative peripheral-artery disease, 7 shoard a decrease and I no change in skin-flow; 1patient with th6 disease showed a decrease in skin blood-flow. \o significant alterations in muscle blood-flow were observed. 467. Manner of Smoking (225). D. L. Simon and lglauer (1960, 1963) reported that skin•t,empernture changes in the finger and toes of subjects smoking cigars or pipes showed similar drops to those observed following the smoking of ciga- rettes; and they drer- attention to the fact that these, and other circulatory effects, occurred even though none of the (habitual) pipe- or cigar-smokers inhalecl. &wnini, Conti and hegro (1963) measured cutaneous and muscle temperatures, arterial pulse, and EKG in normal subjects before, during, and following the smoking of I or 2 cigarettes (see above, 453), a pipe or a cigar; they found that cigar- and pipe-smoking were comparable in their effects to cigar(:tte-smoking, that is, a lowering of finger temperature and no change in, or a weak lowering in, muscle temperature, a hile artariolar pulse was reduced in amplitude without change In wave morphology. In 4 healthy moderate smokers, who smoked I cigarette per honr for 5 hours, the vasoconstrictor effects reportedly became prognessively weaker (flaimbbck, 1961). $'he possible im- portance of repetitive smoking has been recognized. J. 1I. Harris (quoted in Modem 1led., \iinneapolis 82: 30 ft.,196-1), taking noted of the marked decrca.~e in temperature and amplitude of the capillary pulsations in the extremiti" upon cigarette,smoking, coircluded that there must ob• viously be a reductiom in the osygen supply to the areas covered by the prripheml capillaries; and he as4erted that repetition of this from 20 to 80 times lxr day must surely produce sufficient anoaia to do a certain amount of damage. He expressed the belief thatt this facet of smoking deserves a great deal more than passing cousideration. We ourselves had earlier written (653a) that, in principlc, it would not be sur- prising to learn that repetition of such a pharmacological stimulus as bmokinR, many times a day over many yeata, could result in cardiovtl.~tular disorders, functional or organic (see also Clough (1956, 1957a) (856-055a) Dock (1905).1 468. Effect of Deep Respiretion, i'eychic Stimuli, etc. (225). In normal subjeNs, xham smoking produced no srgnifi• csnt effect on the diftital circulation, exrept that due to rea• piratory changes noted in plethysmography (J. h7eund, 1057; Freund and Ward,1960). Hurch ar.d De Pasquale (1961) made a special study of variables which offered diii'iculty in the evaluation of the peripheml vascular responses to ciaarette-smoking. Their subjects were 15 normal men (e whites; 9\egroes), all eetab- Produced b-i Pal Council for 0003023 Tobacco Resarch-USA, Inc.

i I 112 TOBAt'CO-F.?iPER1DIElvTAL AND CLIh1CAL RTUDIE3. 9UPPLEAlENT I lished cigarette-smokers who inhaled. Digital blood-flow was weneureu uy a rheoplethysmograph. 'l hese w•orkers found that one of the most important of these variables was the psychogenic effect of cigarette-smoking, since offering a sub- ject a cigarette, or light irig the cigarette, resulted in peripheral vasoconstriction. They considered that tbL- most probably was due to the orienting reflex; but might be in part due to a conditioned reflex, inasmuch as the anticipation of smoking recalls certain learned sensations; furthermore, the vascular s,vstem can be conditioned to constrict to certain stimuli. Sham smoking and deep inspirdtion also resulted in peripheral vasoconstrictiun; but to a less marked degree. However, the effect of moderately deep inspiration can not be ignored when studying the effect of nicotine ih cigsrette-smoking on digital blood-flow. 469. Effect of Food or If`asting (226). lA'o new data.) 470.EAect uf Ph3sieaf :Acti.iny (226). JICo new data.) 471. Effect of Environmental Temperature (7l0). Smok- ing in the narm-room, the cool-room, and after 24-48 hours of abstinence from tobacco, caused decreases in foot blood- floa which fell into the sAme gieneral range [l:ckstein, Wood and N1'ilkins (1957) (226b)). Essentially these same results were again reported by J. E. Wood (1960), who added that, although reactive hyperemia of the foot was decreased by cooling the environment, unilaterally s.mpatbectomized limbs failed to show this response, while the contralateral limb did show the respont;e. This worker concluded that the acute effects of smoking on blood-flow of the foot, at rest or after a period of ischemia, were vasoconstrictor, and of the order of intensity of that prodticed by a cool environment. These effects were not observed in the calf. 472. Effect of Chilling ot the Extremities (220). It is per- haps relevant here to mention the case, reported by E. Davis and Landau (11)O1), of a female patient sensitive to cold, whose finger-tip temperature was nsually 3°C below that of her wrist. Smoking I cigarette reduced her finger-tip t:empem- ture up to 3° more, and the capillaries narrowed. 473. Effect of Heating the Extr'emities. Hirvonen and co- workers (1961) investigated the effect of smoking on vasodils- tstion of the skin of the lower eztremities when ure arm was 'heated; the subjects were 44 healthy• cottscripts, and toe tem- perature was measured. Snaoidttg 2 cigarettes prevented vaso- dilatation in 10 cases, and slowed it in 14; in 10 cages, smoking had no effect, and, in 10 subjecto, the temperature rise after smoking was more rapid than in the control tests. 474. Effect of Nicotine Content (227). According H. L. l:arpman, Payne and 14'inspr (1980), the amount of peripheral vatoconstriction is related to the nicotine and other vasocon- stdctn•e elements in the cigi+retter, as lcell as to the reactivity of the blood-vessels of the subjects. See also Simon and lglauer (1961, 1963), above. 457. It will be recalled (above, 453) that Drettner (1905) studied peripheral blood-flow after smokitag I strong and I extra-mild cigarette, and found that blood-flow• in the hand u-as reduced in both casea Blood-flow in the tm,cal mucosa decreased more often on sntoking the strong than the extra-mild cigarette, but this was not always trur.. 475. Nenwus Factors (228). G. Yf. Roth and Shick (1960b) presented data sbowing no vasnconst.ection (no decrease in toe tRmneratrlM) Oioicinn rinnrnttn.avnn4ino Gr tnmM.. y theectomy were complete, although smoking decreaced the skin temperature 'of the fingers. H. L. Jiarpµthu, Pacne atid Winsor (1960), rodi8tg that a sympa¢hectomisecf digit failed to constrict after tubatmo-smoking, eirplained that one site of nicotine action-the gantdia-had been removed. Aar intact nervoots supply to the limb appears not be nmcr- sap• for the increase in skeletal-mtrscle nutritive blood-flox during cigarette-shtdoking, since 3 of 4 syinpathectotnir,M/ hmbs showed an increase in muscle blood-flow during smoking; and, acsuntjnF that skin blmd-flotr did not dn•res,r in sgmpathectomiked limbs during cil.%rette-amoking, Coff• man and Javett (1961-62) concluded that this increa,,ed skde• tal-muvele nutritive bloud•flow• was dot a simple shift of blood from the va ocoruitiTtctil Nkin tu tErr musr•lr. \a"'I diup• pearanee-rates were sdudicd by CoBman and ;favett (11103) in 4 patienta with lumillar sympathectomies. The average control clearance constant, K, of 0.033 increased to O.Oa7 during the smoking of 2 non-filter, regular-sised cigarettes (p < 0.2j. The disappearance-rate increased in 3 of the 4 subjects, as also did blood pressure and puLse-rat'e. Toe arod calf temlmni- tures did not decrease, excr)t in the one patient whose dialv peatance-rate did not increase. See also Zannini, (:onti and Negro (1963), below, 476. 4i6. Effect of Smoking on Peripheral Circulation in Disease (2''-8). In the normal subjects studied by 7.annini, Conti and Negro (1963), cigarette-smoking consistently m- sulted in a lowering of finger temperature and in the amplitude of thearteriolyr pulse, but muscle temperature'rarely chanked, at most in the directi'on of a weak Iowering.,Thtam workers alro studied 36 patieitt.a with different degrees of arterial in- eufficienes•, and found thal the vasoconstti'ctor action of smoke did not appeafr more intense in Ihese patients than in normal subjects, and that the more affected vascular area-a esperienced the constrictor effect to a lesstx degree than other areas. In certain patieitts, mostly t:hcrse with thromboangiitic obliterans (TAO), values for cutaneous temperature w•ere incmased, instead of being decreased or unihanged, after smoking. The authors speculated that constriction of ve~~e'o in other aress resultcd,iqt a larger flow of blood into the affected limb, the vessels of which remained insensitiire to nicotine. Fbllowing sympathectotm•, in many eases (more than 50S;J, amoking w•im, foil ved hiv either an increase in the pulse.a•m•e amplitude or an in ace in the cutaneous thtrmometn• in the enervated limb. In healthy subjects, the combined load of physical work and sthoking did not change the escape-curves of radioisotope "KsCI injected i.m. into the lateral head of theqast-rocnemius musrle; whereas, in I patients with claudicatory troubles caused by TAO, the alope of the curve was lowered under identical conditions (Kolar, Sova and Budeshuk3-Komarkove, 1960). It may be relevant to mention here that kfehuQick and amn•iates (1981n) have described aortogram, feirlotal-arteriu- gram, ®nd brachial-artoriogram studics in patients with TAO, from which they conchuded that the arteriograf>hic features were sufficiently charaeteriFtie to repretxnt one more arltu- ment that lluert.rr's dirrase exists as an entity distinct from other forms of arterial disease. With respect to them other forms, J. Freurpd and Ward (1980) made repeated 'studiea of 14 patients with peripheral vascular disea,ee, but failed to find in them the reduction in skin teriiperature, radiosodium clearance, and venous o.~~µrn saturation after cigatrfte-stnoking which they dmd noted in normal individuals. In patients with rather marked peripheral Produced by Thc Council for Tobacco RESearch-USA, Inc. 0003024 00

CARDIOVASCULAR S1'STEht 113 vascular disease, only minimal changes m utooa-now numwY- ments occurred, and these were not statistically significant in the group as a whole. A. J. Barnett and Boake (1960) noted a lack of consistency in blood-flow changes in the foot or calf of the affected leg In 8 adult male patients with occlusive arterial dise.ase (atherosclerosis) following the smoking of I cigarette; and they concluded that it is likely that cigarette- smoking has no effect upon the already low peripheral flon• in severe atherosclerosis. Kalaci (1960) studied 9 men afiicted with intermittent claudication based on femoral arterio• sclerosis, and reported that smoking I cigarette made no ap- preciable difference in blood-flow to the legs. In 6 patients with bilateral arteriosclerosis oblitemns of the lower extremi- ties tested ln• Coffnzan and Javett (1983), nuua•1e capillary blood-flow and total blood-flow in the cal( did not change with cigarette-smoking; sErin-temperature changes w•ere small and variable; blood pressure and puLge-rates increased. 477. Influence of Drugs on tlre Smoking Effect (229). In the case, reported by E. Davis and Landau (1961), of a female patient sensitive to cold, whose finger-tip temperature was usually 3°C below that of her w-rist, in whom cigarette-smok- ing further reduced finger-tip temperature and caused capil- lary narrowing, neither skin•temperature fall nor capillar,v narrowing resulted when the test cigarette was smoked 45 min after oral administration of 8 mg of the sympathomimetic drug, nylidrin (Arlie>lin). On taking the drug alone, skin tem- perature rose, and the capillaries became congested. Following administration of 0.25 mg reserpine daily for 14 days to 10 university students, the maximum ehange in finger temperature was found to be -4.0°F and the maximum change in toe tempemture, -^.2°F (B'eatfall and Watts, 1961, 11962b). The mavimum changes in untreated subjects were, respectively, -8.4 and -2.7°F. The change in finger temperature was significant at the p< 0.1 level. 478. Mechanism of ihe PeripAesal l'aaoeonstrfction Follo+oing Snroking (230) The amount of peripheral vasocwnstriction is related to the nicotine in cigarettes (Karpman, Payne and Winsor. Ifl80); but, while nicotine produces peripheral vasoronstrict.ion, studies designed ta quantitate the dcrrease hi peripheral blood-flow due to cigarette-smoking must take into coruaidera- tion other associated factors which may reduce the flow (burch and De Pastluale, 1961). The sympathetic fibers were said to be the pathways through which the nicotine exexte.d its pharmacological effect (G. M. Roth and Shirk,1960b), since the lack of vasoconstric- tion in svn:pathectomised limbs following smoking demon• strated the necessity of an intact sympathetic nerve supply to the blood-vessels (see 475, above). Reterring to clinical observations which have shown tlut the smoking of a ciltarette causes a large fall in skin tempera- ture and a diminution of bbod-Hoa through the hand, J. Ii. Burn (1960b) stated that it appeared that these effects wrre due to the release of, noreiHnephrine from structures in or near the artery wall. With respect to the cardiovascular pharma- cology of nicotine, la'awas (1163) asserted that its sympatho• mimetic properties may be explained solely on the basis of the amount of nor0pinephrine the drug releams. Page and 1feCubbin (1982) suggested that it could a•ell he that sensi- tiaation of the adre,nal gland is caused by minimal amounts of nicotine, and that the peripheral constriction vrhich follows smoking result_s in part from enhancetl secretion of cateehol. aminm DeTakats (198,9), among others, regarded cate- ..uv.a,....... .,..~. .... ....,....., .. r, .f... ..n....o.. „r r.d.ornn nn the circulation. 479. Peripheral 1'enous Cireufnliwn Eckstein and Hors1ey (1960) studied the responses of the peripheral veins in matn to the i.v. administration of nicotine, apparently the first investigation of its kind. Venous pre.sure• volume curves were obtained plethyFmographica{h- from the right forearm of 12 healthy meu, and, in 5 of these subjects, venous pressure was measured simultaneousl,v in the ante- cubital vein of the del.-9dc:nt left arm. AfteP control values were obtained, 1.0 mg nicotine in 5 ml of normal saline was injected rapidh• through an indwelling needle into a foot vein. Observations were continued until control values returned In 7 subjects, the nicutine injection was repeated atter i.v. administration of 5.Q mg phentolamine methnnesultonate, an adreneritic blocking drug. !n I1 of the 12 tests fol,loning nico• thie injection, venous constriction occurri^d. The venous volume at a tmnsmural pressure of 30 nun 11g averaged 4.0 ml per 100 ml of forearm tissue during control periods, and 3.4 ml alter nicotine (p <'0.001). The venoYis volume at 30 mm Hg after nicotine averaged 3.2 ml before, and 3.4 ml after, phentolamine, a difference dvhich was not statistically signifi- cant. Peripheral venous pressure increased in 2, decreased in 1, and remained unclaanged in 2 of the 5 ea~periments in which it was measured; arroge figures before and after nicotine were virtually equal. The naturally-occurring venous volume averaged 2.8 ml per, 100 nil tissue before, end 2.0 ml after nicotine (p < 0.05). The venous constriction in 4 of the 5 ex- periments was sufficient to push blood from the fdreartn. [Tlre reader may wish to compare the effect of nicotine on isolated human veins by consulting Nlaloff (1932) (176a).1 Solti, Iskum and 7.oltan (1961) studied the effect of nicotine on venous pressure and vein tonus in 10 healthy subjects and in 8 patients suffering from chronic circulatory unsutTicfency. Venous pressure and vein tonus were ccimultaneoukly re.eorded, the former being measured in one at=rt, and the latter in the opposite arm by isolating a vein section with no side branches hy means of prnxitnal and distal tie•ofTs aith adjustable ntbber wedges; heparin was used to prevent blood coagatlation, and a needle was inserted into the isolated section nnJ bour.d with a second Alorits•Tabar apparatus. L%•. injection of I mg nico- tine tartrate over a 1-min period consisterrt.ly caused an in- crease in venous pressure of 6-18 mm HrO, and in vein tonus of 7-12 mm HA An', incres,se in vein tonus couid be detected after 2 min, while the Increase in venous pressure occurred later, usually after 5`1 mrn. After 20 min, both pressure and tonus increase had, )n neral, subsided; aqd, after 30 min, they had, as a rule, d' pleared• No substantial diFerences In response were noted between the healthy and the circulatory- deficient subjects. The mectianisrn of the vein constrietion was further studied in S, eaperOtnents in which nicotine .vas in• jected directly into the vein section. No effects o<rrurrMt until the ligatures were loosened to pemiit the nicotine to enter into the general circulation, after which the increases were again observed. In 6 other subjects, the %vall of the isolated- vein section wate infi,ltrated, with procaine before injection of the nirotine; increa.se in vein tonus a•as then con.ddcrabl}' less, and, in some cases, did not occur at all After the effect of the procaine had ceased (30 min), injection of nicotine tvas repeated, and Increases in vein tonus again obtained. The authors concluded that, the increase in venous pressure tvas not simply a secondary result of actions of nicotine on other areas of the drculatory system, but was induced bi• an increa.°r in sympathetic tonub. Solti and co-workers (1962) also mnas• Produced bv The Cotmcil for 0003025 Tobacco Rescarch•IiSA, loof

114 TOBACCO-EXPP.RIAlENTAL AN1) CLIItICAL STUDIF•S. t3UPPLIE-NfEtiT I uved the effect of slow i.v, injection of 0.5 mg nicotine on systemic veuous pressure in the venn cubitalis, and reported a 15-25% rise, which was considered to be refle.e in origin. For details of these experiments, see Solti et al., (1963); below, 480•B. 480. EBect of Smoking and/m .t'icotfne on Sperial Circulations in .1fan (231) 480-A. Retina. Haimb(iek (1961) reported experiments on 50 cLnically sound individuals between the " of 16 and 76, all of whom were moderate smokers, but who refrained from srimoking un the morning of the test day. Systemic arterial pressure and retinal arterial preaure were measured, following which each subject smoked a cigarette in his accustomed manner; pressures were measured after starting smdcing at 2-, 5-, 10-, and 45•min intervals. \fean upper-arm blood pres- sure rose from the control level of )03 f 13 nun Hg te a peak of 107 f 13 in 2 min, and gradually dropped to slightly below control level by 45 min. XIeap retinal arterial press~re rose from a control level of 52.5 * 6.8 mm Hg,to a peak of 57.0 f T.3 at 2 min, and gradually dropped to slightly below control level by 45 min. Both increases were highly statistically sig• nificant. The linear difference, taken as an expression of con- strction or peripheral intra-cranial vessels, rose to +3.8 * 3.6 at 2 min, which was calculated to be highly significant. Four subjects who repeated the test on 5 days gave relatively consistent results. In 4 subjects who smoked 1 cigarette per hour for 5 hours, the vasoeonstrictor effects became prngres- sively weaker. According to Rohn~ (1962), fundus photo. graphs revealed questionable vssoeonstriction after cigarette- smoking in 5 of 13 subjects, and a marked vasoconstriction after smoking in 1. In a medical investigation by C. J. Ellis and colleagues (1964) of 146 patients with retinal vascular occlusion, the ahtoking habits of 133 were obtained, and 97 of these were compared to 97 matched controls. The number who were smokets at the time of their veuous occlusion (50-51 % of the group) was slightly higher than the number in the control shriex (39-40% of the group), the diH'ernaihe being statisticslly non significant. A similar comparisoai was made between 28 patients with occlusion of a retinal artery,, and a control popu- lation; and the number of :.mokers within this groul. (24 or !16 0) was significantly higher (p < 0.01) than the number in thP control group (11 or 39%). These findings were said to raise the question of the relation between smoking and occlu- eion of the retinal artery. Following slow i.v. injection of 0.5 mg nicotine, blooti pres- sure in the retinal vessels rose slightly and tempomrily, paral- kl to the moderate rise in systemic blood pressure (Solti et al., 1962). For details of these experiments, see Solti et aL (1963), below, A80-13. t180-B. Srain. Solti and colleagues (19M measurecl c•erebral blood-flow snd venous pressure (by direct puncture of the bulbus venae jugularis) before and 15 min following slow iv. injection of 0.5 mg nicotine, and found tlust, althouteh venous pressure rose in the brain to the extent of 20-30'`r. the amount of blood passing through the brain, and the resistance of the cerebral vewels, did not changesignificantly; the risrm in venous ptmure was considered to be reflex i.r origin. The actne eQert of nicotine on cerebral blood-flow and cerebral v cnauP Im~.ure was further reported by Solti and associates (19631, who studied 14 patients (smokers and non-smbkeat) without heart failure or renal di`ease. Cerebral blood-flow was measured by t.de :% lv iuruani; ccIc•u,a; . ..,V_ N....,... .. . ... . , L. direct puncture of the interrw) jugular vein; systemic veiN,u, pressure was recorded from the antecubital veiu. After iujw. tion of 0.5 mg nicotine into the antecubital vein over a I+eri1.) of 4 min, cerebral venous pressure rose 20-30%. the inrrraw starting in 2 min, and reaching a maximum within 8 min, a ith return to normal by 15 min. Antecubital venous pneseure n,W 15-25%, the rise occurring some minutes later than that 44 cerebral venous pressure, with return to normal in 15 ntin Cerebral blood-flow shoa•ed no marked change; but, Ix.buw of the moderate rise in blood pressure, there was some incns•e in cerebral vascular resistance, although not to auy significant d Lt~'bow (1963) found no Fil;nifirant differences between non. smokers and chronic cigarette-smokers with respect to cerebral blood-flow and cerebral oxygen utilization; for further cMlaib of these subjects and findiup,.., see below,1197. For the efl.rt of ciRarette-smoking on constriction of periphe•al intra.crauial vessels, see Haimbock (1961), above, 4g0•A. Discussing the medical management of eerebral vaw•ular insufficiency, Fazekas (1954) stated that the efTectr of tolwI.+U on the cerebral circulation are not known, but that snwkiuR should be discouraged because of its arcepted hazandE, apart from its possible influence on the cerebral blood supply. 480-C. Lung. Pagan-Carlo (1959) reported vascular meaY• urements of smokers and non-smokers, the caliber of the lwl• monary blood-vessels being measured in posteru-anterior roentgenograms made with the subjects standing erect; this was said to be the first attempt to detect a.n)• ALruifirant changes in the caliber of the arterial shadows in the chest X-ray. The studies were made on 694 as)•mptomatic smukerx and non-smokers, comprising 257 non-smokers, 133 light smokers, 204 moderate smokers, and 100 heavy smokers. Thc most striking difference was found in the size of the main hilar vessels (distal end of the descending branch of the right pul. monary artery), namel.i•, 0.5R3 inch for non-smokera and 0.664 inch for heavy smokers of more than 30 cigarette+ daily. The difference, significant by statistical criteria, was inler- preted to indicate h.-.1mrtrophir dilatation of the pulmonary arteries. Measurements iu the other zmies studied (perihilar. mid-zone, and peripheral) were higher for heavy smokers than for non-smo!.ers, but differences were not statistically sqmifi• cant. There were no discernible diBerentws beta•een light aad moderate smokers and non-smokers. It may be useful to mention here the measurement of pul- monary capi!lary blood-.rolume, as desctibed by Datc-. Christie and Varvis (1960), based on measurement of the lung-difTusing capacity for carbon monoxide at differing alveolar oxygen tensions, which may be vitiated in the cav of heavy smokers with a relatively high concentration ol carbun monoxide in the blood before the experiment is begun. 480•D.1lfuscle. Kolar, Soya atd Iludesinski-I:omarkova (1960) attempted to ascertain whether or not the smoking of one-third of a cigarette would affect the capillary field ol a working muscle, by means of measurement of esrahe-curer- of radioisotope "AaCl injerted i.tn. into the lateral head ot the gastrocnemius muscle. ln 5 healthy subjects, the comhinnl load of phyrical work and ruuoking did not change the e,raiM• curves; however, in 7 patients with elaudicatory troubleq cau.xd by lluerttcr's discam, the aoi>p of the curve w~Lq hmemt under identical conditions. l'sing rate of disaplmaranrr nd a mdioi.otope from skeletal ntuKelr as a messnre cd itu mnritive blood-flow, CofTman and Javatt (1901-62) found that 14 of a Produced h-r The Counca for 0003026 Tobacco l'Wsira,"ch-USa , InC.

