Tobacco Documents Online

Acknowledgement The AMA-ERF Committee for Research on Tobacco and Health wishes to thank the manv individuals both in this countrv and abroad who participated in the rNsearch which resulted in this publication. Special thanks goes to Edward Domino. \i.D.. Professor of Pharmacologx-. University of Michigan Medical Sc:hool. who assisted the Committee in summarizing Section IV. Central and -\utonomic: \ervous Sx-stem, following the death of Doctor Seevers. We also wish to thank Dr. John C. Ballin and Dr. Ira Singer who served as scientific ~ secretaries to the Committee. and Mr. Leo E. Brown who provided the final stimulus for the production of this publication. Special appreciation is due Ms. Barbara Newkirk. Mrs. Susan Remlev. and Mrs. Shirlev Sullivan of the AMA secretarial staff. and to the personal svcretaries of our Committee members. To Mr. Ralph Linnenburger, Director. and to Ms. Irene Parks Foster. Production Editor of the AMA Department of Creati\e Servic:es. and \ir. Joseph Giacalone. we are indebted for the design and the t%-pc,graph}- of this publication. TIMN 0115739 T200230 I iv

' AMA House of Delegates Action December 1963 "A Res r(:h Studv on Toba(.(u and Ht~,illh - devoted tn the study of human ailmwnts th,+t mav be :aused or aggravated bx sniokiil,. I}I,. t)arti(:ul r element or t+le(nentti that m,i\ he tVl causal r aggraxatin' agents and thr• m('( of their a(:tloll.- T'Il\rIN 0115740 T200231 ~•

Contents preface <ontributor` ~ommittee ~lemh "~hip <<)mmitttt ~tatt,n„ ~r ycitinn Section VI Sumn~1ti~~n F\cwtiuii. 3 Abstr'l( l, 4, tiummar\ Abstracts 52 tiO ti Carcinu~enr~sis Summarv Abstracts 45 i, Ih t ?~ Section X'II Respirator\ S\stt"» "~1 1 ,9 Su mmar% "1 3 31 Abstracts 172 49 Authors Index Secaion III Cardio~~ascular S~~stem 59 Summarv 77 Abstracts 262 367 Participating Institutions Index Section IV Central and .~utonomic 171 \en-ous Srstem 177 Summar}~ Absirm t, 145 182 231 Section V Gastrointestinal Tract Summarv 235 Abstracts 72 237 TIMN 0115741 T200232 vii

P r i ~ v S _,i.110 ~I'4.p A ('up% ri:;ht • P+'3h\ ~tnwrii ~ n \trcii ,ti .~:,m i,itiwn Erlui,itir~n inil Rr-<ui li Fmuml.ilinn LC -H- -IriH1 Reprints of published research reports are not available through the Publisher. Please contact each author directly. Grantee's name is in bold face type in abstract. r t American Medical Association Education and Research Foundation 535 \orth Dearborn Street Chicago. lllinois 60610 TIMN 0115737 T200228

Contributors American Brands. Inc. Bron•n and Williamson Tobacco Corporation Li;gett Group Inc. Lorillard. A Division of Loex%,s Theatres. Inc. Philip Morris Incorporated R. J. Rex•nolds Industries. Inc. T2pp234 TIMN 0115743 x

) , TOBACCO AND HEALTH Compiled by the AMA-ERF Committee for Research on Tobacco and Health 1978 American Medical Association Education and Research Foundation TIMN 0115736 T200227