CARDIOVASCULAR SYSTEM group U1 10 IIUrWal tlnUJelltl DIIUN{A1 an 4/tACaLtl W muscle nutzitive blood-Boe• during cigarette-smdking, deApite small changes in total ealf blood-flow and the ueoal decreases in akin temperature. The increased skeletal-muscle blood-flow during,cigarette•smoking is apparently not a siYople shUt of blood from the vasoconstrieted skin to the muscle. In these ey.7eriments, Na'nl was injected into a lateral calf muscle; smokiqg consisted of 2 unfiltered, regular-sized cigarettes; and the total blood-flow in the cali, including both skin and muscle, was measured plethysmographically (f',offman and Javett, 1963). Na'a'I disappearance-rate increased in 5 of 6 subjects receiving i.v. infusion of 4 mg nicotine bitartrate, and the average increase for all subjects was statistically signifi- cant. Plethysmographic calf blood-flow Ineressed in 5 and decreased ih 1, the average increase again being significant. In 5 subjects tested with sham smoking of an unlit cigarette, n'I disQppearance.rates did not change. Additional details of these studies may be found above, 461 and 475. For further accounts of the effects of nicotine and/or smoking on muscle blood-flow, see Rediscb. Aleekeler and Steele (1960), Rottenstein et al., (1960), Znnnini et al., (101i3), and Drettner (1965), above, 458; Bremer (1962), above, 454; and Rediseb,Meckeler and Steele (1960) and Redisch (1962a), above, 466. 480-E. Nasal Mucosn. Blood flow in the nasal mucosa of 21 healthy persons decreased more often on smoking 1 strong than I extra-mild cigarette, but this was not always the case 115 ~L/A~NIN, /JWI/. u.WY•YVN Y/ ~Ul IM,/1I NA1 Y1OV 4//NNW~.4 in these subjects; see above, 453. 481. 1'ascular Nistopa¢ho(ogy of Smokers ond Non-Smokers Based on a at.udy of 989 necropsies on men, Rilens and Plair (1962) described various vascular lesions in relation to smoking practiees; their findings are given in detail below, 1455. See also Auerbach, Hammond and Carfitdcel (1965a, b), below, 1397. 482. nEVIE{{6 (232) The action of smoking and/or nicotine on the human cardio- vascular system has bePn reviewed, more or less briefly, by a considerable number of writers (Kumlin, 1959; J. H. Burn, 1960c; G. M. Roth,1960; Roth and Shick,1960b, I9G1; Lupu, Popescu and Enescu, 1961a, b; Schievelbein, 1982; Arroyo, 1964; Dmowski, 1964~ Giovanelli, 1964; Konttinen, 1964; Bing, Cohen and liluemchen, 1965; Ferreira, 1065; among others). Two of these were parts of published symposia (hurn; Bing et al.j; and, while the remainder of t.hese short, and necessarily incomplete, articles were without doub¢ interesting and useful to the casual reader at the time of theit journal appearance, they must remain ephemera. Vati Proosdij's (1960, ch. 5, sect. 3-B) review of the effects of smoking on the heart and blood vessels, being contaiued within bard covers, is somewhat less ephetderal; although, as we ourselves appre- ciate full well, even hard-covered reviews °daqe", all too rapidly in this fast-moring field of clinical investiptation. Produced by ThO, Council for Tobacco IZesearch•USAA Inc, 0003027

A / RESPIRATORY SYSTEM 483. RIiaPIRATtON 484. Fish (233) J\o new data.J 485. Frog (233) See Cberoigovsky (1962), below, 508. 486. Safamweder (233) 487. Tartle (233) 488. Sna1z (234) 489. Luard (234) 490. Bird (234) INo new data.) 491. 1louse (234) Intracerebral injection of nicotine ia mice resulted in ime mediate convulaione, followed by hyperventilation; none of the auimals tested showed spnea (Gokhale and Gulati, 1962). 492. Rat (234) Following convulsive dases of n:cotine in rats, convulsions always preceded the period of almea, but never followed it (Gokhale eud Gulata, 1962). After i.p. injection of I mg/kg i~ anesthetiaed rats uuder artificial respiration, centra: apnes appeared, and lasted for about I miin (G. Schmidt, Rrdmaon and dal Ri, 196;i). In bivagotomised animals, this dose of nicotine caused fleeting hyr>erventilation, followed by reapiraton• standstill in expira• tion, accompanied by excessive and continued excitation in the effererit vaRus (Schniidt and da) Ri, 1964; Schmidt, dal Ri and Lendle, 1964). A similar reaction was observed following injections of 1-3,ug nicotine into the fourth ventricle (Schmidt and dal Ri, 1965). Applied topically to the area ventrolateralis of the medulla, 1-10 pg nicotine elicited a marked respiratory stimulation. (For further aspects of these studies, see below. 302, 519, and 321.) 493. Gufnea Pfg (234) [No new data.) 494. Rabbit (234) l.v. injection of 0.1-0.5 mg/kg nirotine in rabbits caueed a brief inspiratoq• apnea (Takasaki, 1956; tiakano, Taluuaki, Ota and Nobuyuki, 1957). A similar result followed injection of the drug into the carotid artery (fakasaki,1956). Injection of 0.2-0.3 mg/kg into the pulmonary artery of urethaniaed rabbits resulted in immediate reflex apnes in the inspirat©ry position; on injection of the drug into the left auricle, refiex ap'nea did not occur, the onh• result being an increase in nes. piqation (Takasaki, Yokooand Xagasaki, 1959). Georaiev and associates (1962) grafted ordinary tobacco (1iulFarlan and Virginian) bu Dafura stramonium stalk, thus pr©ducing nicotine•free tobacco (see below, 1026). When tested on rabbits and cats, smoke from cigarettes made from this tobacco did not accelerate respiration (Georgiev et al., 1962; Rasev, Zografski and Tomov, 1964). 495. Caf (235) As reported in the preceding section, smoke from cigarettes enade from nicotine•iree (stramonium-grafted) tobacco did bt'ot accelerate respiration in eats (Geotltiev et al., 1962; Ra.aev et al., lt#'if). R. A. 1litchell and co-workers (1963) reported that direct aµ"plicatinn of nicotine or acetyleho6ne on the floor of the foiurth ventricle in the region of the area postrema decreaced ve+spiratory rate and frequently tidal volume in 3 of 10 r.ats Iuud 15 of 16 dogs. Respiratorv deprehion usualh• appeared after one or two breatha, and, in dogs, often progressed to iipnea. Hypoventilation occurred in I cat and 2 dogs at con- centrations of nicotine down to 50 µR/ml; and, in 2 cats and 10 dogs, at concentrations of 100 µg/mI or more; 7 cats and 1 dog did not respond at a concentration of 1000 µtt/ml. Repeated applications of the drugs, or cauterization of the region of the atea )wstrema, abolished the response; and stimulation coulci not be achimed by topical application of nicotine to other arcas of the fourth %rntriclc, dorsal melulla, or extravrntricular portion of the choroid 1>fexus. Studies were also made by perfuRion of drug.- throuKh catheters inserted into the fourth ventricle, the in'rTusion fluid consisting of a mock comiwsition of rnrrbnn:pinal fluid (CSF). When the catheter tips were laid in the subarnchnoid sinur on the lateral surface of the meduilu in tne rtq!inn where the 6th and 9th cranial nerve-roots entered the ntedulla, rapid injection of nicotine intn the stream of mork ('tiF increflsed tidal volume, and frequently increased ra9e; the latency of the mponse in a fresh prelmration n•a-- 3-12 xti• (uQuall.• one breath). Signifi- cant rcspiratory reslanwe. uere ohtnined im mw animal< following a quick injection of 5 Wt nicotine (lOD In 3 of 5 eats tested for drulrihreshuld, respiratow tes;pwnsr. were obtained with ;,6,-tn.r ci.mu to 3 pg (10 pg/mI); diluted to I µg/ml, nicotine fnili•J to nffert twpiretion when teiven in 1•ml volumes. Regunnd tmrfu•ion a•ith solutions containiuF eosin demonstrated tlwt respiratory rcxlwn:r~ were elicited only when the ventrolaternl surfacee of the brainstem a-em stained. In cats, Lv. injection of U.(1.i-0.5 mg/kg nicotine caused a brief espimtory apnea IivitinRa few seconds, followed M• strik• inR accelen.tion ir, rate and depth of respiration (Takasaki. 11e Produced bV Thn~, GOu11611 tOf 0003028 Tobacco fZeseardt-'JSA1 I~~~,

t .. RESPIRATORY SYSTEM 117 i 1956; Nakano et al., 1957). Following a large (0-2-0.5 mg/kg) ... .............., dose, °secondarv nrnsnnvnri lvbti). In cats under urethane, however, it was reported by Takasaki, Yokod and Nagasaki (1959) that injection of m.1- 0.3 mg/kg nicotine, i.v. or into the right ventricle of the heart, resulted in reflex apnea, followed by rapid, shallow breathing (in contrast to the "striking acceleration qn depth of respira- tion" previously'reporoed by Takasaki (1958)j. In open•chest preparations under artificial respiration,, puhnonary-artery injection of these doses of nicotine resulted in a brief marked decrease or disappearance of the pht'rnic discharge,folloned by an increase in frequency and amplitude: Nicotine injection into the left audcle caused only an inerease of phrenic dis- charge, without disappearance (see below, 520). No effect on respiration of chloralosed 'cats was obser+red after injection of 0.01-1.0 mg nicotine iyitartrate into the submanillary duct (Eminelin, Muren a6rd Strbmblad,1954)'. 496. Dog (7dB) The respiratory responses of dogs, along with those of cats, to direct application of nicotine to various axess of the medulla, :+eported by R. A. Mitchell nnd essociates (1983), have been described in the section immediately above. With respect to vascular or intracardiac administration, Wagemnann and co-workers (1957) injkcted 2-10 pg nicotine i.v. in unanestbetiaed dogs, and reported that the lower doses caused a deeper inspiration, and the expiratory apnea whs frequently prolonged; the subsequent 3-5 bre,aths were usually irregular. With the higher doses, seveial deep breaths pre- ceded the apnea. In dogs under urethane and morphine, in- jection of 0.1~i.3 mg/kg nicotine, i.v. or into the right ven- tricle of the heart, resulted in reflex apnm ("primary apnea,") lasting about 10 see, followed by rapid ahaGoN breathing (Takasaid, Yokoo and NageRaki, 1959; Taisesaki et al., 1959a). In open-chest preparations under artificial respiration, these doses of nicotine injected into t.he pirlmonary artery caused the phrenie discharge to deenease markedly for 10-2p sec, or else to disappear completely; theneafter, there was an increase in frequency and amplitude. Nacotine injection into the left auricle caused only an increase of phrenic d'uscharge; without disappearance (see below, 520). Following the rapid shallow breathing, there occurred in these dogs the so-called, "secondary apnea", which was not always seen in cats or rabbits (fakasakib et al., 1959a). Infusion of 15 Icg/kg/nun nicotine in anesthetized dogs resulted in am increase in respira- tory minute-volume; infusion of b pg/kg/miry had no signifi- cant effect ((Schmitthsnner et al., 1957) (238)1; and accom- panying the increased respiratory minute•volume were a greater arterial pH, elevated artedal and coronary-sinus blood-oxygen content, and reduced artetial carbon dioxide (Forte et a7..1989). In anesthetized dogs inhaiing dgaretwamoke through a tracheal tmnnula, increased respiratory amplitude preceded the pressor effect, and typical apnea appeared at the peak of the preaor effect (de 1lfoura, 1959). These e$'ects aere as- :.ribed to nicotine, since they did not appear on inhalation of smoke front a plant other than tobacco. 497. Alonlcey (237) (Vo new data.j 496. Man (237) From a correlation of results of spirometric tcsts and blood garometric observations, Lupu and Racoveanu (1901) divided ~c.. habitual smokers into3 aroups in termR of ;uouwciency: i. sinokers for a period of 14 years, with respire• tory insuf6ciency revealed by functional tests; 2. smokera for a period of 25 years, with respiratory insufficiency at the lesRt exertion, and hemoglobin saturation reduced by 8<i; 3. snlokers for a period of 400 years, with very pronouD^ed mpira• too•y insufficiency,(h)•per,capnia and hypoxia) observed even during rest. See 523, below, for an extensive account of pul. monary function in relation to smoking habits and to riFa- rette-smoking. <<n 1 I of 19 normal subjects exhibiting a type-I pattern of abbormal ballistocsrdiograms on smoking (see above, 441). 6 shoa•ed a decrease;, 4 no change, and I an inctrase in respira• tory rate; the meah respiratory rate fell from 14.5 to 13.5 per min (hock, 1a83a); Threc of the subjects shon•cd no chai.ke, 2 an increase, and 6 a decrease in depth of abdominal respira- t.ion. All showed a decrease in respiratory motion of the thoi•ax, the decline averaging 47%. In 9 of the 11, the mid- position of the respiratory curves moved towards the expira• tory position. It ia these changes in respiration evoked bv nicdtine which, in part, lead to apparently abnormal II(`G smoking-tests; and Dock (1963b) remarked: "Even though ballwstocardiography remains caviar to the profession, it is cheerful to know that much of the mystery surrounding the respimtory variation in the Starr trace has been dispelled." A total of 58 maternal cases and a correspc uding number of infant ce<ses were studied by Heron (1962), who reported that smoking in the first stages of labor is .-ery liable to produce infants whose respitation is depnested, and who cxinsequently take longer to establ`usit normal respiratory excursion and tone. Carlon-monoaide blood concentrations were found to be higher in maternal and fetal bloodshi patients who smoked. 13evan and Nfurray (1963) reported that lobeline injected i.v. igtto 20 normal subjecte caused ventilatory depressicsi in 17; the mean respiratory depression for smokers aas 31.4 0 and for non-smokers in the group, 18.Iryo (p < 0.03). The authors considered that the increased response to lobeline seen in smokers probably reflected an additive action of lohe. line with its pharmacological analogue, nicotine. 499. ComparoRtive Bffert on Respiratton of Nicotine and t'.crlsin Deritnlivrs (238) Io his in•.eetigation of the pharnmm!od,n• of some pyrrolidine i1'-sttl+stituted nomicotine derivatires, 'Mattilla (19tC;a, b) found that nicotine, rpornicotine, and etltylnornicotine stintu- lated respiration in the cat, while ahylnornicotine, acrty1• noroicotine, carbaminoylnornicotine, and beneoybiornicotine had no effect. 5M. ''~MtO,f,1(affipP(RepfOtfd) DoSPF (23fl) Following i.v. injectiofi of 1 mg/kfi nicotine in rats, 3 addi• tional injections of 3.0 mg/kg over I hrnar no longer produrnd a change in central respiration (G. 5chmidt, L•'rdmann and dal lti,,1903). In bivagAtomited aninmis, in which this dose of nicotine caused fleeting hyperve»tilation followed by respira- tory standstili in expiration, these reactions decreased ahen ntcotine was injected repeatedly, and parl of this tschyph;i• axis could be overcotne by increatdng the dose of nicotine (Schntidtand dal Ri,1m;Schmidt, del Ri and Lendle,196!). Itats made tachyphylaetic to nicotine administered Lv. also failed to respcmd to nicotine injected into the fourth ventricle (Schnmidt and dal Iti, 1flti5). See also R. A. Mitchell et al. (1983), above, 495. Produced bv T" ~ CU+ncil for Tobacca IM 0003029 0

118 TUBAC•CU-E\PEItI31E\TAL AND CLI%IC.1L STUDIES. SUPPLEMENT I snr "'o new data.] 502. Influenre of dif Vagus (239) In rabbits and cats, primary (reflex) nicotine apnea failed to appearafter bilateral vateotomy (Takasaki, 1936). In anec- (hetized cats and dogs, bilateral vagotomy just below the hilus of the lung did not affect the reflex apnea resultant upon nicotine injection, but bilateral vagotomy above the hilus abolished the nicotine apnea (Taka.,*ki, Yokoo and \agasaki, 1959). In dogs subjected to the perh/sed-head technique of Heymans, vagotomy abolished the apnea caused by i..•. injec- tion ol nicotine, although re,;phatory,acceletation remained (Takaeaki, Katsuda and \ags~ski,1959). Injections of 190-525 pg/kg nicotine increased the raUe and depth of breathing in 4 vagotomized rabbits (in I animal, the depressor nerves had also been cut) (1lott, 1903). The decreased respiratory responses observed by li. A. \fitchell and co-a•orkets (1963) in soqte eats and mos; dogs following application of nicotine to the floor of the fourth ventricle in the region of the area 1»ulrema (see above, 495) could still be elicited, in reactive animals, after the vagus nerves and the carotid sinus had been cut. G. Schmidt and associates used mele rats mtder urethane and aprobarbital anesthesia and artificial respiration to stud.v the effect of nicotine on the electrical activity of the efferent vagus and phren+c nerves. In bilatemllyvat;otomized animals, 1.0 mg/kg nicotine f.v. caused fleeting h•rl~rrventilntiony followed by respiratory standstill in expiration, accompanied by excest+ive and continued escitation in the effereutt t~agus (Schmidt and dnl Ri, 1964; dSchmidt, dal Ri and Lendle,19rr1). These reactions decreased when nicotine was injected re- peatedfy. An increase in artorial pressure was considerad not to be responsible for the vagus-ncrve excitation and the aeuma, since both effects persisted when the blood-pressure response to nicotine was completely blocked bv phentolamine. Hoa•- ever, the apnea and stimulation of the vagus nerve were abol- °..ned by hexamethonium (Sclunidt and dal Ri, 1904, 1965; Schmidt, dal Ri and Lendle, 1961) and by procaine (Schmidt and dal lti, 1905), but not by atropine or reserpine (Schmidt and dal Ri, 1961; Schmidt, dal Ri and I_.^nalr, 1'J5-1). ;Fc: further details of these drug effects, see below, 319.1 Afferent stimuli of low intensity applied to the vagtrs caused an inhibi- tion of discharges Iroru the efferent va>;tts, and similarly rr- duced the contiuuous efferent excitation elicited by nicotine; nfferent stimuli of higher intensity gave rise to a contitauout, discharge in the opposite desrendina vat¢tts (Schmidt and dal Ri, 1964; Schmidt, dal Ri and l.endle, 1963). lnjections©f 1- 3og nicotine into the fourth ventrirle also produced regpim- tory standstill and an earessive excitation in the efferent va- gus nerve (Schntidt and dal Ri, 1965). 003. E•(lert of Dnrrebral(ort (•239) 504. E1fert of Stellate GangJfon Ab)atton (239) 503. BJfect qf GlossopharUngeal •\'ertr Sertion 09) 306. E.(fert of 1'pperCemica7Canglion Block (240) [No new data.j S0?. Effect of.\•itotinc on RespiratorU Itr-Rexes (240) 508. Cnrotid SinuP (240). Respiratory stuuulation via nirn- tine excitaticm of carotid-a.inus ehemoreceptors, and/or its elwnnrn fnllna'inn rqmtid t9Pnern•ntinn (24/b-hl, have Arnn been noted in the rabbit (nau•es and Mott, 1939), cat (Shchegoleva, 1961), and dog (Takasaki, Katsuda and \aFa• saki, 1959). In reactive animaL•~ (cats and doAs), howecvr, respiratory responses to medullary (area postrenta) applir,a- tion of nicotine could still be elicited, even after cutting of the carotid-sinw and vagus nen•es (R. A. Mitchell et al., 19Git; see above, 495 and 502. Follo.ring denen•ation of the rarotid- sinus area in both rabbits and cats, the apneic response to nicotine remaiued unchanged (Takasaki, 1956). 1Cr~ylov (1960) studied Ihe effect of nicotine, cyanirle, and aretylcholiue on the chemorecefNOrs of the carotid sintt.~ in cats, the sinus-re9exogenie zone being isolated from the rest of the anintnl, and perfused, and action-currents of the sinus nerve rvg6tered. Addition of fti0 µg nicotine to the periut:ion fluid resulted in a sharp ttimulus to the chemorcceptor.• (in• crease in frequency and amplitude of oscillation) lasting $-4 min, and then gradually d'mrini:hutg. 11•ith pas..,gc of 400 pg/mi nicotine through the sinus, the action-current+ in• creased in frequency and amplitude for about 3 min, and then, with continued flow, sharpl.• decreased, and levcied off ot their initial values or below. On this backfiround, the scn: ititi•- iq• of the chemoreceptors to acetyIcholine a•as no longer effective, but the sensitivity to cyanide remained. About 10 rnin after passage of nicotine wasstopped, the reaction of the shemoreceptots to acet.•lcholine and nicotiote was completely restored. Shrhegoleva (1961) found that the chemorcveptors of the carotid sinus in old eats were more sensitive tu nicotine, injected either i.r. or directly into the isolated carotid sinus, than those in young animals. After denen•ation of the carotid sinus, the same doaro of nicotine did not cause dyspnea Cherrtigovsky (1962) attributed to ICravtchinsky the ob- senation that applications of tampons soaked in 0.2 % nirat- thie to the trunk ot the aorta and to the site of it-3 bifurcation in frog:c affected the respiration: nicotine applied under the trunk of the aorta and its site of bifurcation stopped the respiration, but accelerated it .+hen placed above the trutpk of the aorta. Kulaiev and 13eller (cited by Chernigovsky, 1962) observed cessation of respiration, followed by cardiac arrest, cn applying 100 pg/mi nicotine directly to the vascular labyrinth in frogs. From these and other observations, C'hemi- govsk.• considered that it seemed to estabhsh beyond doubt that the chemosensitive ltroperties of branchial vesseL were immediate precursors c.f thr chrmorereption of the caCotid glomus, and that a clear-cut moqtho),mnetic relationship taren the bronchial arteries and the ckentoreceptor appat•a(w+ of the glomus caroticum in manmtnls is also quite apparent. With respect to the effect of other dtugs on this nicotine action, retrograde injections into the lingual artery of cats of 20-40 pg of the non-barbiturate anesthetic, the diethylamide of 2•tncthot)•A•alh•1•phenosyacetic acid (G•2950fr), dimin• ished the electrical activity in the carotid-sinus nerve indutvtil hr injcrtion of 125 ~ utt nirotine sulfnte (Itrhding, Shnpinp and t;igg, 1962). From esperments on dogs under chloralo.4e- morphine anesthesia, in which nicotine in dorru ranging front just threshold (0.3 µg) up to sulttanta:dntal (100 µft) was ht- jected into the common carotid, and varyinR do>sm of heso. methonium injected i.v., liyck (1961) concluded that hexa- methonium caused a competitive block of the chemoteceptor response to nicotine. The respotue to 100 vg nicotine conld nnt be blocked by m•en near lethal doses of hemmethonium. (Hexamethonium did not block the response to cyanide in any A'.1'•biisot.ropcl•N'•isoamrl• t'' diethrlamfnaethvl• dosage.) utea (Y-280) is also a gatqtlion•bluckinR agent. In dogs under 1>entubarbital anesthesia, i.v. administration of 6 utg/kg 1'-2);6 Produced btr The Gouncil for 0303 0 :Tobacco Research-USA, Inc. 0 0

RESPIRATORY SYSTEM 119 ~ ~ n _• m n. ....:.... ....r....<i . ,,b~ and 2 mg/kg hexamethoniunl completely blvcked the respira• tory effect of this dose of nicotine on the a•hemoreceptors (Gardier and Traber, 19d5). Also in dogs, inf'iltration of the carotid-sinus area with Neohntergan (pyriEatnine) abolished all reflex respiratory effects (I,ecomte and Iio<kam, 1952). In dogs under morphine and chloralose anesthesia, 0.3 ml/kg acetic acid injected into the carotid sinus and glomus blocked the hyperpnea and bmdycandia of a subsequent injection of 10 pg/kg nicotine (PbrssAsz, Such and P6rss4sz-Gibisaer, 1957 a). In "drug•antagonism" experiments in which nicotine was injected first in order of time, high doses of the latter drug blocked the respiratory-stimulating effect of amphetamine on the isolated carotid-sinus preparation of the cat (Reinert, 1957-58). Following nicotiniration of the caintid chemorecep- tats, sensitivity to cyanide remained, but ecetylchoGne was no longer effective (Krylov, 1980). Using an ingenious technique developed by them (see below, 509). Comroe and tiortimer (1963, 1964) fourid that carotid- body hyperpnea produced in dogs by nicotine was greater than that following aortic-body st6mulation. 509. Aortic Reflex (242). 1'oting that previotis investigators had compared the effects of chemical agents upon the aortic and carotid bodies, either by selective inactivation or perfu• sion of one chamorecept:or arrn,a, Comroe and Mortimer (1963, 1964) devised a technique of separating the Aortic and carotid bodies temporally by placing delay-paths (eonsisting of coiled plastic tubing) in both common carotid arteries of anesthe• tized dogs. Thus, drugs injected through a catheter placed In the ascending aorta reached the aortic chemon5ceptors within a second, but required an additional 20-60 we to reach the carotid bodies; and the eltect of aortic•body stimvlation was complete beforn carotid-body stimulation began. In these exyeriments, carotid and aortic pressureceptors remained in. nervated in all animals, and were exposed to approximately the same pressures. Lrs;ng this method of ane$•sis, carotid- body hyperpnesinduced by low (4-14 pg/kg) concentrations of nicotine was found to be girater than that following aortic- body stimulation. In a typiehl result shonn for nicotine in a dog breathing spontaueously, hyperpnea, hypertension, and tachycanlia began within 2 sec (aortic-body response); 60 see after injection, greater hyperpnea occurred, accompanied by bradycardia and hypotensioro (carotid-body response) (Com• roe and Atortimer, 1964). In a fea instances, non-typical carotid•body and sortic-body responses occurred (see above, 313). 510. Chemoreceptor Reflexes From the Lungs; Pulmo- nary Artery t242). llaw•es and \lott (1959) report t adult rabbits injected with nicotine showed a change in breathing which differed considerably from that observed on injection of cyanide. Further hnmlysk indicated that receptors in the lungs were involved in respiratory rerywnrrs following i.v. injection of nicotine. Such injection of 100-400pg nicotine caused an initial increase in rate of respiration, and almost Invariably a decrease in depth. This response occurred much sooner after the injection than that due to cyanide, front which it also differed in that the depth of reapiration was rot incrensed. Moreover, vaftotomy abolished the rapid shallow breathing induced by nicotine. Injection of nicotine into the cavity of the left ventricle in 3 rabbits failed to elicit the rra- pitntory re.rywnse choracteristic of i.v. injections. In 3 animals, the carotid bifurcations were denervated before nicotine was ~ ~. .,...:.i .-...n...• -.. .t:. . .. .t. . • a ... .. .;.... of nicotine, and was abolished by vagotomy. The authmx next sought to determine xhether, in addition, nicotine excited the chemoreceptora of the carotid body. When the dose of nicotine was increased, responses other than the characteristic rapid shallow breathing appeaMd. It proved difficult to carry the analysis of these changes to completion; but, in 5 vattoto- mised animals, there was an increase in both the rate and depth of respiration. This was abolished by denen•ation of the carotid bifurcations; but, in 2out of 3 such animals, injection of nicotine then caused prolonged respiratory• pauses. Takasalti and his associates have relwrted experiments which led them to conclude that the receptors for nicotine apnea lay in the distributed area of the pulmonary artery (Takasal•i. Yokoo and Nagasaki, 1959), more precisely, be- tween the pulmonary-artery bifurcation and the pulmonary vein (Takasaki, Katsuda and Nagasaki, 1959). Iievan and Murray (1963) have presented evidence for a ventilation- modifying reflex from the pulmonary circulation in man. Lobeline injected i.v. into 2D normal subjec•ts caused a ventila• tory depression in most (17), and concotnitant bmdycardia and hypotension in a smaller number (10), these effects occur- ring within the ann-lung circulation-time. These frndinp,e were considered to be strong evidedce for a ventilation-te;tulating reflexogenic area between the large veins and the pulmorsry circulation in man. Nicotine r,va~, said to have had similar action to lobeline on the pulmonary-artety reflexogenic sone (Bevan, unpublished results). In the group of subjects studied by Bevan and Murray, the mean reFpiraton• depression for smokers was 31.4% and for non•smnkers, 1S.1r•c (p < 0.03). The increased response to lobeline seen in smokers was con- sidered probably to reflect rui additive action of lobeline with its pharnnacological analogue, nicotine; although other expla- nations were said to be possible. Sume structural requirements of drugs (including nicotine and various derivatives) which stimulate this pulmonary-artery reflexogenic sone in cats have been studied and presented by lievan, 'Murphy and Hardy (1964). 6I1. Chemoreceptor Reflcxes From the Ear. MietkieN•aki (1956) perfused the vexcels of the rabbit-ear (5co)ated from the animal except for a nervous connection). and found that nicotine acted strongly on the chemorcr•eptots of the rabbit- ear, causing a sudden fall In arterial blood. pre:ccure and weak augmentation, followed by reduction, in re.-lriratory move- ments. In the perlused rabbit-enr, connected with the body only through the auricularis pnagnus nerce, stimulation of these afferent. fibers elicited a reflex fall in blond preTmum and riRe in respiration; 10-•10 µF nicotine had a stimulating action, which, on repeated injection, decreased and 6nally disap- peared (Lembeck, 1967). 512. Chemoreceptor Reflexes From ljte Spleen 1242t); Intestine. Anikina (1961) prepared urethaniaed cats, in which a section of intestine, isolated except for its nervous connections, was perfused via its ve;sels. Addition of nicotine to the 1MrfusinR fluid resulted in r.light stimulation of respira- tion. It is not clear from the rrlxbrt what effect monoiodoacetIe aeid, n•hich was continuously perfused through the intestine, had on this nicotine action. For a theoretical dibcusdon of the influenee of monoiodoacetie and on cbe~,meccptottc, see aI„n•c, 339•F (Chernignvrkii (1943); Lebedova (1954)J. Of some interest here is the effect of nicotine on distension- iw,ensitive nxK•hanorrs•epton, located In the mut•osa of the fmsl) intestine, descrilxd by I'aintal (1957), below, 621. Produced by The Councii for 0003031 Tobacco Fesearch-I;SA, Inc.

0 I i 120 TOBACCO-EXPERIMENTAL AND CLINICAL STUDIES. $IIPPL'.EhfE\T I a.0. VCfCL.q; a.u~..w~..~..~.'...., !: ! •••••••• nn ottteri- ments in dogs (descri~ed above, S50-D, injection of 150 pg nicotine into the arterial side of the perfttsion circuit of the isolated central nervous system caused intense respiratory stimulation in the r+ecipiehtt animal, lasting 2-4 min after each injection (Taylor and Page, 1951). G. Schrgtidt, dal RS and Lendk: (1964) considered that the respiratory effects of nico• tine observed by them after i.v. injection of nicotine in rats (see above, 492 and 50) might have been induced by an excitation of chemosemsit.ive areas in the cen,tral nervous sys• tem. The respiratory responses in dogs and cats, mediated through the soper8cid ) chemosensitive areas on the medulla, have been described by R. A. lfitcl,ell and co-morkers (1063), above, 495. 515. Sneezing. Application of nicotine to the na3al mucous membrane of guinea pigs caused frequent rneerirsg (Janes6, Jancs6-GSbor and Takdts, 1961). This "nicotinic sneezing" was inhibited by ganglionic blocking agenta and nicotinolytic drugs, and also by repeated application of nncotine; but these substances were ineffective against sneezing, induced by cap• saicbte IN-(4-hydm.%)•4metho..ybenzyl)-S•enetbylnon-trans• 0-enamide) orfonnalin; and sneezing induced by a light touch n-ith a nylon thmad upon the nasal mucasa was not disturbed. (Inhibitory effects of the antagonistic dntgs.nianifested them- selves by diminishing the number of sternututory discharges.) The sterautatory action of nicotinic stimulants was said to be mediated by a distinct neural me.chanism,' which could be selectively blocked by ganglion-blocking and nicotinolytic substances, whereas, in other respects, sensibility remained unimpaired. In rats, the development of a refractory state could also be achieved by i p. administration of nicotinolytic agents. hocal apolication of 0.2% physostigmine to the nose of guinea pizs also blocked the stimulating action of nicotine. 515. Coughing. G. A. Harrison (1962) described a method for comparing the duration of r.etive respiratory responses (coughing, laryngeal spasm, ot breath-holdir.g) tN irritation of ti:< respiratory tract during anesthesia with different agenta; the irritant stimulus was one puff of cigarette-smoke applied at a level of anesthesia which was approximately the same for all patients (who w•ere free of cardiovascutar and respiratory disease, and who did not smoke more than 20 cigarettes per day). The reflex responses were found to be more prolonged with cyclopropatte and thiopentone than n ith nitrous o%tide alone or halothane with nitrous oxide. In 5 cases, i.v. injection of 1.2 mg atropine sulfate before stimulation failed to modify the responses during cycloptropane anesthesia. $16. Other ReApiratory ReBexes (242). Stubnng an in. hibitory reflex from the nose in rabbits under urethane nar- eosis, in which impulse activity in the large mucrleA of the legs and in the trspiraton muscles was recorded, Franken. haeuser and Lunden•old (1949) found that blowing cigarette. smoke on to the nose caused refiex inhibition of "spontane"s motor activity" in 60-70% of ttials. 517. Effet•t of Adrenaa Renwtnd (243) In dogs, bilateral adrenalectomy almost prevented the ptr*,-or response to inhaled cigarette-smoke, but did not alter the respiratory effecLs (de \Ioura, 1959). 518. ReapEratorp WErt of Nirotiae in Alteno Stat.ea of Oz•rfgrn and Carbon.Uiozide Tenaton (243) The threshold dose of nicotine to cause nicotine apnea in rats was lowered from 20f) µg/kg to 100 pg/kg by 25-30.^o carbon dioxide (isngntcBea et at., ivaoi. S19. Influence of tNlur brupa (294) The poly pnea resulting from i.v. injection of 50 f.g/kg nicotine In chloralosed dog- was not affected b) prior adminis- tration of asapetine (J. \fereier et al., 1956). The apnea in rats injected i.v. with 1.0 mg/kg nic•otine remained uoaifected by I mg/kg phentolamina' (G. Schmidt and dal Ri, 19Bt; Schmidt, dal Ri and Lendle, 1964). Pretreatment with reser. pine had little or no eHect on the apnea or vagal excitation induced by nicotine in this specice. Tbis was not the case with other species, however. Unanesthetized rabbits reacted to i.v. injectiwt of 1 mg/kg nicotine acid tartrate with a brief tachy. pnea, followed by a short ri,pnea, and theu returned to nuntuJ respiration (J. J. Reuse, 1960). The same animals were then treated with reserpine (for details of dosage, see above, 130-C), and, 24 hours later, two typee of results were ob• served. With moderate doses of reserpine, the tachypnes following nicotine injectioh was still manifest, but the apnea was absent or less marked. One month after reaerpnic, the responce to nicotine had returned to normal. After strong doses of reserpine, an initial tachypnea a•ns followed by a permanent spnea and death of the animal. The modifications in the respiratory action of nicotine were considered to be due to a central action of reaerpine. Atropine bad no effect on the respiratory apneas caused by nicotine in dogs (Tal:asaki et al., 1959a) or in rabbits or cats (Nakano et al.,1957). In rats, atropine failed to show a specific inhibition mf nicotine apnea, extremely high (10 teg/kg) doses being required to reduce the effect of nicotine (Schmidt and dal Ri, 1964; Schmtdt, dal Ri and Leudle, 1964). Nicotine (p:+mary or refies) apnea was blocked by tetra- eth)•lanunonium (TEA) in rabbits and catas (\akara et al., 1957) and in dogs (Takasaki et al., 1959a). Nicotine apnea was also blocked by heaan.ethoniurn in rabbits and cats (Nakano et al., 1957) and dogs (Takasaki et al., 1959a) ; and, with both TEA and hexamethoniurn, larger doses were ree quired for cats and rabbits than for dogs. In rabbits, adtr.inis• tration of 18 mg/kg hexamethoniunr abolished the respiratory stimulation elicited by 140-525 µg/kg nicotine (Mott, 1963). In rats,.be.®methoniam in high (10-2D mg/kg) dosage i.v. exhibited an antagonism to the apnea and vagus excitation Induced by nicotine (Schmidt and dal Ri,1961; Schmidt, dal Ri and Lehdle, 1904); these effects of i.v. nicotine were also completely abolished by an injertion of 150 pg hexametha alum into the fourth ver.tricle (Schmidt and dal 13i, 1905). In dogs, 2 mg/kg hexamethonium i.v. completely blocked the respiratory eftect of 20 pg/kg nicotine on the chemoreceptors; the respiratory response to this dose of nicotine was rrdured by 6 mg/kg P•268 Lv. (Gardier and Trsber,1965); see above, S0& Following atitninistrstion of S00 mg/kg cotittine to doga, i.v. administration of 125 µg/)tg nicotine produced au inctra~e in respiratory rate (Borselleca, Ilon•rnan and Mcl:emris, 1962). Slow injection of 10 mg Neoantergan (pyrilatnine) into the carotid artery of dogs markedly reduced the hyperpneie responfie to subse.quentlyinjected 100 pg nicotine (Lecomte and Roskam, 1952). Neither nicotine nor diphenhydramine apnea was blocked by procaine in uretbaniaed rabbits and cats (Nakano et al., 1957). In dogs anesthetized with urethane and morphine, nicotine apnes was blocked by procaine t1'altasaki et al., Produced bv The Councii for 0003032 Tobacco fiesearch-USA, Inc.