Dedication This book is dedicated to Maurice H. Seevers. M.D.. Ph.D.. who served <)s Chairman of the AMA-ERF Committee for Research on Tobacco and Health from the time of its inception in 1964 until his death in Mav 1977. Doctor Seevers was Professor and Chairman of the Department of Pharmacologx- at the l'ni.•ersitv of Michigan Medical School from 1942 until his retirement. He distinguished himself as an authority on drug and drug abuse not onlt- at the medical school level but as a consultant to state. federal and foreign governments. private industrv. medical publications, the American Medical Association and health organizations around the world. During his career. he served as a member of the Surgeon General's Committee on Smoking and Health. the President's Committee on \,larihuana and Drug Abuse. the White House Conference Drug Abuse Panel on Narcotics and Drug .'rbuse, the National Research Council's Committee on Problems of Drug Abuse. the AtilA Committee on Alcohol and Drug Dependence, the AMA Council on Drugs. and as a consultant on Drug Abuse to Japan. Thailand. Austra4ia and the United States. The AMA-ERF Committee for Research on Tobacco and Health is pleased to have had the privilege of serving with Doctor Seevers and herewith dedicates this publication to his memory as a physician, scientist, educator, leader and humanitarian. Richard 1. Bing, Zi. D. Stuart Bondurant. ~I. D. Earl A. Evans, Ph. D. Robert J. Hasterlick, M. D. Paul Kotin. tii. D. Marvin Kuschner. M. D. Paul S. Larson. Ph. D. Richard D. Remington, Ph. D. Ira Singer, Ph. D. TIMN 0115738 T200229 ll]

Preface In fanuarN of 1964. the American Medical Association Education and Rrsr,u-c h Foundation (AMA-ERF) entered into a five vear agreernent with six tubmc co companies' to conduct a comprehensive program of research on Toba( co ~)ncl Health. The research was to be devoted to the studv (if human aiiments that ma,' be caused or aograt-ated bv smoking. the particular elc,ment or element." that may be the causal or aggravatino agents and the mechanisms of their action. The six participating tobacco companies pledged to contribute a total of ten million dollars to the AMLA-ERF to finance this five-vear research effort, The ALIA-ERF Board of Directors appointed an eminentlv qualified Scientific Committee' to develop guidelines and suggestions on research policies and procedures. identifv significant areas of research and screen applications for research grants. The agreement was renewed in 1969 for another five vears, terminatin, in December 1973. On July 18. 1972, it was altered to eliminate indusM 's financial commitment in 1972 and 1973 with no new grant applications being accepted in 1973 although grants funded in 1972 were continued to com- pletion. Betcveen 1964 and 1975, 844 researchers in 85 United States and 13 foreign research institutions produced 795 publications and reports on the relation- ships of tobacco and health on (1) Absorption. Distribution. Metabolism, Excretion and Toxicology: (2) Carcinogenesis: (3) Cardiovascular S%-stem: (4) Central and Autonomic Nervous Sy'stem; (5) Gastrointestinal Tract: (6) Re- production: and (7) Respiratorv S}'stem. The content of this publication includes a summary of the research projects conducted in each of the above sections. abstracts of the research projects and the names of the participating researchers and institutions. It is hoped that this information will stimulate additional research in the field of tobacco and health. The American Medical Association and its Education and Research Fmunclation wish to express its gratitude to the Tobacco Industrv for its tin,cnc.ial support, the members of the Scientific Committee, the participating institutions and the 196 researchers without whose dedication and intellect this hook ivould not have been possible. James H. Sammons. M. D. Executive Vice President American Medical Association Education and Research Foundation ' see page x 2 see page xi T200233 TIMN 0115742 ix

The summarv statements in this volume draw attention to the research which has been done in each area supported through the funds awarded by this project. The summaries are intended to be neither complete nor comprehensive but to indicate trends and results. The Committee is proud of and satisfied with the work that has been completed under the sponsorship of the American Medical Association's Projec:t for Research on Tobacco and Health. Important contributions have been made to basic medical science as well as to problems associated with tobacco usage. Valuable information has been obtained relating to distribution, metabolism, excretion and toxicity of nicotine absorbed bv the human bodr via cigarette smoking. In the area of carcinogenesis, the Committee restricted the number of awards because cancer research was being generouslv financed bv the National Institutes of Health and other agencies. Nevertheless, the demonstration of potent co-carcinogens in tobacco tar and the potential ~alue of the measure of inducibilitv of arvl hvdrocarbon hvdroxalase as a de- terminant of susceptibilit}• to lung cancer represent some of the more significant contributions in this area. Emphasis .vas placed on the impact of cigarette smoking on the physiology of the cardiovascular, respiratory and central autonomic nervous svstems. The Committee believes that the bulk of research sponsored by this project supports the contention that cigarette smoking pla}•s an important role in the development of chronic obstructive pulmonarv diseases and constitutes a grave danger to individuals with preexisting diseases of the coronarv arteries. On the central and autonomic nervous svstem important findings %vere made related to effects on behavior and on biochemical mediators elicited bv nicotine. Gastrointestinal tract studies include new mechanisms by which nicotine may influence production of peptic ulcer. In studies in reproduction important insights were gained into the mechanisms of higher center control of releasing factors for pituitary hormone. The Committee wishes to express its appreciation to the American Medical Association and the tobacco industry for sponsoring this effort and to the manv ;rantees and institutions who participated in attempting to develop in- formation and find solutions to gaps in knowledge in this complicated area of scientific inquiry. Mav 27, 1977 T200237 TIMN 0115746 _ Seci Abs Exci Sun Abs A. 11 1; 1! 2( 2 2: 2: 2, 2` 2( X,V

Statement AN(A-ERF Committee for Research on Tobacco and tlealth On Ianuar~. 31. 1964, the American Jledical Association-Education and Research Foundation established the Committee for Research on Tobacco and Health. At its first meeting. the Committee agreed to commit available resources to support projects of high scientific interest undertaken b% in- vestigators in universitv and institutional centers. These studies were to he devoted to human ailments thought to be caused or aggravated by smokin,, and to the particular elements that might be the causal or aggravating agents. The Committee planned to allocate its resources primarilv to three broad categories of research in which there %vere important gaps in knowledge of the effects of smoking: 1) cardiovascular. 2) respiratorv and 3) central and autonomic nervous systems research. the latter because of its relationship to habituation. In all of its funding endeavors, the Committee attempted to support innovative research. Although the individual research projects were av,-arded on the basis of specific targeted investigations inevitably manx- investigators found interesting and fruitful areas for digression which developed during the course of their research. Agreements between investigators and the American Medical Association-Education and Researc'. Foundation required publication of reports in the open scientific literature. During the ten vear active life of the Committee, the membership varied from five to ten. The Committee met on an average of four times per vear to consider applications for new grants and to review the progress of the research being funded. The Committee was active in recruiting scientists to participate in Tobacco and Health research, an area in which they had not been previouslv scientifically active. As further encouragement, a post-doctorate fellowship program was instituted in order to stimulate young scientists to participate in this research program. Throughout its life, the Committee maintained liaison with the National Institutes of Health and The Council for Tobacco Research. U.S.A. keeping th(,se organizations apprised of current trends in the Committee's work and to minimize duplication of effort. The Committee is grateful for the free exchange nf information and the close liaison which was established with these organizations. To further stimulate informational exchange, the Committee convened three workshops. These also served as a measure of progress among its grantees. The workshops proved to be highly successful. In addition, a scientific presentation of the work of the Committee's grantees was held in conjunction with the American Medical Association Annual Meeting in San Francisco in 1968. This meeting constituted a report of progress and in- formation to the profession. At that time a statement was issued to indicate tl;a; the research completed under the aegis of the project had not altered the conclusions of the 1963 report of the Surgeon General. T200236 TIMN 0115745 X111