>rESPIItATORY SYSTEM 121 1959a). Procaine imatilled into the pericardial ese did not block mcotme apnea, except in the following cases: ih rebbite, the nicotine apnea was inhibited or blocked by procaine in a relatively small dose, through paralysis of the p'hrenlc nerves; in dogs, the nicotine apnea was inhibited or aboUshed by pro- caine in large doses, due to absorption of the procaine into the svstemic kirculation (Takasaki, et al., 1959b). The spaea and stimulation of the vagus nerve produced by i.v. injection of 1.0 mg /kg nicotine in mta was completely abolished liy 50-100 r,g procaiue injected iuto the fourth ventricle (Schmidt and dal Ri, 1*5). \'•Pentameth3•lene-Q-pheno:Qisopropylamine Sbol?shed the presarr effect of 50 ug/kg nicotine in chloralosed doge, but the nicntine polypnea persisted (Polonovski, Schmitt atid Pelou, 1U53)• Neither nicotine nor d'qrheuhl•dramine apnea was ebol'ished in urethanized rabbits and cats by premedication with irnid- szoline (Nakano et a4, 1957). Following administration of pbenethyldlguanidine to dogs, 1he effect of nicotine on respiration still persisted (AsbL•ar, 13urrier and de Peralta Ramos,1958). 520. Pflrettlt Nerve Qnlt Diaphragm (242) Nagasaki (1953) recorded the action-current of the phrettic nerve of rabbits following i.v. itqection of nicotine, aud noted that the diaphrngm,, receiving continuous impuises from the respiratory center, fell into a tonic contractive state ("byperp- aoeic apnea"); then, as periodicity of the impulses teturned, flyperpnea followed r23a). In a subsequent report, TAknsaki. Yokoo and Nagasaki (1959) described phrenic necve dis- charges in cats and 'dogr in response to nicotine adwiniatta- tion. Followbrg puhaonar.Y-artery injeetions of 100-300 µg/ kg nicotine, the phrenic discharge was markedly decreased for 10-20 sec, or else disappeared completely; thereafter, t6ere was a marhed increase in frequency and , amplitude. Injection of the drug into the left auricle caused only an increase of pbrenic discharge, without disappearattce. Using seiesthetized male 11'tstar mts under artificial respiration, G. Schmid:, Erdmann and dal Ri (1963) recorded action.poten- tials in phiznic nerve and diaphragm. After i.v. injection of 1 mg/6•g nicotine, central apuea appeared, then diaappeated after about I min of regular, somea•hat Paevaled phtenic ac- tibn; the romuscular transmission remained unchanged. Additional t.gectionsef nicotine no longer produced a change ht central respiration (tach.phylaxis), and only a slowly in- creasing neuromuscular paralysis took place. After i.v; injec- tion of 7 mg/kg nicotine, the physiologic action of the dia- phrabnn practlcally diisappeared; a mechanical contraction no lohger ensued. With supratnaximal electrical stimulation of the pF,renic nerve with imjrulse-ftrquency series of 30 inypultes/sec and 50 impulses/sec, however, an almost unaffected muscular action resulted. By simple proof of indirect muscle irritability under supraronmmsl stimulation conditions, the aut.hors de. duced a central respiratory paralysis; but registration of cen- trAl respiratory action showed, instead, that thc central respi• ra8ion bad been somewhat stimulated. 1Yhen the intensity of stimulation of consrant frequency was increased atrpnhse, the neuromuscular ttamanission of submaxtintal stimuti was more readily blocked than a suptaamaeimai stimulation: iV•ith nirntine doves greater than 10 mg/kg, muscle respone to s•ipramaaimal stimulation failed. These arorltets stated that the facld that only part of the nerve-fibers are stimulated in physiological innervation and in submaximsl stimuEation, while all of the nerv•e-fibeis take part in a supramdaimal RtimnlatioPl. nffrrn an tivit;• of submaximal and supramaximal stimulation^to bloc6'ingageats /e.g., nicotine/ of neuromuscular traasmiaviuu. Schmidt, dal Ri and Lendle (1904) later studied the a,.ffecQ of nicotine on the electrical activity in the efferent vagus and phtenic nerves of the rat; and their results led them,t.d eon• aider that the effects of nicotine observed (see above, 492, 502, 513, and $19) might have been induced by an e.teitution of cheaiosemtitive areas in the central nervous system. The isolat,zd mammalian phrenic-nerve-diaphragm prep- antion has been utilized by a number of workers to study either neutomuscular transmission per se, or else the site of action of neuromuscular blocking agents on the nerre-rmo,tor end•p'late--amuscle system. The activity of nicotine a•as tRSted by J.,T. >;ami)ton l1Jd'3) in a series of experiments with the rat phmnir; nlen•e---~diaphragm preparation at four different pHs, and the results indicated to him that there was a quan• titative rel'ation between activity and degree of ionaze.tion. (For the theoretical implications of this, trec above, 227.) In these eaperiments, organ•bath pII was varied from 6:73 to 8.05, and nicotine monomethiodide as well as riicotine tras tested;,alod the findu`ngs were taken to show that it 4 the univalent nicotinium ion, rather than the un-iottieed base, which, acts at the nedtromuscular junction (Barlow adtd Hamilton, 1962b). A number of isomers and homologues of nicotine were also tested for their ability to block trrflnrrmis- sion in this', preparation (Barlow aud Hamilton, 1962a); for details of which the rea{ler is referred to the original tirticle. Barlow and Hamilton (1965) presented a quantitative c.om- parison of the activities of the (-)- and (+)-isotqeri+ ;of nicotine at a variety of sptes in the peripheral nervous~s On the rst-diaphrsgm preparation (block), a ratio of p.96 0.08 was obtaaned, based on 4 results-the only site of those tested (aside from inhibition of ox red-cell acetylchofiu• estetgse) at which the (-)•isomer was not considerably the more active or effective (see below, 978). In their comparison of the pharmacological activity of nicotine and related alkaloids occurring in cigarette-smoke, Al. S. G. Clmmr•k, Rand and A'anov (1965) dissected out guirtea- pig hemidiaphragnu together with tLeir phrenic nerves, which were then set up in an orRan-bath and stimulated with rectangular pulses of supramaumal strength (1-5 V) and 100 µsec duration at the taate of 6 pulses per min. Effective concenttstions of nicotine or the other active alltaloi,fs eauaed a slow•ly developing blockade, the rate of progression of which was proportiotdal to the concentration; for purpose of com- parison, the degrre of blockade was measured 5 min after adding the dt•tag. In this comparison, with the blockade-ttc- tivity of nicotine taken as 100, the activity of nornicotine was found to be 73; metanicotine, 0.8; anabasine, 50; m)rowttiae, 12; nicotyrine, 0.8; 2:3-dip.•ridyl, 4; 3-metbv1-6-(3-p,rrid~9)- tetrah~•dro-1:2-oxazine, 0.7; dih~•drometanicotine, 0.8; .Y- nmt hylnnabasine, 3.5; cot ininp, 0.t:; nornirut yttine, 0.9; pyrid,vl methylketone, 3; pyridyl pnopylketouc, 3. Some cxpetiments were also performed on the rat phrrnic•nrn•r•diat-hmcm, using cotlne, nomicotine, annbasine, and n., 2~minr. Rcla- tive Iw ncies differed from those obtained with the Intinea- pig preparation; norninotine and anabasine were equipotent, and about twice as potent as nicotine, while myosntine was about one-third as potent as nicotine. M. F. Cuthbert (1964) reported relative potcncies to nicotine for tytamine methiudide, dopaminc u,rthnbromide, and notepinephrine methochloride on rat tdin•niri env- diaphragm preparations. Following degenexative section (10-71 days) of the phtenic Produced bv Th Councif for 0003033 Tobacco Rcscarck-USA, Inc.

122 TOBACCO-EwPER1AIEATAL A\D CLINICAL ltTUl)1F.S. SUPPLE.IIETT I nerve, isolated rat.diaphragm in Tytode's solution responded to nicotine by contracture (lihoola and Schacbter, 1901). Cambridge (1961) also reported that denervated rat-dis• phragm muscle responded to nicotine by a contraction, AlcCarty and Chenbweth (1962) found that certain poly- propylene glycol adducts synergir.ed with nicotine to depnbzs response to nerve stimulation of isolated ra4phrenic•nerve- diaphragm preparations. (The compounds tested were poly. propylene glycol polymers in which the terminal bydrox)l group:, of polypropylene glycol were replaced with a diethyl. enetriamine moiety.) Using the guinea••pig phrenic-nerve-.diaphragm prepara- taon, Beani and co-workers (1964) found that 20 µg/ml nicotine did not modify acetylcholine release from the hemi- diaphragms at rest or ondirectly stimulatkd at 59/sec, leading to the conclusion that the neuromuscular blocking action hed only a post-jt?nctiodal origin. Beani and Bianchi, in a series of papers, attednpted to study the nature of nicotine and other neuromtrceulnr blocking agents. The antagonism by neostigrnine of the effects of nicotine, id-tubocurarine, hex• amethotdum, pempidine, and dimethy lphenylpiperazinh6m iodide (DbiPP) was mid to be in accoidance with the by. pothesis tbat an0st of the nruromuscular blocking agents have a much more complex mechanism of action than is so far believed (Bea,ni and Bianchi, 1964a). NVhen responses of the preparation to intrtlvhscular injection of ecetylcholine were recorded during the establishment of newomusculat• block produced by nicotine, the acetylcholine twitch was found to be reduced praportionAteh• with the indirect contraction from nerve stimula,;,ibn, leading to the concittsion that the site of action of nicdtime is chiefly at the post-junctional membrane (Beani and B9anchi, 1964b). Interactions of nicotine, d- tubrrurarine, :)AiPP, hexamethonium, and pempidine were studied in an attempt to ascertain if the combined effects could be inter~,preted on the basis of one or more characteristics of their individ"tal actions; but this proved not to be the ease (Isaani and Bianchip 19fi9c). 321. AIecharifs'm oJ Aetion of tAe Retpiratory IWccls of A'icotine (lh5) The classical sequence of respirator; events following administration of niiot,ine to small tnamrlralsruay be descn'bed as an initial brief snest of respiration (°pitnary" or refiex apnea), followQd itf, turn by stimulation, a secondary, pro- longed period of apnetl, depression, and eventually, with lethal doses, by cessation of respiratory movements and ultimate central paralysis (oh-e Silvette et al., 1962; P. S. Latson and Silvette, 196Sb). Respiratory failure in nicotine poisoning has been dettmnnt.nted uneqmvocaUy to be due to peripheral curare-like patal~sis of the respiratory muscles, during which the tespiratory center continues to functSon. With the other events of the nrspiratory picture folloa•ing nicotine, boa•ei•er, the renpiratoiy center appears tn be ui.~olved, either directly or eia nicotine action on nicotine-sensitive chemoreceptors in afferent branches of respiratory reflexes, for example, in the carotid body (b98), aorta (S09), auricles (2f2b), lungs and/or pulmonary dt•tery (51D), spleen (242a), intestino (312), and ear (511). Nicotine may also stimulate respiratioa through stimulation of cerebral chemotrceptors (S13). Reflexvs may also be invdh•ed in the respiratory effects of nicotine, for example, from the pulmonary deflation receptors via vagal afferent fibers (Paintal (1955) (242b)J, from the tonpue via the aublingual nerve (242a-b), and from the ear via thr great auricular nerve (511). The inhibitory reflex from the tio.v excited by inhalation of tobacco-emoke (242a), and the Producc;f hif Th-? Col,nci{ for TobaGco Rcscaidi-liaA, Ittc. coughing reflex caused ny ctgarerrr-atuuee tiuusiew,, ii,v,:j, are not specific nicotine effects; and thk "nicotinic sneezing" of feuinea pigs following application of nicotine to the na~al mucous knembrane is said to be mediated by a peripheral neuromechanism (Jancsb. Jancsb-GBbor and Takdts, 19G,1). Primary nicotine apnes has been termed both "expiratory" and '•hy1serlmeic", deftending upon the animal species uKed (dog, cat vs ratrbit) (Nakano et al.,1f).57) (246b). The central component of this primary apuea has been variously inter. preted; the condition was said to be due to a paralysh, of the respiratory center, and again, not due to a direct effect of nicotine on the center, but rather to ®n effect on receptors distributed in the area of the pulmonan' artery. Takasaki d.nd his associates have extensively investigated the mechanism of the reflex apnka caused by nicotine. Takan-d.i (193G) fir,.t postulated that the epnea was caused by activation of the pulmonary respiratory chemoreflex; and Takasaki, Tokoo and Nagasaki (1959) reported experiments which led them to believe that the receptors for the nicotine apnea lay in the distributed area of the pulmonary arterv, in the lung between the pubjronary-arier•y bifurcation and the putmonary vein (Takasaki, Katsuda and Nagasaki, 1959). Experiments indi. cated that nicotine spnea was not due to the direct effect of the drug on the respiratory center (Takasaki, Katsuda and Nagasaki, 195f1), nor were receptors in the heart resfwnsible for the respirart.ory reflex caused by nicotine (Takasal•i et al., 1959b). After bilateral vagotomy and denervation of the carotid• sinus arm both the prlmarq• nicotine apnea and respiratory stimulation by nicotine disappear, leaving only the secondary nicotine apnea unaffected. The mechanism of secondary nicotine, apnett is still not thoroughly, elucidated, but it has been suggested that it has a central origin. Them seems little doubt .hat nicotino may also exert its classical artion--stimulation, foUoned by depression- directly on the respirr.tory "ceater" or "centers", although, from lack of data, it is difficult, if not impossible, at this time to integtate most of the teapiratory phnnrtacology of nicotine with current cbncepts in reslrirator}• physiology. Central stim- ula+ion of the vespiraton• center nwy be deduced from the re- sults of compaqat§ve experiments using small and large doses of nicotine, in animals with intact or severed carotid-sinus nerves (240b; 247a). Also, Schmidt, F;rdmann and dal Ri (1903), recording action-potentials in phrrnic nerve and diaphragm in rats, demonstrated that the central respiration was some• vrhat stimulated, not pamlyeed, following Lv. injection of 7 mg/kg nicotine, after which dose the physiologic action of the diaphragm had practically disappeated. With stnailer (1.0 mg/kg) , doses of nicotine, fleeting hyGierventilation, followed by rrspiratory standstill in expiradon, and accompanied by excessive and eontinued excitation in the efferent vagus, were observed; andl these effects of nicotine might have been in- duced by an excitation of chemnfenFitive areas in the central nervous st•stein (Schmidt and dal Ri, 1984; Schmidt, da) Ri and Leitdle, d9B4). lhrther studies by Schmidt and dal Ri (1965) indicated that, in the rat, the "central" effects of high doses of nicotine, evidenced as apnea and stimulation of the vagus s6en•e, were elicited from a site adjacent to the fourth ventricle, and that the pharmacological properties of the receptor elenients there are similar to the X•receptors of vegetative )tatiglia. According to H. Schmitt and HElAne Schmitt (1903), in• jcction of nirbtine Into the diencephdlon, mesrm•ephalon, or medul6 ohkrriRats had no eNect on rrspiratory movenrent- In anexthetised cat~ (.ee above, 153). In Iheir study of respim- 0003034

RESPIRATOkY SYSTEM 123 tun teatxrue,ea meuwmu uuuugu u.r euNu...,... .~. ._ tive areas on the medulla of cats and dogs, R. A. Mitchell and co-workers (1963) found that direct application of nicotine on the floor of the fourth ventricle in the region of the area postrema generally had an inhibitory effect (decreased rate and ttrial volume), and thcv rt+ponces could not he elicited by topical application of the drug to other areas of the fourth ventricle, dorsal medulla, or e%traventricular pnrtion of the choroid plexus (see above, d9S). Central paralysis of the respiratory center as s late result of nicotine has been demonstrated in cross-circulation experi- nunts; but, in the intact atimal, the peripheral curare-like paralysis of the respiratory muscles invariably occurs before crntr.+t reapimtore paralyAs or failure (F.. Fischer et al., 19601. 522. Innapletual Preasure; Putmonary Reaistance (:Intniala) (247) No change in intrapleural pressure was observed in 26 ex- periments on 13 anesthetized dogs receiving i.v. infusionc of nicotine at a rate of 20 µg/kg/min (R. K. Larson and \fur.*ay, 1963; Larson, Fukuda and \lurray, 1965). Arterial pH, Pco, and Po, , obtained during the initial control period and mid- aa,v through the nicotine infusion, showed no changes which could be attributed to the effects of nicotine (Larson, Fukuda and Alurray, 1965). In dogs anesthetirxd with chloraloce-urethane, paralyzed with succinylcholine and artificially ventilated, ebemoreceptor stimulation by injection of 2-10 µg nicotine into a carotid artery caused a decrease in volume in a by-passed tracheal segment in each of 9 artims Is (range, -3-31 qo), and increased total lung resistance distal to this segment in each of 3 animals (range, +42-57 %); cooling or cutting the vagi always blocked these effects (Nadel and 11'iddicombe, 1961). For an account of the effect of nicotine and smoking on pulmonary function in man, see below, 523. 523. aert of Nicotine and Smo6* on Pufmonary F'unclion in Alan (248) Among the "Guides of Evaluation of Permanent Impair- ment" published by the A. Nf. A. Comnuttee on Rating of Mental and Physical Impairment lcalled hereafter in this arction, Conunittee (1965)) is one on "The Respiratory System" (JA.XIA /9¢. 919-932, 1965), which contains a fair amount ot descriptive and critical material on certain pul- monarydunction tests. There are many such tests, as the Committee pointed out; and the reader will dkscover that a wide variety of them has been used in the attempts of even more numerous workers to evaluate the effects of smoking on ventilatory function; so many, in fart, that one finds it dif6cult, if not impossible, to collate or correlate, in any meaningful way, the findings of the many investigators; and it is to be hoped that future workers will find it possible (as it is surely desirable) to limit the number and variety of pul- monary-function tests and to standardize procedures, testing as w-ell as smoking, to the end that a useful consensus can be arrived at. The need for such a consensus is apparent from the conclusion of the USPHS Surgeon General's Advisory Com- mittee (Smoking and Health, 1964, p. 292) that the relation- ship between cigarette,amokinp, and abnomtal results of pulmonary.funetion tests is more difficult to evaluate from the published surveys than is the relationship betwren s3-mp- tnnts and cigarepe-amoking Isee belmr, 1261•Aj. The Cotn- t tittee's (19R'n) comments are thus welcome and valuable. It rccommends the use of not lesa than two of the following tests of ventilation: (I) 1-ser forced espiratory volume (FE:\'t,e); ~- ......u ~.. . . . . ~ ...... voluntary ventilation often termed °marmum breathing capacity" (\I13C). These testi, the Committee said, should be reported in liters or in liters/min. The first should also ba expreu.rd as a percentage of the predicted value for FhV, (Tables 2 and 4 in the Committee reportJ; and this percentage is reconunerded for rating Iturhoses. The third of the recommehded tests w•as said to be mm valuable one, which usua0y correlates closely with complaints of d}-spnea. Vital capacity (YC) was not renmimended for rating purposes, sinre the result mhy be normal in those xith se.•ere respiratory irnpaimient=-and, by extension, in smokers showing respira- tory signs atnl/or flniptam- (see below, li;6l-A). The C'omniittee (1965) also recognized that there is a wide variation in the resvlts of tests of ventilatory function among normal individuals of the same aex, age, and height, and that the tests themseh•es have certain Iimitations. Performance may be affected by the subject's physical condition (disease or training), and also I-a consideration of more than usual importance in comparirons between smokers and noa-smokers and in acute smoking-tests-) by his motivation and co-opera- tion. As to the latter, other writers have warned of the pos- sibility that some smokers having smoked or pufled on a cigarette immediately before being given a pulmonan•-func- tion test might therefore have had their mea-cured ventilatory efficiency significantly influenced (13alchiun et al., 1962), thus giving reaaon to regard somewhat crytically the reported statistical differences in findiugs in light and heavy smokers (Carlens and llahlstrom,1A64). On the other hand, the diBer- ences In results of pulmonary-function tests between smokers and non-smokers were said to be greater than can be accounted for by acute effects from a recently-smoked cigarette (Smok• ing and Health, 1964, p. 293). In addition, the non-smoking environment must be kept in mind; and Spicer and co•w-orkers (1962) have pointed out that it is approptinte to remember how hazardous it ih to assess pulmonary irisufGeiency on the basis of a few measurements performed on some arbitrary day in a subject's life (see below, 127i). The tests or measurements used by the several investigators to amss the acute or chronic effects of smoking on ventilatory function follow: Vital capoeity (VC), described V the Committee (19&5) as the largest volume ot air measured on complete e.epiration after the deepest inspiration. w•itaout forced or rapid effort (Giusti et al., 1960; Sw•ann and Hatch, 1960; Rothfeld et al., 1961; Guidstnith et al., 1962; ]tarvonen, 1at12a; ,lartt, 1952; Revotskie et al., 1962; t1'eiss et al., 1992; Zwi and Goldman, 1962; Zwi et al., 1954; Bouhuys, 1963b; Cander and lilu- menthal, 1963; Chevalier and Iaumholz and co-workers, 1963 et seq.; Hensler and (aron, 1963; l.ibdnv, 1963, Mclmne, 1963; Hyatt et al., 1964; Rasev et al., 1964; TersioRlu and Cinemre, IQ64; Tuberculosis Institute, 1961). l'ital caparfty, eTpired or erpirafory (F\'C) (1C. Shat» ro and Paticm.ou,1962; Shapiro et aL, 1964; R. IC. Larson, 1963a,b). 1'ital capatftu, I• or 3-sec titned, or forrrd ():1'C), defined by the Committee (19GR) as the vital capacity lmrfotmed with espiration as forceful and rapid as )wsAblc (11iKdetson and Kohan, ll9fi0; Rothfeld et al.,196);11artt,1962; Shnotiason,1902; Chevalier and I:rurnhols and co-workers, 1963 et seq.; Hensler and Giron, 1983; R. K. l.arson, 1963a,b; Oshima et al., 1964). Total lung ropacity (R. H. Wilson et al., 1960; Chevalier and 1Crunthola and co-wrorkers, 1962 et seq.; Martt, 1962; Hensler and Giron, 1963; Zwi et al, 1964); trsrfnl lung eoRnru (Ccl. Ierino and 1lillin, 1962; CChevalier and ICrumhola and co- wrorkers). f ror'ird b; Th3 Ceunc;f for 0003035 Tobacco Reseaidi'uSA, Inc.

i 124 TOBACCO-EXPERIMENTAL ANI) CLINICAL GTl1DIEF. SUPPLEAtE1T I Naaimum breafAiny rapacfly (N1BC), to wtuch term tne Committee (1965) preferred masin,um uofanlary ventilation (JiVV), described as a volume of air which a subject can breath with volunt.ary niasimal effort for a given time (15-20 see, if possible, equated to 1 min); this was said to be a fatigu- ing test, requiring considerable muscular effort on the part of the subject or patient, but nevertheless a valuable one (I. T. T. Higgins et al., 1959; R. H. Wilson et al., 1960; Rothfeld et al., 1961; Chevalier and Krumhola and co-a•orkers, 1962 et Feq.; Gandevia, 1962; 11'. Shapiro and Patterson, 1962; Shapiro at al., 1964; Zwi and Goldman, 1962; Zn•i et al., 1964; Cander and Blumenthal, 1963; Chosy ct al., 1963; Hensler and Giron, 1963; Libore, 1963; J. R. Webster et al., 1963). lndircrt maximu.ra bre.klhirg rapacity (I\l1iC) (n14wn and Gilson, 1960; 1. T. T. ?3iggins and Cochrane, 1961; Higgins and Oldham, 1962; Lintfeman et al., 1964). Ezpiratory volume, fohted (W. Shapiro, 1960; Shapiro and Patterson, 1962; Rogan, Ashford et al., 1961; Gocke and Duffy, 1962; Bouhuys, 1963a; Brinkman and Coat.es, 1963; Tuberculosis Institute, 1964) or timed (Wilson et al., 1960). The latter is generally e.vpressed as the 1-second forced ex- piratory volume (FEV,.a, that is to say, the volume of air exhaled during the performance of a forced vital capacity in the frrst sec (Ashford et al., 1961; D. O. Anderson and Fenis, 1962; Balchum et al., 1962; Gocl:e and Duffy, 1962; Gold• smith et al., 1962; Revotskie et al., 1962; Simonsson, 1962; Brinkman and Coates, 1963; Chosy et al., 1963; Gandevia, 1963; R. K. Larson,1963a,b; McLane,1963; Weisset al., i963; Hyatt et al., 1964; D. C. Morgan et al., 1!!64; M. Pa5 ne and Kjelsberg, 1964; .1nde,rson et al., 1963); but occasionally FEVo.,, (I. T. T. Higgins et al, 1959; Olsen and Gilson, 1969; IS.aroonen.1962a; IAmine,s et al., 1964); or even the 3rd•sec FEV (R. K. Larson, 1963a,b). According to the Committee (19ti5), FEV,.o/FVC, or the 1-ttc forced earyriratory volume expressed asa percentage of FVC, does not q•iantify accurately the degree of obetrvctrve airway disease. Maximum expiratory volume of the second expimtion (Giusti et al., 196t)): erpir- atory rear7ur uolu»ae (Chevalier and linmiholz and co-workers); and nuuimal expiratory pressure (Zwi et al., 1964), are appar- ently little-used masniements, and thus non-comparable, nor are inspiratory capacity andJor fnspirafary re8ertr twlunie (Chevalier and Krwnhmlz and co-n•orkera). Espiralory,{lo1C-rate (Brit MM. J. $.' 9i 3-9T9, 1961) is usually eqxmxf in some temporal y dationship to the stage oi e.vpira- tion, for example, mid-expiratory Bow-rate (R. K. Larson, 1963a,b);or ma,xsmaf mid-expiratory,Rom rate (ININiF) (S4igder sonand Kohan,1960;Bower,1961a, b; BrWcman and Coates, 19t33; Hyatt et al., 1963; R. K. Larsou, 1963s, b; Zwi et aL. I964); or erpimMry ,(tam-rate during tke third quarter of maai- mal jorced ezpinQtion (Franklin and Lowell, 1961; Zwi and Goldman, 1962; Zwi et al., 1964; R. K. Larson, 1963a, b); or rNOxtmal (or pe¢k) ezpfralory ftourrate (A. L. Fliek and Paton, 1959; Brit. M. .f. R: 9i3-979, 1961; Hallet ar,d llartia, 1961; Fletcher and Tinker, 1961; Read and Selby, 1961; D. O. Anderson and Ferris, 1962; Chevalier and Krumhola and coworkers, 1962 et secj.; Karvonen, 1962a; C J. 1liartin and Hapet, 1962; Mork, 1962; ( ander and Blumenthal, 1963; Gr+egg,1963; R. K Latson,1983a, b; Speizer and Fer+is,1983; Weiss et al., 1963; Zamel et a1.,1963; Zwerdling et al., 1983; HaM.horne,1964; Pelter and Thomson, 196A; J. T. Sharp et al., 1964, 1965; Anderison et al., 1965); or terminal erpiratery flotr-rofe (J. T. Sharp et a1.,1965). Fairbairn, Fletcher, Tinker and Wood (Thorax 17: 165•174, 1962) reported that the Peak Fbtv Meter appeared to be a less satisfactory screening test than the forced expiraton volume. In a comparison by R, a e-°•-a vnh,mo and r"k flow in clinical practice, the Peak Flow Jfeter seemed to be a more sensitive index in measuring a response in pulmonary-funetion tests in patients with oardiac and pulmonary disease. These findings and conclua[ons may or may not be relevant in the comparative assessment of pulmonary function in smoken and non-smokers. Residual vofume (Chevalier and I:rumhola and co-n•orkerr, 1962 et seq. ; Dlartt,1962; Heenrler and Giron,1963; Zwi et al., 1964), often expressed as the ratio to total lung capacity (residual volumeltotal lung capacity) (R. H. Wilson et al., 1960; Chevaliet and Iirumholz and co-w•orkers; Bouhuys, 19fi3b; Hyatt eta1.,1964; Zn-i et a1.,1964). Fundionaf reaidual capacity (Rothfeid et al.,1961; Chevalier and Krwnbolz and co•crorkern; Dauwiseau et al., Iftba, b; Lovej.,y si•d naiitre- band, 1963, Zwi et al., 1964). Several d•orYers have studied more sensitive tests of airway resistance and htng compliance. It reemed preferable to Comroe and Nadel (1962) to measure airway resistance directly, rather than make inferencea from changes in vital capacity. They themselves and their associates used the body plethystnograph method, which measures airway re-i„tance separately from any changes in pukvmnary+ti4sue resistatce or pulmonary cwmplianee, and they converted their data on airways resistance into airuny ronductance, which is the re- ciprocal of resistance (Nadel and Tierney, 1960; \adcl, Tierney and Comroe,1960; Nadel and Comroe,1961; Comroe and \a9el, 1962; also Lovejoy and Dautreband, 1963). Airway resistance (Hunailter and Buhlntann,196D; W. Shapiro, 1961, Cellerino and Billia, 1962; Chevalier and KrumlM)lz and co•workera,1962etseq.; Damo6esu et aL,1962a, b; Guerrant, cited by Swinetord, 1962; Lovejoc and Dautrcband, 1968; Zamel et al., 1963). Pubnonary compfiana (Zwi et al., 1941; l:rumhols, Chevalier and Ro.-, 19Gia). All the above measurements may be expressed quantits. tively, and thus (in principle, at least) the results of workers using the same method of "uation under similar test-oondi. tiong should be comparable, which is not the case when the changes in pulmonary function are expressed in general or functional terms, such as ventilation or ventilation "halues" (Guisti et al., 1969; Carlens and Dablstrom, 1963, 196•1) or ventilatory capacity or requirements (Gandevia, 1962, 1963; Fletcher and Tinker, 1961). Stili other workers have used expresRions in which quatn:tation i,: inherent, such W total air exchange (Guerrant, cited by Saineford, 1962) or air- velocity index (C. J. Martin and Ilallet, 1962), but tvhich nevertheless cannot be correlated w ith the more usual ancas- urements of ventilatory function excFpt in a general way. This survey of pulmonary tests and the method of presenting results Illustrates the desirability of future standardisation. In addition to the recommended tests of ventilation, the Committee (1965) included among procedures useful in eval- uating impairment of the reapiratnr.• system such pulmonary- function tests aa blood-oxygen level studies (see below, 585. Oxygen Saturation of Venous and .lrterial Blood; SM. Oxy- gen Uptal:e). The reader may al.so coas-uk 548. Puhuonary Clearnnce, below. 543-A. Normal Smokers re Non-Smokers. Apart frcmt random samples of populatinnA, normal subjects tested have been drawn from the folloe•ing slxvi6c occupationa, whirh may be differentiated into (1) 1ho.w in which industrial ex- posure to atmospheric hnllutiuu ir- pttyumed to Ix prr-rnt to a more or lm marked degre.•; (2) those in phirh the environtnent itself is more or leca "normal"; (3) tlo••r characterited by heavy labor; or (4) by a high degree of t e { Produced bv ThL Council for 000303b Tobacco reszch-USA, lnc,

RESPIRATORY SYSTEM phyAcal traininR. In the 6rrt category are ooal-miners in West Virginia (Ferris and Frank. 1962: Hvatt et a/.. 19641 and miners in the United Kingdom (l. T. T. Higgins et al., 1959; Higgins and Cochrane,1961; Higgins and Oldham,1962; Ashford et al., 1961; Rogan, Ashford et al.,, 1961; D. C. 5lergan, Pasqual and Asbford, 1964); welders (Hunnicudt et al., 1964); foundry-workers (Higgins et al., 1959); transport- workers (Mork, 1962); road-tunnel employees (Speizer and Ferrk, 1963) ~silroad•yard workers (Battigelli et al., 19&f); steel-miU workers (McLane, 1963); and chemical-industry workers (Gandevia, 1963). In the second category are fae- torv-+corker, (Franklin and Lowell, 1961; Chns~• et al., 1963; Hawtborne, 1964); bank employees (G. li0wer, 196,Ia); military personnel (Hensler and Giron, 1963); and medical penwnnel (G. flower. 1961b; Shapiro et al., 1964; Krumhols, Chcvalier and Rorw, 1964, 1965a). The third category in- cludes lumberjacks (Kan•onen, 1962a) and longshoremen (Goldsmith et al., 1962). The fourth category i9 reprer.e:ated by Navy "frogmen" (W. Shapiro, 1961; Shapiro et al., 1964). t'entilator.- function of certain populations in England has been compared to that of populations in Denmark (Olsen hnd Gilron, 1960), Norway (\fork, 1962), and the Netherlands (Lammers et al., 1964). A number of workers have attempted to correlate pul- monary function in smokers and non-smokers with age, sex, and smoking habits. \ieaavrements of ventilatory capacity in smokers reportedly fell with increasing age (D. 0. Ande>gon and Ferris, 1962; Brinkman and Coates, 1963; R.1{. Larson, 1963b; Pelzer and Thomson, 1964). Some investigators have found significantly lower ventilatory function in women srnokers than in women non .mtokers (Read and Selby, 19'61; R.1>:. Latson, 1963s, b; J. R. Webster, 1963; Af. Payne and Rijelsberg,1964); but others have reported decreased ventiia• tory function in male smokers, but not in female amokers (G. Bawer, I961a, b; r'iiggit~sand Cochrane, 1961; D.0. Anderson and Ferris, 1962; Ander~on et al., 1965; Hawthorne, 1964 Tuberculosis Institute, 1964). Cander and Blumenthal (19&3) had no ready explanation for the more rapid decline of pul- inonan• function (vital capacity; maximal breathing capacity: maximal expiratory flow-rate; distribution of ventilation) in men than i., a•omen, but suggested that differenees En smoking habits between those members of the two sexes who are now in the fif.h decade and older may well be tha single most important factor contributing to the different rates of decline of lung function. Higgins and Cochrane (1961) bad previously offered as a possible explanation for the fittding in women, that those women with high indirect maximal btrathiing cspacity (I\1BC) were more likely to take up smoking. Aemrding to some workers, there was no significant relation- ship between ventilatory function and the degree and/or dum- taon of smoking (Ashford et al.,1981; Ferris and Frank, 1962; Hensler and Giron, 1963; Bouhuys, 1963b; Libow, 1963; J. R. Webster, 1963; Weiss et al., 1963); but others have reported finding a significant correlataon, especially with degree of smoking (Higgins et al., 1959; Franklin and Lowell, 1961; Anderr-on and Fe.rris,1962; R. ii. Larson 1963b; J. T. Sharp et al., 19U, 1965; Anderson et al., 1965; Higgins and Oldham, 1962). Impairntent of pulntonary function was less in cigar- and /or pipe-smokern than in cigarette-arnokers (Olsen and Gil- smn, 1960; 11'eiss et al., 1963; Tuberculosis Institute, 1960. The effect of stopping xmoking on ventilatory function ia uncertain, with the alleged impa'vment said to be reversible (Read and Selby, 1961) or non-reversible (AlcLane, 1963). 1Cith respect to pulmonary function, there was said to be no difference between atuokers, non.smokers, and ex-smokers 125 (Hascthorne,1964); again, ex-smokers were found to he ipter- mMliBtn iwtw•vnn amnkwn nn.t nnn.cmnt.n.v Iw•.-:~~ ... ~~ .nn•.. and the degree of pulmonary impairment was said to depend on the age of the ex•sbtoken= (Tuberculosis Institute, 1964). The following detailed account of the reports of the several investigators (arranged by groups of workers in chronological order) will, unfortunately, only add to tbe ambiguity of the above general state:nents, while confirming the over-all im- pression thatcigarette-smoking isassociated w•ith a reduction in veutilatory functimi (Smoking and Health, 1964, pp. 38, 292). A comparison of miners, foundry-workers, and others, in Staveley, Derbyshire, showed that the mean maximum breathitVg capacity (aIBC) of the smokers was lower than that of the non-smokers, and heavier smokers recorded a lower AIBC than t.he lighter ones (1. T. T. Higghns et al., 1959). Arxi, in a survey of respiratory symptoms and ventilatory capacit}, in a random sample of 600 men and 200 women tiving in the Rhondda Fach, South Wales, smoking was found to be associated with an increased prevalence of symptoms and lower indirect maximum breathing capacity (IA9BC) in the men, but not in the women (Higgins and Coehrane, 1961). DiBerenees in smoking habits could not, however, account for the differences between miners (and ex-miners) and non- miners iit the population surveyed, as these differences re- mained after standardization for smoking. A 5-year follow-up study of ventilatory capacity in a random sample of men living in this mining district of Wales, including 98 non-miners and 154 miners and ex-inh:ers, was reported by Higgins and Oldham (1962). In the non-miners, a greater decline in 1i1113C was obst n-ed in smokers than in non-smokers, and this decline was greater in the hea%w (15 gm tobacro and over, daily) than in the light 'r,rnokers. In this group of non-miners, the decline in IhfIIC for non-smokers, light smokers, and heavy smokera, was, respectively, 0.489 * 0.714, 1.524 * 0.319, and 3.338 f 0.120 liters/min; in the group of miners and ex•miners, the dec)hjes were, respectil~ely, 0.950 * i.oi I, 2.164 t d1.485, and 2.t160 :k 0.•t281iters/min. Three possible explanations of these findings were advanced: sntoking re. duces the ventilatory cnpacity; those with a lower ventilatory capacit%• smoke; or sonde common factor eauses bcth moking and a Iow-er ventilatory caparity. According to Higgins (196,4b), um-aker's ]nngs are 6-7 ; eats older than the calendar years of his body. B;• eompariso-j, R. 1C: Larson (l963a) de- clared that the pulmonary 'urpairment of smokers was equivalent to 2 decades of ageing. R. H. dt•ilron, rfeadbr and co-urorkers (1M) studied the puknonarj• patholegic physiology of 14 persons who had never smoked and of 14 who smoked 20 or ntore cigarettes daily for o mean of 18 years; body-surftue, height, and age were paired as closely as feasible, and all of the subjects were without signs and sstinptoms of lung disease on physical ex• amination. The tinted expiratoy volume, the total lung capacity, and ,11RC were found to be decreased in heavy sntokers, while the ratio of the residual volurne to the total lung volume was increased. The total and membrane-diffusing capacities were decreased in the smokers; and the authors concluded that cigarette-smoking decreased the pulmonary function to the extent that thet'e persons (that is, smokers) could not be considered to have normal pulmonary function. The above findings were published In abstract by Aleador, it'ilson and associates (1959), and also formed the subject of an Annotation im The Lanret (8: 86, 19Q0). cw•ann and Hatch (19t`0) also compared the pulmonay peHormance of 12 male cigarette•stuokers with that of non• Produced bv The Counfl for 0003037 Tobacco Reseaich-USA, Irtc.

126 TOBACCO-EXPEIU\IE?CTAL ANI) CLIXICAL sTd'll1ES. FCPPLE.IIEXT I smokers of about the same ages auu au:r. eluded: minute-volume, breathhng frequency, tidal volume, and ox•vgen consumptinn, at d+est and follow~mg 4 and 8 kg- meters/sec of exercise, together with simultaneous messure. ments of percentage uptake of ether, carbon unonoxide, and acetylene from inspired air (the test gases providing indices of ventilatory efficiency, diffusion capacity, an,d pulmonary- blood flow, as contributors to o.•er-all cardio-respiratory gas-exchange capacity). Tidal volumes and breathing fre- quencies were not sensibly different in the two groups; median vital capacity was lower in the group of smokers. The two groups breathed equal volumes of air at rest and at 4 kg-M/sec exercise; at 8 kg-ll/sec, the median ventilation- kste of smokers was 10% higher than for non-smokers. Oxygen consumption was the mme for both groups at all levels of activity. Percentage uptake values for the test gases were reduced in emokers; and, converting these percentages to effective rates of ventilation, diffusion, and blood-flow, the authors showed that the smokers had poor ventilatory efficiency and substantially~ reduced diffusion capacity, compared to non-smokers. These deficiencies constituted no functional handicaps at rest or under mild exercise, but there was evidence of the beginning need for compensatory increase in ventilation by the smokers under moderately heavy exercise. According to a later study by Battigelli, \Iannella and Hatch (1964), cigarette-smokers in a groi)p of 364 loco- motive repairmen and railroad-yard workers (average age, 50 years) showed decreased pulmonary function and per- forroance, eompared to non-smokers in the group; and the cigarette-smokesa also sbowed a higher frequency of tespiua- tory complaints (see bebw,1261-A). Wigderson and Kohan (1960) estimated ventilatory per- formance in a group of 113 male patients between the ages of 17 and 99 years, and reported that smokers had reduced ventilation values (1• and 3:rec timed vital capacities; max- imal mid-eapiratory flow-rate). In a comparison between men in Denmark and in agri- cultttral areas in the United Kingdom made by Olsen and Gilcan (1960), the mean indirect maximum breathing capacity was found to be significantly higher in the Danish than in the United Kingdom subjects (106 vs 92.1 )'/min); and the authors noted that thc-re were more non-smokers among the Dauieh men. and many fewer cigarette-smokera than in the United Kingdom sample, and that only in the small group of non-smokers was there no physiological or clinical differ- ences between the two samples. In the Danish sample, there was a rigniticantly lower LIiBC, and pooier single-breath nitrogen-dearance in the pure cigarette•smokers t.han in the eigar :mmkexa, despite similar tobacco cotuuntptions. ln a study of 4,014 coal-workers at 3 Scotti.ch collieries, ventilatory function (as mea.cur-ed by FEVi.n) of the non- smokets in all age-groups was found to be significantly higher than that of the smokers, even when acivuul was tukcn of differences in physique and age; among the sn4okers, however, no consistent reletionahip was rerotded between current consumption of tobacco and ventilatory function (Ashford et al., 1961). In an investigation of 9,758 mihers at 8 liritish collieries, it was found that the effects of smoking on the forced expiratory volume did not vary significantly from colliery to colliery (Rogau, Ashford et al., 1901). Seasonal variations in the tneasurement of ventilatory capacity were determined at two collieries by D. C. 1lorgan, Pasqual and Ashford (1964). At both collier•iea, there was a decreace in average FEV,.o measured during January, contpared to June, and this remained true when the men were subdivided ac- ~-.+:. +n cmnkinv hahits (a significant fa0 in numbere of cigarettes smoked during January, as compared to Julle, Nau~ noted); mean eeasonal difference woa greatett for ewsmoken, least for non-smokers, w•ith smokers, falling in betweeri. There was a high degree of consistency in the classsi8oation of inen in terms of smokers, non:anokero, and ex-smokers on the two occasions. Gregg (19114) had previously noted that, when tested on repeated occasions during the year, there sra® a striking tendency for the expiratory flow-rate of Rmokern to be lower in the winter months; in contrast, the flow-rates of non-smokers remained renharkably constant in summer and w•inter (see below, 523-B). The maximal mid-expiretory flow (DINIF) describes the mean flow-rate in liters per see dnring the middle half of a forced expiration, and ia an ii.dez of air•fiow impsirmrno. In a study of adults employed in a bank, NiN1F of 13 male non-smokers averaged 4.98 :L- 1.48 liters/sec, compared to 2.86 z& :.37 in 54 male (2l)k pack-year) smokers; but, in a compariaon of 36 female non-smokers and 22 female cinokerx of the same degree, there was no significant difference in flow-rate (G. Bower, 1961a). Itt a group composed of 98 young medical students, hospital employees, and physicians, agek 20-29 years, and in a secaid group consisting of 84 bank, employees all over the age of 40, \i\IF was found by Bower (1961b) to be significantly greater in non-smoking men than in men who had smoked cigarettes for at least 5 pack-years. In a group consisting of 78 women bank-employees over the age of 40, no significant difference in NI\IF between nnm smokers and smokers was observed. In a field-study carried out ht Britain and reported in the British lledical Journal ($: 973-979, 1961) on 781 men and 782 women, aged 40-64 years, decreased expiratory flow•.rate3 were ob_ ed in male cigarette-smokers at all level. of smoking, but, in females, this occurred only at a consumption of 25-h cigarettes pe da.N (only 6 women smoked this much). D. D. Reid (1963) oconcluded from this study that cigarette- smokiutg in men is associated with a significant l,twering of the peak expiratory flow-rate below the level achieved by non-smokers. Af estimated by the lYright Peak Expiratory Flow-Meter, :mpaired ventilatory capacity was more frequent in smokers than in non-snmkers in a random srunple of 513 men aged 30-59 yeats (Fletcher and Tinker, 1961). In their study of male factory-workers aged 40-00 yeacY+, Franklin and Low•ell (1961) found that 104 "heavy smokers" had a mean value for the rate of expiration during the third quarter of forced expiration which was abwt 20% lesc than that of 59 "light smokers", the difference being significant at the 1170 level. In most rases, this reduction in flow-rate was not associated with dyspnea. The authors believed this change in flow•-rate to be similar to that present to a more marked degree in patients with chronic obstructive emphr•y et•mn (-ee 1x•Mw•, 1276•U). Read and Selby (1961) determined maximal expiratory ffow•-rate in a group of 302 persons aged 16 years or more, comprisiing twn-xmokera (who had never smoked more thtin I cigarette a day for 1 year), ex,mmokers (who had ceasld smoking at least 3 months earlier), and sntoketx. Among males, smoking, in the absence of symptoms, led to smote reduction of ventilatory capacity. The proence of cough, or cough and sputum, va.t a>;eociatetl with further impairment of ventilatnrc function; thefe same s~ymtptoms were auociated with loss of ventilaton• capacity among non-smokers. Thte ventilatory loss appeared to be largely reteroible ht Qto:e who ceased smoking. jaccord•ung to 'Mci.ane (19ti3), pubnin• Produced hv T1e CoUICil for 0 0 0 3 0 38 Tob~ccu I~eS,arch-i1SA, InC~ i 4 _-J

ItE:SPIRATORY Sl'l1TG:U nnrv imnairment did not seem to be reversible in ex-sitokers.J Among females, there were similar trends, out t.ne resuns were said to be not nearly so clear-cut as in males. W. Shapiro (1961) studied ventilatory mechanics in 25 exceptionally bonditioned Navy "frog-men" , non-smokers, 14 ordinary seamen non-smokero, and 36 ordinary seamen chronic smokers, and stated that the data obtained suggest that athletic conditioning favorably altered the mechanics of the chest-disphrsgm bellows system, whereas smoking prod duced slight airway obstruction. In another series of 27 experiments, Shapiro and co-workers (1964) studied pres- snre--volume-flow relationships during maximum-effort expirations and inspirations on 7 superbly conditioned non- smoking athletes (members of a U. S. Navy underwater dem- olition learn, aged 30 * 4.7 years; body-surface area, 1.97 4: 0.15 sq m), 5 non-atbletic non-smokers (medical personnel, aged 27.2 f 2:7 years; surface area, 1.98 * 0.14 sq m), and 7 nun-athletic smokers (medical personnel who had smoked at least I pack of cigarettes per day for 5 year~, aged 31.3 f 2.8 years; surfdce area, 1.87 * 0.17 sq m). Th~e athletes had larger mean vital capacity and maximum breathing capacity, as well as higher air-flow during the first half of forced inepira- tion, than the other subjects, although di$erences were statistically significant only for vital capacity. Differences between non-athletic non-smokers and non-athletic smokers were not significant. The effect of current cigarette-smoking upon the FEV,.o and on the peak expiratory flow-rate was studied by D. O. Anderson and Ferris (1962) on 532 male and 607 female sub- jects in the city of Berlin, New Hampshire. In mateg, there was an almost regular decrease in forced expiratory volume within each age-group with an increasing number of ciga- rettes currently smoked; in females, there was little difference between smokers and non-t:mokeas, except at 35-44 years of age, when the forced eapiratory volume r<its significantly lower in smokers. With the exception of the age-group from 25-34 years, there was a progre.asive der.rease in both sexes in the mean peak expiratory flow-rate with 'uureasing age; furtbermore, this fail was prngressively accelerated with incteasing cigarette-smoking. In a report stemming from the Chilliwack Respiratory Survey of 1963, Anderson, Ferris and Zickmantel (1965) reported that FEV,,o and peak expir- atory flow-rates showed almost a consistent deterioration with increased current ctgarette-sn.oking; this deterioration was not as clear, however, in the ca-e of females as; of males (r•ee below, 1261 •A). Ferris and Frank (1962) studied a small group of 25 roal-tniners from West Virginia, and found no significant correlation between the amount-of smoking and measurements of pulmonary function; long-term heavy smokers (2 or more packs a day for 2a-30 years) were ran- domly distributed. When smoking and duration of employ- ment of 53 road-tunnel employers were examined in terms of maxbnal expiratory flow-rate, the differences found were not significant, but tended to go in the "anticipated direction" (Speizer and Ferris,1983). A survey of industrial workers in Vernon, California, by Bnlchum and co-workers (1962) revealed that, beginning at age 30, smokers were more likely to give abnormal results in the FEVt,, test than were non-smokers; percentages were, respectively, 4.4 and 6.1 at age 20-29, 13.8 and 7.8 at age 30ti39, 22.6 and 7.4 at age 40-A9, and 41.1 and 21.4 at age 50v"9. Chevalier and associates (198.,1983) reported that residual volume, total lung capacity, maximum breathing rapacity (D1BC), maximum expirator,r flow-rate, and airway resia- 127 ance, showed no significant difference between 7yroung L/a1e b1LLUAtlID tlt/V J LIVU-:11WbNb. Lu .... "-..' 2 . 'r 9 smokers and 9 non-smokers, who were similar in~ body- surface erea, age, and occupation, again no difference was found in lung volumes, airway resistance, or maximuro voluntary ventilation between the two groups (Krumholz, Chevalier and Ross, j963, 1964). Subsequently, Krumhola, Chevalier and Ross (1965a) compared pulmonary compliance in young smokers and non-smokers; the subjects were 2D normal medical personnel, of whom 10 had Fmoked at leasat one package of cigarettes daily for 5 years or more, while the other 10 were non-smokers of similar age-range. Afean pul- monary cempliance for the non-smokers was found to be 0.241 t 0.017 liter per cm of water, which was significantly difYrrent (p < 0.01) from tbc mean valuc for the emnkers of 0.177 t 0.034, although both values were within the normal ranges reported in thk literature for the continuous cycling method used in the study. Mean body-surface area was the same for the two groups; and dividing the mean compliance value for each individual by the functional residual capacity at which it was measured to give the specific compliance, did not change the level of significance between the two groups. The authors discussed several possible etiologic mechanisms, and they themselves oonsidered that impaired distribution of the tidal volume in the lungs of smokers was the most likely explanation. For the oomparison by Chevalier, Krumhols and co.wockers of pulmonary-function measure- ments and responses to exercise between a group of smokers and non-smokers, see below, 528-G Gandevia (1962) found the mean maximal breathing capac- ity in 22 smokers (ave9age age, 32.1 d: 11.6 years) to be 103.9 liters/min, compared to 133.3 l/min in 15 non-smokers (aver. age age, 32.8 * 9.5 years). (For the responses of theQe fub- jecta to exercise-tests, Fee below, 523:-C.) Gsndevi9 (1963) also reported results of studies of ventih.tory capacity (FEVI.c) and histsmine reFpon:e (FEVt,o measure,l before and after 1-min inhalation of 20 mg/ml histamine base, uring an efficient nebulizer with compressed air) in workers during ealwsure to isocyanate vapor in polyurethane-foam manu. fe.tore. Approximately half the subjects studied wem found to show increased binnohial sensitivity to the histamine aerosol; all were smoken. Smokers and/or positive histamine reactors tended to show a greater decrtate in ventilatory caimc+ty during a arbrking day than did non-smokers or non-reactorr. No significant differences in FE1',.o with reyvect to Fmnk- ing habits were found in 428 men aget' 40-59 yearE, living in 9 Jerrey City Housing Units (Gorke and Dufty, 1962). J. R. Goldr.mith and co-workers (1962) performed pul- monary.function tests (FEV,.o; total vital capacities; Puff- meter) on 3,311 members of the Longhorrmen's Union in the San Francisco Bay area, and found that cigarette-smoking had a slight (about 5%) effect on them tests; a biostatistical discussion of the data hors been furni:qhed by Hecbter (1962). Revotskie, Kannel, Gbldcmith and Dawber (1962) presented a table of ratios (without interpretation) of observed to predicted pulmonary-function-tests values (FEVt.o; total vital capacity; Puffmeter) by sJnoking pattern and respira- tor7 findings for men and wome i in the Framingham, Mass. study. Zwerdling, Goldsmith and Massey (1963) reported a study of tnaximal expiratory-flow testa in a sample of the Framlogham population, with an application of component analysis. Four cla:ne,c of variables were ineluded, thtuie reflecting (1) physiologic test results (14 pulmonary-function text%); (2) age, sex, and physique; (3) clktieal 9ndingx; and Proaucca bv Tnr~j coun~li ~~+ 0 0 0 3 0 39 Jobacco Researcli-uSA,

I 128 TOBACCO-F.A:PERIDfENTAL AND CLINICAL SiPUD1M. BUPPLk.'*fh1T I ('4) enpoeure to c1guresrc-au,vs:.%. ;.... a^•^ - subjected to component anslysis, a method by which an attempt is made to transform a large number of inter-related variables Into a smaller number of tnneorre]ated variables. In practically all subsets of variables and samples tested, the first three components were associated with two ssltectR of pulmonary function and with body-weight, and accouo,ted for approximately tbttie-fourthe of the total variaoce. [For further details of the Frsmingh>am study, see To6aeco (661a); also Dhwber et at, 1962; Kagan et al., 1962, bebw, 1394. According to Karvonen (196v'a), who studied a grodp of 815 lumberjacks, aged 40-59 years, in eastern Finland, no diffrarucr in vital capacity was found hetcreen smokers and non-snutkers; however, FEVo,.,S and the peak expir6tory flow-rate were considerably poorer in smokers than in non- ttrnokers. In a comparison by Martt (1962) of 30 non-smokers with 25 subjects who had smoked for more than 10 years (average age of all subjects, about 28 years), tl,ete was no significant difference between the two groups in total vital eaptcity, . 1-sec vital capacity, total lung capacity, or residual vol- ume. Mork (1962) compared male trausport-norketc, aged 40-59 years, in Bergen, Norway, and in London, and found the peak respiratory flow to be lower in the latter group. The groups differed both qualitatively and quantitatively in their smoking habits; there was a higher proportion of, pure cigarette-smokers among the men in London, and the average consumption of tobacco was higher; but the ditferencec ob- served in ventilatory capacity were not eliminated by stand- ardising for smoking. (For further details of this studp, see below, 1261-A.) Zwi and associates studied cigsrette•smol•k,g mid pul- monary function in 10 healthy young adults who bad regularly smoked cigaretSes and in a control group of men who had never smoked; the groups were matched fo• age (20-35 yr.ars), phy:acal characteristics, and fitness (2avi and Goldmarr,1962; Zwi, Goldman and i.evin, 1984). The mean values for v ital capacity, functional tpaiduai capaeity, and total lung capacity were, respectively, 240, 60. and 2U0 ml larger in non-smokers than in smokers, while the value for resit:ual volume was 60 mi smaller. The ratio, residual volume/total lung capacity, was 1.7% smaller in non-smokers; the lung-clearance index (an ir.dex of mixing) was slightly lower in the non-smokera. Maximum voluntary ventilation and manimum mid.expua- tory flow-nlte were slightly lower in non-anwkers; expiratory flow-rate during the third quarter of maximal forced expira- t9on, and maximal expiratory pressure, were lower in the non-smokerR. Exercise-tolerance tests showed that pulmonary compliance was higher in non-smokera than in smokers at rest, immediately after exercise, and during voluntary hyper• ventilation; heart-nue was more rapid at rest and during esereise in smokers, and arterial oxygen saturation w as lower during strenuous exercise. Maximal expiratory non-elastie resistance at rest was higher in the smokers. By the statistical technique of diserimiuant anal,is9s, physiologic indices for smokers and non-smokers were obtained using the foUowir•g variables: vital capacity; residual volume/total lung capacity percentage; compliance; non-elastie resistance; and arterial oxygr a saturation and heart-rate during exercise. The differ- ence between the means of these indices was significant at the 99% level. The authors concluded that cigarette-smoking altered pulmonary function in the direction of chronic ob- structihe lung disease within the fnst 15 years (sooaj after mmmpnoina the habit, end that the changes were progressive over many years. Bouhuys (1903a) measured expiratory volumes in a group of 74 healthy male subjects, and also obtained smoking histories from 98 control subjects, but did not correlate the two variable.s. Ilouhuys (1963b) al~a recorded lung volumes and pulawonary nitrogen clearance during oxygen breathing in 80 healthy rnales, 24-es years of age. No significant correlation was, found between smoking (in gm tobacco smoked per week) and vital capacity, residual volume/total lung capacity .ratios, or pubnonary-clPSrance delay per. centages. A probably atatisticslir sigh,i6cant correlation existed between smoking and lung•clearance index in sub• jects 45 years of age and older; and this impaired intrapul- monary gas distribution bo u1dF, ra,bjccts r.-as not thnaidPral to be a physiological consequence of ageing, but rather re- lated to long-term effects of inhaled noyous agents, such as tobaccu-smoke. [But R. K. l.anson (1963b) and 1. T. T. Higgins (1964b) attributed such puimo¢,ary impairment to physiologic ageing.J As pan of a survey of 1,317 men, aged 40-65 years, who were working full time in the Detroit area, Brinkman and Caates (]983) obtained a Fpirogram on each man, from which were calculated the forced expiratory volume, FEf,',,o; FEVI.a as a percentage of FEV; and maximal mid-expiratory flow. All measurements fell with increasing age, and were adversely aFecded by bronchitis Isee Bti,kman and Coates (1962), below,12111 -A and cigarette-smoking. Commentina on the cigarette-smoking aspect, the authors offered the opinion that, although ventilation iu cigarette-ianokers is decreased even after one,cigaretle, it is not po.<ible to demonstrate a cauee-and-eBect relationship, since a man who rmoker is probably constitutionally aud p•ychologically different from the non-smoker (see 1129-B, below). In a group of industrial workeis, aged 20-80 years, with no history of chest disease, FEt',.o mid A1BC were reported to be similar in smokers and nou-smokera (Chosy, Gee and Renkin, 1963). Heoaer and Giron (1903) studied lung function in 163 senior Air Force officers, aged 38-57, and found no significant difference in total lung capacity between the 113 habitual cigarette-smokers and the 50 non-smokera in the group. liowever, vital capacity was significantly lower, and residual volume significantly higher, a.mong the smokers; and 1.sec timed vital capacity, 3-sec timed vital capacity, and maximum voluntary ventilation were all significantly lower In the smokers. Among the nnoker+y nn statistically signiGcant difference could be demonstrated with increasing cigarette• consumptimt. R. K. Larson (19fi3s, b) performed a total of 418 satis- factory studies on a random series of smokers and non- smokers (subdivided into sex, smoking, and age categories) on whom forced expirntory spirograms were obtained, and calculations nnade of the ratio of FEl',.o to expiratory vital capacity; the ratio of the 3rd-tec forced expiratory volume to expiratory vital capacity; the peak flow-rate; the mid- expiratory flow-rate; and the 3rd-quarter expiratory flow-rate. No rsignificant diference betwm, smokers and non-smokers tras found in the 17-29-year age-group; but timed vital capacities for all age-groups of more than 29 yesrs in both a.xes were infer{or in smokers when compared to those of non-smokers. Measured by the mid-expitatory and 8rd- quarter flow-rates, these differences were statistically signifi- cant bPyond age 29; the most consistent difference was manifest in the mid-expiratory 8ow-rate. The incidence of Produccd by The Council for Tobacco R~«arch•11SA, Ino. 0003040

RElTP11tATO1tY SYSTEM grossly abnormal spirogrAms was almost limited to smokers fl:arson, 1963b). Increasing exnnaton obstruction in smokers correlated even better with cumulative smoke exposure in peck-years than with age. The deleterious effect of cigarette- smoking on ventilatory function was approximately equiva- lent to two decades of ageing (Iamn, 190U). This was a more severe conclusion than that of 1. T. T. Biggine (1964b), who stated that ,mokers' lungs are only 6-7 years older t han the calendai years of his body. In contrast to R. K. Larson (1983b), J. R. Webster (1963) fortnd, in asymptomatic and presumably healthy women, that the correlation between pack-years and functional pul- monary impairment in individual caeea was not significant, and that some individuals maintained a normal functional ca(.raeity with up to 36 pack-years of smoking. This latter worker made maximal voluntary ventilation determinations in 88 woman smokers with more than 20 pack-years each and in 45 non-smoking women, and reported the maximum voluntary ventilal;on of the smokers to be significantly lower than that of the non-smokers (p < 0.01). A comparison of 11 non-smokers with 28 chronic cigarette tanokp,ts failed to reveal any differences in vital capacity, ]t1BC, and arterial oxygen saturation, nor were any correla- tions found between these variables and the duration of smoking; but Libow (1963) noted that other studies had re- ported definite decreases in pulmonary function with chronic smoking. AlcLane (1963) investigated cigan;tte-smoking and pul- monary ventilation in 100 healthy men between the ages of 40 and 60 without medical history of any diseases which would have resulted in reduced pulmonary ventilation; the r.ubjects had worked for more than 20 years in fabricating divisions of an integrated steel mill; b0 of the subjects had smoked 20 or more cigarettes per day for more than 20 years, and the other 50 statesi that they had never used tobacco (the mean age of each group was almost identical). Average v ital capacity for the smokers was 3.78 liters (range, 2.6-4.85) and for the non-smokers, 4.23 (range, 3.3-4.9); predicted vital capacity for smokers and for non,smoke>g was identical (4.28 liters). FEV,A for smokers was 9.91 liters (range, 1.85- 4.42) and for non-smokers, 3.46 (eange, 2.65-4.5!!). Thus, cigarett.e-smcking in this population bad reduced pulmonary capacity about 11 %. in 3 smokers with significantly reduced puhmnary ventilation. abstaining for as long as 16 months did not improve the values, indicating that i•npaument does not seem to be reversible. According to Read an Selby .lby (1961), however, ventilatory impainnent was reversible in those who crosed smoking. Studying 287 men aged 53-69 years reporting routinely for check-up to the Philadelphia Pulmonary Neoplasm Research Project over a 6-week period, Weis~, Houoot, Cooper and Carnshan (1963) found that the prevalenoe of abnormal vital capacity and FEVI.o correlated with type of nnoking and degree and duration of cigarette-fmoking. When FEV1.o was calculated as the percentage of the observed vital capacity, there was a relation to type of smoking, but no relation to either degree or duration of cigarette-smoking. Smoking habits were not well reflected in abnormality of the msavnum expiratory flow-rate. Ex-smokers occupied an intermediate position between non-amokera and current smokers. There was a higher percentage of men w it.h abnormal FEV1,o among cigerettc+smokere than among cigar- and/or pipe-amokers, regez-'less of whetuer this function was related to predicted mean normal or to obsemed vital capacity. Zamel, Yousset and Prime (1963) studied airway resistance 129 and peak expiratory flow-rate in 12 bealthy- non-smokers (6 male. 6 female) a¢ed 18-•32 vpArxi and in 12 rronra/P amnkON of similar ages and the same sex distribution. Airu•ay resist- ance (body-plethysmograph method) was found to be higher in the smokers than in the non-smokers, but the difference was not statistical:y signi8cant. However, when judged against the predicted values for the non-smoker8 and the smokers, the difference became significant for the lab;t,er. Inhalation of isoprenaline (isoproterenol) lowered airw•ay resistance in both groups to a highly significant degree. Peak expiratory flow-rate averaged 530 liters/min for the non-smukerre and 491 for the smokers; inhalation of isoprenalane increased the flow-rates to 542 and 513 liters/min for ioon-smbkens and smokers, respectively, these changes being statistieally sig- ni6cant. Peak forced expiratory flow-ratex were determined by Hawthorne (1964) on employees at several factoriee in Gla.agow, and the Sndings expressed in terms of per-cent of individuals having flow-rates below the average for non- smoking asymptomeatic subjects. ln the 15-45-year age- bracket, the percentages were 44.5, 40.6, and 48.7, respee- tively, for male smokers, exx•smokers, and noa-smokers, which were said to indicate no adverse effects in the first two categories. Corresponding percentages in the "year age• bracket were 69.9, 59.5, and 54.2, which were taken to indicate poorer performance in smokers. Among women, no meaning- ful differences were found, and the author speculated that this might be due to the inability of most aromel to blow effectively into the Peak Flow .lleter. (For an analysis of clinical evidence of respiratory disease in these subjects, see below, 1261-A and 1271-A.) As judged by spirometric measttt rements in 100 welders and 100 non-welders, more of the former than the latter had abnormal puhnonary-tunction tests; but, when the non- smokers were compared, there was little diference, between them (Hunnicutt, Cracovaner and AI-vles, 1964). However, welders who smoked cigarettes showed twice as dtuch ob- st.ructive pulroonary impairment as welders who did not smoke, and three 'r,imes as mach impairment as nom•welders who smoked. The incidence of respiratory symptnms followed the incidence of impaired lmimonary function among the several groups. In the course of their study of tespiratory dieraar in a random sample of bituminous toal-miners and rac-miners, 45-55 years old, in a geographical unit in southern West Virginia, Hyatt, Kistin and \lahan (1964) grouped 266 miners accordinq to both years worked underground and smoking habits, and then associated each group with pul- monary-function findings. ln the 0-10-year-underground group, there was a progressive impairment of respiratory function with increasing pack-years tanoked, judging from the maxitnum mid-ezpiratory flow (DiDIEFn-in) and the residual volume/totsl luag t•apacity per-cent; FE',V,.o and vital capacity were lower in the 30•plus-pack-year group than any other groups. Among the 11-30• and 30-plus-yetirunder- ground groups, there was impairment in function in the former group compared to the 0-10-pack-year group, but no further intpairment with greater smoking. Although smoking definitely impaired pulmonary function, impairment of lnd- monary function by years worked underground was Rhow•n to be clear and separate from the Anoking effect. 1.ammers and associates (1964) reported finding } higher prevalence of respiratory symptms, and lower FEtio.,a, in 414 Englisl: cotton-workers In Lancashire, compared to 9R0 Dutch cotton-workers in Almelo, the A'etherlands. Examiua- Proc~t~ccc~ Tobacco f~es~a~ci~ U , 0003041

, TUBACCU-EXPER1ME1TAL A\I) CLINICAL STUDIES. SUPPLEMENT 1 130 tion of smoking habits in relation to these findings showed Unil UIC/C MC/C /CMC1 CA- 4//u I/VU'.~IUVhI'.4 fM.n~u ' and their smokers connumed more tobacco than the English; but 85% of the English wnokprs smoked manufactured cigarettes, whereas 57% of the Dutch smoked a mixture ©f manufactured and hand-rolled cigarettes or the latter only. The authors concluded from other data that the most likely explanation of the unfavorable picture presented by the English workers was the much higher level of air-pollution in l.ancashire (see below, 1261-A). In a survey of pulmonary symlitoms and function in 4,922 persons in rural southeastern Oklahoma reported by Lindeman. Shaw and Bloss (1964), cigarette-smokers tended to have Ibover indirect maximum breathing capacities than did non- smokers. (For an account of respiratory signs and symptoms end their relationship to the smoking habits of these subjects, see below, 1261-A.) In their investigation of air pollution and respiratory diseases in the Tokyo-Iyokohama area (see below•, 1261-A and 1266), Oshima and associates (1964) noted that a decrease in 1-sec vital capacity was frequently seen in ciprette-smokers or in allergic subjects who had respiratory symptoms. Respiratory symptoms (see below,1261-A)', lung function, and smoking habits of 5,140 adults over the age of 16 years laving in or near the town of Tecumseh, Michigan were re- porled by M. Payne and lijelFbti.rg (1964). In comparison with non-smokers, men who smoked cigarettes luui, on the average, values of FEVI,o decreased by 0.2 liter at the over-all tlnean age of 40 years; in women hho smoked cigarettes, the compatabie reduction was 0.1 liter. These differences amount to 7% and 8 0 of the values in noti-stnoking men and women, respectively, and +xre stated to be significant rtt the 1 io level in men and the 5 0 level in women. In a study of maximum espimgor)• peak flow, Pelur and Thomsnn (1964) found that it decreased sharply with age in smokers, but not in non-smokerR. J. T. Sharp, Paul and a.~ociates (1964, 11>85) reported findings from respirator,v-di.ea.* surveys conducted in 1960 and 1961 on 1,887 white males bitwren the ages of 43 and 59 yeara who were emliloyre3 of an electruc company in Chicago. In general, heavy smdkers showed significantly lower spitvmetric mea.aurements than light smokers, who, it• tutrn, had more spiromctric imfmirmeot than non-smokers (Sharp et el., l9G•f). Vital capacity, FEV,_o, marcimal ex- p'iratory flow-rate, and terminal expimwn, flow-rate were obtained for 1,641 of the subjects; and mean .elue.< were found to be significantly lower in light smokers (1-19 citce- rette.a per day) than in non-smokers, and significantly lower a4nongheavysmokers(20+ lrer day) than among light smokers (Sharp et al., 1965). Cigarette-r•mokhtg and the presence of persistent cough were a.-rociated with spirometric alteratimR: suggesting early airway obstruction. Even in men who denied Fiknifieantt respiratory nymptoms (including persistent cmtgh) spirometric measurements were significantly decreased in smokers, compared to non-smokers; and this nidicated that smoking was associated with ventilatory impairment even in subjects not exhibiting overt symptoms of chronic bronchitis (J. 7'. Sharp et al., 1961, 1965). (See 1261-A, below, for the prevalence of rettpiratory signs and symptoms and of chronic bronchitis in this urban industrial population.) In a sample of Turkish population studied by Teraioglu and Cincnire (1964), no relation between cigarette-use and vital cahacity w•as, found; the subjects apparently included lwth men and women. According to I3i0rek (1tt68b), 200 twin pairs in Sweden, who were discordant with regard to smoking habita, were .1 • .f ,1.. ..R .1:.. .,9 .....i 1..1... ..~. ...-. ::0.. . .. ~:,-..-. .., with preliminary results showing that the pulmoriary function in smokers was impaired. The reader is reminded that studies of pulmonary function in patients, with respiratory disorders (below, 523-B) and following eaercise tests (belowo 523-C) usually contain pte. sumably healthy subjects as controls or comparisons; and he is therefore advised to consult the following two sectiotu for further data on ventilatory function in normal smoker• vs non-smokers. 523-B. Sm'.okers vs Non-Smokere with Pulmonary Disease/Msfunetion/Sympao.us. Hallett and llartin (1961) measured m.9xims! e,riratory flow-rate ('ttEF) on each of 710 patients admitted to Firland ItuberculosisJ Sana- torium, Seattle, Washington, and reported that the number of cigarettes smoked, or the qpantity of other fontis of tobacoo used, was not significantly related to abnormal MEF. In one group, conoprisinµ men Ie.Y than 59 years old who had stopped smoking 2 or more year.• before, the incidence of ah. norrnai A1EF was significantly high (p < 0.05), but no such relationship orcurred in older, men or in women who had stopped smoking. Evidence of the diffuse obstructive pul- monary syndrome, as detected by a low MEF or air-velocity index, was found in 91%of 45 tbtberculous patients considered treatment failures; this incidence was unrelated to age, sex, or smoking.habits (C. J. )tfartin and Hallett,1962). Gregg (1963) found a marked diBerencein peak expiratory 9ow.rates betrren non-smokSra, asymptomat6e s,mokets, and smokers wilh '/smoker's cough"; howvver, there were a num- ber of heavy smokers whose flow•-rates were as high as those of non-smokers of cornspondu`ng age and heiL+ht, whereas some moderate smokers had a cough and low flow-rate. Douhuys (1963a) measured foned expiratory volume in 31 teatile•w•orkers with :,yccinosis [see below, 128-t:C] and iu a control group of 74 healthy male subjects and 94 male sub- jects with evident or susperted cardiopulmonary disease; regmssion analysis showed that the decrease of ftlrced eapirs- tM'1• volume with ap was significantly larger in patients with b)&-inosis than in both control groups (the textile-workers smoked ri;;niflcantly less than a, group of 98 control subjects). Celleriuo and Billia (1962) noted, and furnished additional data. that cigarette-smoking reduced somra•hat the useful lung volumes, and increased, respiratory aincay r, sistance, even in healthy individualc; broncho-pneumopathic hetients suffered the same changes, but more severely. These writers described the use of a chemical filter which vras said to have abolished in great part the objectionable changes (see below, 1623-E). ]n an article entitled "Obstp•uctive F:mphysettta in Ciga- rette Smokers", A. L Flick aind Paton (1959) prquaunl data on the smoking habits and niaidmal expiratory flow-rates of 22'i male patients at the Sea¢tle \'eterans Administration Hospital, according to which httln-smoking men showed mini• mal changes in the rates to ap',e 70; mnny tmnker= showed maderate to marked decline itj the ratea with increasing age. and heav y smokers showed morh loss than did light Rmokcm Smokers who left short cigarette-butts showed more lo&t in maximal e.tipiratory flow-rntes than those who left longer butts. In this connection, it dnby be walled that Franl:Jin and Lowell (1981) believed tliat the reductnm in exzpir.dory flow-rate in heavy smokers was similar to tinli ptr:-cnt so a more marked degree in patit!ms witb clu,.aiv ulR.truetive emphysema. Produced h,f The Council for 0003042 Tobacco Research•i;SA, Inc.

1tPSi'IKATUItY SYSTEM 131 I C,,,•wvt wnrirnrc wl.nw m"miramontp nf mtlmonnrv fnnr- tion in noemal,individuals have been reported in the preceding section (523-A) have also included data on patients with various fdrms of puknonary dtsease (Brinkman and Coates, 1963; J. T. Sharp et al., 1964, 1965; D. L. Anderson, Ferris and Zickmantel, 1965). 523-C. Smokers sa Non -Smokers Follo..ing Exercise Teses. Gandevia (1962) described a 5-min exercise test, in which the ventilatory respodtse to a known amount of work was measured, and the result, expressed in terms of ventilatory requirement at a standard work-level of 300 kg-\I/min; no significant difference was found in this respect between 22 smokers and 15 non-smokers (29.8 * &2 vs 27.8 f 5.1 liters/ min, respectively). Chevalier and Krumhola, and co-workers, in particular, have interested themselves in csrdiopulntonary responses to exercise of young smokers, tjon-sntokers, and ex-srnokers (for an account of the heart-rate khanges observed, see above, 433; for pulmonary diffusing capacity, see below, 547; for oxygen uptake and/or debt, see below, 718). Chevalier, lirumholz and Ross (1963) also studied the effects of carbon-monoxide inhalation on the cardio-puhponary responses of non-smokers to exercise, and their data suggested that the differences in response to exercise eaisting between smokers and non- smokers might be caused, att least in part, by the effects of carbon-monoxide inhalation. Chevalier, Bowers, Bondurant and Ross (1962, 1963) evaluated the effects of cigarette- smoking on some of the ventilatory responses to exercise in 18 young male smokers ancj 14 non-smokers. Exercise con- sisted of a standard 5.min test performed using a Godart bicvcle-etgometer; and, folloQVing the standard exercise-test and a rest period, the subjects pedaled continuously at in- creased resistance for 2-min periods until exhausted. Pulmonary-function characteristics (vital capacity; total lung capacity; residual volume/total lung capacity; airway resistance; FEV,,o, per-cent of total lung capacity: maximum expiratory flow-rate; maximum breathing capacity, per-cent of predicted normal) in the two groups of subjects showed no significant differences. In another investigation by Krum- bolz. Chevalier and Ross (1964) on 18 house-staff physicians, aged 27-37 years, of whom 9 had smoked at least 1 pack of cigarettes per day for 9 years or more, and 9 of whom had not smoked at ail for at least 5 years prior to the study, a 5•min exercise test was pedonr.ed on each subject, the ..•ork•load being the same for all. 0xygen uptake was measured for the 4 tnin before, the 5 tnin of exercise, and the 10 min after exer- cise; and, although the mean increased total oxygeen uptake for the exexcise period was about the same in the t wo groups, the ozygen debt accun•,ulated was significantlv greater in the smokers than in the non-smokers (see below, 718). Heart-rate was determined each rain throughout, but the mean rates showed no significant difference between the smoking and non-smoking groups at rest, during exercise, or after exercise (see above, 433). Breeth-holding pulmonary diffusing capacity (D,,) was measuted just prior to the 5-min exercise period with the subject sitting on the ergometer, at 2 and 4 min of exercise with the subject continuing to exercise, and 3 min after exercise had ceased. The resting DL was significnntly lower in the smokers, and this reduced level of Dt, was main- tained throughout exercise in the smokers, although the per-cent increase over the resting Dy remained the same for both groulvs (see below, 547). Pulmonary-function studies, while showing differences in the two groups, were within ac- cepted normal limits for a general population. Total lung canacitv a•as greater in the non-smokers thsn in the smobers (7,870 f 1,060 vs 6,650 :h 690 ml; p< 0.05), as was the vital capacity (5,720 *; 830 vs 5,0G0 f 380 n,l; p < 0.05) aud inspiratory capacity (3,610 f 350 vs 3,040 f 500 ml; p < 0.05), with the remainder of the functions (inspiratory reserve volume; expiratory rrserve volume; tidal volume; functional residual capacity; residual a~olume/total lung capacitv; maxi- mum voluntary venulation; maximum expiratory liow•rate; airway re=istance) showing no significant difference. Residual volume showed a strong tendency to be larger in the non- smokers, but the differences were not statistically cignificant (2,160 :k 690 vs 1,600 t 570 ml; p < 0.10). The authors pointed out that the differences observed may not be inter- related, but• instead, may be multiple different effects of the sarne stimuli. Krumholz, Chevalier and Ross (1t165b) aLio studied changes in cardiopulmonary functions related to abstinence from smoking; the subjects were 10 young chronic cigarette-smokers, all of whom had smoked more than I pack of cigarettes daily for more than 5 years, and their responses to the standard bicycle-ergohieter e%ercise•test were measunad before smoking was discontinued, and after 3 and 6 weeks of abstinence. After 3 weeks of,no smoking, expiratory peak flow and D,, were found to be significantly increased, and heart- rate, oxygen debt, and the ratio of oxygen debt to total in- crease in oxygen uptake produced by the evrrrise were signifi- cantly decreased. After 6 weeks of no smoking, functional residual capacity was decreased; inspiratory reserve volutne and maximum voluntary ventilation were increased; and airway resistance was significantly decreased, when the functional residual capacity changes a•ere taken into account. Pulmonary compliance tended to be increased after 3 weeks, with the tendency continuing after 6 weeks. Swann and Hatch (1960) and Zwi, Goldman and Levin (1964) also performed exercit;e•tolerance test+ on smokers and non-smokers; their results have been summarized above, 523-A. For the eAects of arute smokinR and exerci_r tests on ventilatory function, see Hunaiker and Buhlnrann (1900), Rothfeld et al., (1961), Sinionrson (1962), and Carlemt and Dablstrom (1903), below, 523-D. Also of interest in connec- t5on with exercise are the effects of athletic or physical training or conditioning on pulmonary function, for which see.qhapiro and co-worke•s (Shapiro, 1861; Shapiro et al., 1964), alavr, 523-A, and below, 523-D. 523-D. Pulmonary Funcviion Following Smoking or Nicotine Administratinn. According to the Advisory Committee to the Surgeon (:eneral of the 1'SI'HS, the acute effects of eigarette-smoking upon pulmonary function are expressed mainly through incrrase in airway resistance, which is not severe enough to prottuce clinically evident manifesta- tions; the smoker is not immediately aware of any increa`ed difficulty in breathing, nor are the pulmonary•-function tests used in surveys sufBcientlt• sensitive to detect the acute effects (Smoking and Health, 19W, p. 293). ]t does not appear that nicotine is the cause of decreased airu ny conductance after rnioking (Comrce and Nadel, 1962; Simons.con,1962). Inhalation of aemsols of isotonic saline and of nicotine bitartrate (2 mg/ml) had no significant effect on airway conductance (\adel. Tierncy and Comnoe, 1960; Comroe and Nadel, 1962). Vollowing i.m. injection of I mg nicotine (at least 6 hours after the last meal and 3 hours after smoking) in 31 normal subjects (of whom 13 were smokers), ventilation per min was unehan~.•ed in 10 (of whom 6 were smokers), was incteased in 1S, and dectro.4cl in 3; the increases were supposedly due to a stimulating effect of nicotine on the Produced bv Th~ Council for 0003043 Tobacco ficscaich-USA, Inc.

132 TOBACCO-EXPl:ltlAfENTAL AND CLINICAL STUDIES. SUPPLEMENT I canbt.id chemoreceptors (Giusti, )\icoletti and Guerini, 1960). 13udt no consistent effects ol the nicotine ullecuon were noteo on the vital capacity or ma3dmum expiradoq• volume of the second expiration during a test of rqaximurn ventilation per miti. lp a few cases, there was an indication of an increase or a decrease in bronchial-muscle tone. No curare•like effect on the aatdliary respiratory muscles was evident. Because other studies had sbown that the inhalation of a t.•ariety of phatata- oolpgic,ally inert, submicronic particieg causes a similar de- crease in gDrway conductance, Comroe and associates believed that the changes following smoking wen: caused by the inhala- tion of the very large number of smnll particles contained in cigArette-einoke, and not to its nirotine content (Nadel, Tierney aqd Comroe, 1960; Comroe And Nadel, 1962). Fur- thermore, the decreased airway conduetance folloNing inhala- tion of cignrette-smoke was similar, regardless of the nicotine contp.nt of the cigarette-smoke (2.3 vs 0.5 mg in the smoke from 47 nhhi of cigarette length) (Comroe and Nadel, 1962). Nadel, TiernPy and Comroe (I860) used the body- plefhysmpgrapb to measure the achrte effects of cigarette- smoke ob airdvay conductance in 15 notmal healthy nort-su9okere and 21 smokers, the ipieasunements being e>;- pre~ed at;, the ratio of sirwa.y conductance to thoracic gas volume (TGV)., After 10-20 inhalations of cigarette-amoke, the, airmay aonductance decreased inymediately (p < 0.05), and th3s e(feat last,ed approximately I hour; the average maxi- ma1 decre2lse observed was 50%. Changes were similar in gmokers and non-smokers, and uere neproducible. Smoking a pipe, cigar, or cigarette without inhbling bad no effect on airruny coitductsace. (tiVhea these experimenta were later reported liy Comroe and Nadel (1902), it was said that airway conductarice/lung volume decreased tigni6cantly in 31 of the 36 subjcclo (p <. 0.001), the mean decrease being 31;",. There was no significnnt, difference between the response of the smokers and non-amokerb. If a secortd cigarette were given after t¢e airway conductance had returned spontaneously to control levels, there was again a decrease in airway conduct ance similar to the first, indicating that tachyphylaxis did not occur.j Previous or subsequent inhalation of an aerosol of 0.5% isoproterenol prevented or reversed, the effect of cigarette- smoke (Nadel, Tiemey and Comrce, l 5180; Comror and Nadel, 1962). In 22 patients with caydio-pulmonary disease with a widerange of initial airway-conductance values, cigarette- smokin'g led to statistically significAnt che.nges of similar degree to those in normal subjects. Nadel and Tierney (1960) further indestigatRd the refation of lung•volume history to airway resistance in two normal subjects after the inhalation of substaneea capable of increasing air+cay resistance, as we)1 as in 2 patients with obstructive pultntonary emphysema. in nordnal subjects, airway resistance, mi?ssured during panting at "Fatnetional Residual Capacity", incrrasted after the inhalation nf cigarette-smmoke, sulfur dioxide (24 ppm), or an aerosol of 6% histamine phosphate. After a maximal inspira- tion and relaxation to the original lung vo)ume, the airway resistance decreased significantly in eacb msse. Subsequently, over aperiod of 2 min of tidal breathing, the airway resistance gradually iacreased. Before inhalatiola of "bronclmeonst.ric- tor" subatAnces, there was no significant decrease in airway resistance after a maximal inspiration iend subsequent relaxed expiration. Nadel and Ttemey believed that a tnamtal in- spiration tempotarily decreased smootlh-muscle tone, and that this explained the temporary decrease in air+vay resistance after a maximal ingpiration; thus, lutng-volume history must be considered in the measurement of airaay resistance; and since deep inspiration may temporarily chauge airway t>?sist , ance, methods which require a prior deep inspiratiom cannot uer.%prcuti . .y . ~' . ~ during normal bresthing. In a separate investigation of~the effects of a previous deep inspiration on airway resistance in man,lCadel and Tierney (1961) found that a deep i)tspitation never altered airway resistance, measured at functional residual capacity in the control state, but always cedurerl it for 1-2 min when bronchoconstriction (from cigarette-smoke or other agents) was present. Locejoy and Dautreband (1963) also studied the effects of cigarette-smoke on the airway conductance in smokers and non-smokers using the bodyplethysmographic method. Their subjects were 10 normal persons and 4 patients with chronic respiratory disease, n ho were requested to inhale , cigarette- ruwl.e, usualh• at a ratc of 10 •''0 puffs in 5 min. -lf ter airwsy resistance and functional residual capacity had been estimated beJore and after smoking, the subjects then toqk 5 deep breaths of a dilator aerosol (Aerolone Compound, Lilly:0.W~, isoproteuenol and 0.5%r cyclopentylamine in a vebicle bt 8f1 7" propylene glycol and 2D iwater) directly from a,generator. Following such inhalation, the measurements were repeated; then another cigarette was smoked, and the meaaurementa repeated once more. In habitual smokers, both normal sub- jecte and patients, inhalation of cigarette-smoke induced a mild airway constriction and a lowering of airway eonductancerl'GV ratio. Breathing the dilator aerosol readily abolished the constriction, and, later on, prevented the ciga- rette-snwke from exerting the usual constricting action The non-smokers behaved less uniformly, 3 of the 5 (2 normals, 1 asthmatic patient) showing considerable discomfdrt, some. times after 1 puff; and, during the onset of the aymptoms (bw'eatfug, pallor, nausea, bradycsrdia, etc.), the ratio in. creased, instead of decreasing, as in smokers. Nine of the subjects, all smokers, were requested to smoke a second cigarette after testing their reaction to a first one, and im- mediatclr before inhaling the dilator aerosol; in some in- stances, the second eigarrtte accentuated the initial constriction, but, frequently, the reaction to a second ciga- rette produced less marked changes than to the fibst one. Hun$iker and lluhhnann (1960) determined respimtorv resistance by meaqs of the esophagus-pressure-messurvig method and pneumotachography before and after sstol,•ing a filterless cigarette. In 4 of 7 healthy persons, and in 11 of 23 patients suffering from chronic spastic bmnchitis or chronic bronchial asthma, flow resistance was clearly incteased by smoking (all of the subjects were habitual smoke.ts). W. Shapiro (1960) stated that the forced expin.,ygram has become one of the tnost useful tools in the evaluation of pulmonary function. In 4 normal male subjects tested by this worker, tbe smoking of 2 standard cigarettes (1 inhalation per min until two-thirds of each cigarette were consumed) had no eSectt on mean volumes per see. In a subsequent report by W. Shapiro and Patterson (1962) on ventilatory functions before and after the smoking-test described above, the subjects consisting of 25 superbly trained athletes (smoking histories not given), 11 non,smokers, and 31 smokers who were not athletes, athletic eonditioni,- g was found to be Associated with an Increased expired vital capacity (EVC), wfthout the eame relative increase in FEV,,o. Chronic Smoking wa,a as- sociated with a decreased \lBC. The presence of bo'th factors (athletic conditioning plus chronic smoking) resulted in large differences in EVC, AIBC, and the entire forced eicpingram in comparable men, the higher values being in nod-smokiag athletes. Repetition of the forced e%pirogram at brief intervals yielded nearly constant values, unaffected by acute cigarette- Produced bv 1'he Council for 0003044 lobucco rt~,-a;ciruSll, Inc.

RESPIRATORY SYSTEAf 133 eI11ULlUS. JYY/IUV 4uu ...... . ^^~~ th°/ thv fnr- tora of athletic conditioning and chronic smoking should be borne in mind when interpreting pulmonary data in relation to predicted normal values. Rot.hfeld, Biber and Berhstein (1961) determined vital capacity, timed vital capacity, MBC, functional residual capacity, and ventilation equivalent in 19 normal subjects and 23 patients with various pulmonary dtsorders, before and immediately after smoking 2 cigarettes. They observed a striking increase in the ventilation equivalent (expressed as liters of air breathed per 100 mm of oxygen consumption) in the normal subjects, but foldnd no signifieant in the patients. No significant changes occurred in the~ot~her meas- urrments. With respect to the failure of patients with pulmo- nary disease to increase their ventilation after smoking, the authors considered it possible that the chronic impa',irment of transportation of oxygen from the atmosphere to the blood induced by the pre-earisting pathologic state was so great, compared to the interference caused by smoking, that the latter added little to the total effect, and thus the,ae was ao significant change in ventilation. Damoisesu and co-workers (1962a, b) measured airway resistance in 9 normal adult non-smokers and 5 smok;'ers during a 5-min control period, during 5 min in which nozt-s<mokers inhaled 6 puffs of cigatette•smoke end smokers inhaled 15 puffs, and during a 5-min period after inhalation of an aerosol of isopropylnoradreluslinc. Residual functional capacity was not modified by inhalation of the smoke, but dynamic resiste ance was increased in the non-smokers from an average of 2.76 to 3.22 em water/5tm'/w. Changes in dynamic resistance of smokers appeared to vary according to their ah.customed smoking habits; 2 who were habituated to a diHerent quality of tobacco than that used in the test showed a net increase in dynamic resistance (from 3.53 to b.32), and, in the other 3 who generally smoked the same quality of tobacco, there was little effect (2.17 to 2.33). The resistances accrued decreased after isopropylnoradrenaline. Atropine administened to 3 nou-smokera did not appreciably modify their response to cterette-smoke. Simonsson (1962) made spirometric studies of 16 normal persons and 16 subjects with pulmonary disease before, immediately after, ant'. 45-60 min after inhaling the smoke fmmn 1-2 eigarettes, measuring forced vital capacity (F4C) and FEV,,o. In 28 of the 32 subjects, there was a decrease in FEVI.o immediately after smoking; after 1 hour, 23 of the subjects bad higher values than immediately after smoking, and 12 of the 32 had values even greater tLau the basal vol- unmes. The decreases In both groups of subjects immediately after smol&g were quite small, but stat;asticaqy significant. In the aormal subjects, there was no signi6cant change in P1'C after amoking; in the group with pulmonary disease (bronchitis and/or chronic obstructive pulmonary disease), FVC decreased in 10 after smoking, while, •ib-6D min later,10 of the 16 values were higber than before smoking. The author considered that the inereased airway resistance after amoking u•as non-speci8c (that is, not due to nicotine), and was elicited by reflexes from the respiratory tract, the magnitude of which was due to the sensitivity of the receptors in the bronchi. In bronchospirometric studies carried out by Carlens and Dahlstrbm (1963) on patients (7 smokers; 4 non-smokers) with res,piratory symptoms but without serious lung damage, there was, in almost all instances, an acute reduction of as much as 20% in ventilation 1-5 min after inhalation of smoke troin a single cigarette. In some cas:es, the reduction lasted for 11-15 min, and was more pronounced in smokers than in non-smokers. Oxygen uptake showed a similar reduction after smoking, ano thie wu wew wucu av,a,yw :.. a,. :...-'":L in non-smokers. Vi"hen these patients smoked a cigarette through one side of a double-lumen catheter, the "amokiug lung" in almost all e,ases showed acute reduction of ventilation values, which was most pronounced in the 7 smokers compared to the 4 non-smokers (Carlens and Dahlstrom, 1964). Oxygen uptake was similarly affected. The unilateral character of the effect appeared to exclude a central reflex mechanism (com• pare Simonsson (19Ei2), above). The study of airway resistance and peak expiratory flow- n.te in emokers and non-smokers by Zarnel, Youssef and Prime (1963) b4e been described above, 523-A. In these subjects, smoking of I cigarette caused an acute increase in airp•ay resistance in both smokers and uon-snwl:eb, but morc severely in the Idtter. This effect did not seem to depend on inhaling the smoke, and was raised substantially even in those who did not appear to inhalc. This observation suggested to the writers thaEt a potent bronchoconstricting fraction of cigarette-smoke was absorbed in the upper respiratory tract, which exerted its action systemically, or el5e that the acute bronchoconstaiction might be a reflex response to irritatlon of the sensory nerve-endings in the upper respiratpry tract )see Simonsson (1962), abovel Rasev, Zogra,fski and Tomov (196A) described certain clinical investagmtions with nieotine-free cigarett.es ("Atro- tabac") prepared from tobacco produced by grafting ordinary tobacco on straononium roots (see below, 1026). The group studied included 33 patients with chronic pulmonary, and combined pulmonary and esrdiac, diseases,17 v: ithout and 16 with ventilatory insufficiency; 23 were smokers of many years' duration, and 10 were nonsmokers. The controls were 10 healthy persons and 5 with chronic pulmonary disEases. Tests were made after smoking regular and ":atrotabac" cigarettes; the latter reduced dyspnea in 6 patients, and re• dueed asthma attacks in 3 patients, after 3 days of use; no changes were recorded in the other patients, except for an increase in dyspnea in 1. \ itel capacity was said to have improved in 24 of 33 patients af ter 1"Ahofabaa" cigarette, but electrocsrdiograma were not affected. We may mention in this place that Guerrant (cited by Swineford, 1962) found that the dyspnea and wheezing which followed c@illing the chest or inhaling tobacco-smoke may be accompanied by hyperventilation, an increase in respiratory effort, and in total air exchange; but there was no increase in resistance to air-flow nor in the effort required to exchange a unit of air per sec. 524. satoora htuscts or wU nESrtnAtvns saaccr (218) 625. i'roeheaf Muscle (M) According to Carlyle (1983), the response of tracheal chains prepared from guinea pigs to nicotine, although varia- ble, usually consisted of an initial contraction, which was followed by relaxation; cooling to 19°C almost el'uninated fhe responses to nicotine, as did reducing the calcium ion in Krebs solution to one-twentieth of normal; raising the concentration of magnesium ion to 4 times normal had no effect on the motor response to nicot)ne. A concentration of 100 flg/m1 nicotiae competitively blocked the response to a massive dose of nicotine; on washing out the nicotine, the motor response to nicotine returned. With respect to other drugs, hexametho- nium, mecamylnmine, ptocaine, and byoscine all blocked the motor response to nicotine (he.remethonium blocked sD the acUone of nicotine). I Produced hti The Council for Tobacco I~VJearclrUSA, In~, 0003045

i 134 TOIiACCU-L•»\Pk:It1ME\TAL AN1) Ct.1MCAL -;TCUII:s. FCPPLE5IENT I For the effect of nik•otine on tracheal volume in dogs, see ladcl and tt tddtrombe (19ti1), above, 5t•L. 526. JJroncAiaf Muscle (249) 52s. Isolated Lung (2•19). Reichertz and Grunberg (19G0) recorded action-potentials and meclranical contractions of isolated lungs of frogs (Rorto earnlenta), and reported that the results obtained with nicotine and other drugs agreed with observations on other organs with smooth muscle, and sup- ported the view that the source of the electro•potetitials in frog lung was the smooth muscle itsell. 528. Perfused or In-Situ Lung (249). Although Loomis (1956)(24flb) had sugGtested that smoking causes broncho- spasm, which may interfere with adequate distribution of air, the studies of Rothfeldp Biber and Bernstein (1961) on normal human subjects and }iatients with pulmonary disorders re- vealed no evidence of bronchospasm alter the smoking of 2 cigarettes (see above, 523•D). In a few instances of subjects given 1 mg nicotine i.td., there was said to be some indication of an increase or a deerease in bronchial-muscle tone (Giusti et al., 1960); but the potent bronchoconstrict.ing fraction of cigarette-smoke is not necessarily nicotine (see Zamel et al., 1963). It has been suggested that increases in airway resist- ance (which may or may not represent bronchoconstriction) may be due, not to nicotine, but to cigarette-smoke-elicited refleres from the upper or lower respiratory tract (Simonisson, 1962; Zamel et al.,1963); a central reflea mechanism for the snioking-induced reduction in ventilation has, however, bern denied (Carlens and Dahlstrfitn, 1964). In in-situ studies by Velluda and co•workers (l~&f) on drug-induced bronchospasm, an anti-asthmatic ef)'cct of nicotine was considered to be due to release of t•atecholamines (species and doses not given in the abstract available). 529. Effect of Nicotine on Ner.nus Control of Ihr Bron- chioles (250). S. Stent, Ferguson and Rapaport (1964) studied reflex pulmonpry va-roconstriction in dogs due to stam>;lation of the aortic body by intra-aortie injection of f-2t1 µg/kg nicotine bitartrate. The injection was followed by a significant r^fles rise in pulmonary vascular resistance (PVR), the sensory receptorc for the rrfie% being aortic chemoreceptors, and the efferent paths being sytnNathetio fibers; but bronchoconstriction was ruled out as a factor in P\'R increase. No rise in PYR was elicited ky stimulating the carotid bod-i• alone (for a fuller account of these experiments, see above, 395). The eaistence of reflexes elicited by cigaiette-smoke inhala- tion and resulting in an increase in ainrav resistance hets been affirmed (Simonsson, 1962; Zamel et al., 1963) and denied (Carlens and Dahlstr&nh, 198•1). The relationship of increased airway resistance to btvnehocoastriction is, however, unclear (scc Comroe and Nadel, 19G2, p. 236). • 530. Effect of Olher Drugs on Nit•oti ine/Cigerette•$make Action (250). In the course of a preceding section (523•D) we have given some account of the effect of certain drugs on the increased airway resistance which sometimes follows acute cigarette-smoking; but there seems to be considerable doubt whether such changes In resistance represent true bronchocon- striction, and also whether they are due to the nicotine 1^entent of cigarette-smoke. lnhalati„a of isoproteretto) prevented or reversed the decrease ilt airway conductance which followed cigarette-smoking (hadel, Tierney and Comroe, 1969; Nadel and Comtroe, 1961; Comroe and Nadel, 1962; Lovejqr and Dautreband, 1963; see 523-D). Atropine did not 91q1rc,•iuhit mKXtn y tile auerauua. W alf t% a) hrMalauCl• w~wtuu/; C~Fant ~p smoke inhalation; w,)proprlndradrenaline df••ma"4t tlr. iucreased airway resistance which sometimes follba•rd ri:•y rette-smoking (Dantoiseau et al:, 19G2a, b; see 52S-Dt. 531. E1Ttrt of A•irotine and To6acto-Smokc on Cilin „iu Inhibition of ciliarq• motility or mucus flow follottine et Ix,sure to tobacco-tarf, cigarette-smoke, or it.- cwistitut•„t. har• been demonstrated frequently with exiierimental uM•..t respirrtorc ephhelium from a wide varMtv of atnntal gtMY•a+ (fre-h-water mua~el; chicken; rat; rabbit; sheep; goat; rat; dog; monkey), including ciliated human respiratary epithe lium (reviews: Liberman, 1961; Smoking and Health, )l4la, pp. 26i-2oat. althuugh utua ui the iuvt-stit.-atiuus hat•c trn•n conducted in vitro, the mtiformity of the inhibitory rfftrtF in a number of different experhnental model: was considered tOIk• impressive (Smoking and Hcslth, 1964, p. 263). ln•vit.. experiments also have demonstrated a ciliastatic effect of cigarette-smoke in the chicken (Battista and h;enslrr• 1tiG5), cat (Dalbamn and Rylander, 1964, 1965), attd'Jog (Guillt•mn et al., 1961). H. L. Falk, Tremer and Kotin (1959) described the effect- of cigarette-smoke and its constiiuents on the rate of floft• of mucus over the surface of isolated strips of rat and rabbit trachea (republished hy Kotin and Falk, 1960). After a ahort preliminary perod of aceeleratoon of flow, there orcurrtYl followire the application of whole cigarette-smoke or of it. various fractions a predominant, ur:ually persistent, decrifty in the rate of mucus flow. After,rnacimunt slowing, a I>rri.wl of gradual recovery was generally observed. 11'hen repeatai applications of smoke were made at 20-min intervals at a time when inhibition from previous exposure hr.d reached it: maximamt, the slou•it.g effect persisted. Fiaretion of mioke was capable of obviating the rrnponse only when it was eitfli• ciently efficient to retain all parQiculate matter, so that gas- phase rnaterial alone remained. Both alkaloids and other chemical compounds in smoke were effective in producing the ciGastatic response. Nicotine produced a trsponse quallita- tivelv similar to that obtained fRwn whole smoke. Thr appli. cation of an aqueaus solution of tars, which had been colkrttYl front filters, resulted in the inhibition of mucus flow typically seen with whole cigarette-smoke. i his effect of cigarette• smoke was sinular to tfiat praducetl by air.).ollutants (Ttenter. Falk and Kotin, J. \at. Cancer Inst. Iv9: 9:9, 1959). The relat;on of these findings to the pathogenesis of pulmtnbary neoplttsms is discussed belott•,1571 and 1614-E. J. J. Ballenger and co-n•orkerK used a modification of the rotating ciliated explant technique of Corssen and allen (Texas Repts. liioL Med. 16: 19t-202, 195g) to ecaminr the effects of cigarette-smoking and nicotine on human ciliated respiratory epithelium. Cigarette•smokc solutions haltetf the ciliary beat in such preparations, irreverFibly so if the sohtEimt were left in contact until the effects became marked (liallen. Rer, 19t10). On ciliated respirattirt• epithe)iunt obtained by tracheal curettage from anestha•tieed children undergoing tonsillectomy, cigarette-smoke solution slowed the ciliary beat (liallenger and Orr, 196'3), but nicotine or "nidiaM" nicotine stimulated ciliary activity (Ballenger et al., 19(i5). The so-called oxidized fomt of nicntine was prepatrd hy a11on•iug redistilled nicotbte to Rtand in sunlight for 9-12 hours (Ballenger et a)., 1965). Cultures rontaining rotat,ing ciliated.cell aggregates u•ere transferred to Rose chamber.: of 2•6-ml volume, and mte of rotation was obtained by micro- scopic observation and stopavstch timing. Nicotine perfustates Prodaced hti The Coilncil for Tobacco kiscai cii-U Sl1l h1c, 0003046 I

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