.v\1A-ERF COtiiMITTEE FOR RF.SEARCH ON TOH ir :CO AND HEALTH \F,E,ninted lunt• 1964 Richard J. B,ing.M. D. 1964-1977 Paul S. Larson, Ph.D. 1964-1977 Professor of Medicine L'niversity of Southern California Director of Cardiology and I ntramural Medicine Huntington Memorial Hospital Haag Professor of Pharmacology Chairman Emeritus, Department of Pharmacology Medical College of Virginia Stuart Bondurant, ?bf. D. 1970-1977 President and Dean Albany Medical College of Union University Charles LeMaistre, M. D. 1964-1966 Chancellor University of Texas Southwestern Medical School, Dallas, Texas Earl A. Evans, Jr., Ph.D. 1969-1977 Richard D. Remington, Ph.D. 1969-1977 Professor and Chairman Emeritus Department of Biochemistry University of Chicago Dean, School of Public Health University of Michigan Robert J. Hasterlik, M. D. 1966-1977 Maurice H. Seevers, M.D., Ph.D.** La Jolla, California CHAIRMAN 1964-1977 ohn B. Hickam, M. D.* 1964-1970 Professor Emeritus Department of Pharmacology University of Michigan Chester M. Southam, M. D. 965-1966 Chairman. Department of Internal Medicine C'niversitv of Indiana Paul Kotin, M. D. 1966-1977 t'ice President, Health, Safety and Environment Johns-Manville Chairman, Dept. of Oncology Jefferson Medical College of Thomas Jefferson University AMA Staff Secretaries John C. Ballin, Ph.D. 964-1965 Greenwood Plaza Marvin Kuschner, M. D. 1969-1977 American Medical Association Ira Singer, Ph.D. 1966-1977 Dean, School of Medicine Health ; ciznce Center State University of New York at Stony Brook American Medical Association Deceased February 9, 1970 * Deceased April 29, 1977 T200235 TIMN 0115744 xi

Sect 27. Effect of Nicotine Treatment on the Metabolism of Nicotine in the %iouse Liver in Vitro ...... 14 28. AIteration of Enzt-me Activitv in Rat Liver Following the Acute and Chronic Administration of Nicotine .................................................................. 15 29. Effects of Increased Liver Metabolism of Nicotine on its Uptake. Elimination and Toxicitv in `lice ..................................................................... 15 30. Stimulation of Nuclear Protein Svnthesis in Rat Liver After Phenobarbital Administration ..... 15 31. Comparison on Effects of Phenobarbital and Nicotine on Nuclear Protein Svnthesis in Rat Liver ..................................................................... 16 32. Potent Inhibitory Action of Pilocarpine on Hepatic Drug Metabolism ..................... 16 33. Copper-Binding Activity of Tobacco Smoke Condensate .............................. 16 34. Isolation of Metal-Binding Fractions from Tobacco Smoke Condensate .................... 17 35. Effects of %tetal-Binding Fractions of Tobacco Smoke on in Vitro Activity of Enzymes ........ 17 36. Potent in Vitro Inhibitors of Tyrosinase: Vtetal-Binding Fractions of Tobacco Smoke Con- densate ................................................................... 17 37. Isolation of Metal-Binding Agents from Lettuce Cigarette Smoke and their Effect on 0,-Uptake of Liver Slices ................................................................ 18 38. Effect of Tobacco Smoke Extracts on Mitochondrial Respiration and Anion Transport ........ 18 39. X%-litol Metabolism in Perfused Rat Liver: Interactions with Gluconeogenesis and Ketogenesis .. 18 40. Molecular Structures and Catalytic Activity. I. Catalytic Polarography of Thiamine. Oxythiamine and Thiamine Phosphates ..................................................... 19 D. Excretion ..................................................................... 19 41. Molecular Structures and Catalytic Activity. II. Proton Reactivity and Catalytic Polarography of Histidine and Related Imidazole Derivatives ....................................... 19 42. Excretion of Nicotine and its Metabolites in Dog and Monkey Saliva ..................... 20 43. GastricExcretionofC"-I`'icotine ................................................ 20 E. Toxic:olog}..................................................................... 21 44. Comparison of Pharmacological Responses to Nicotine and Release of Catecholamines from the -\drenals in Dogs and Monkeys ................................................. 21 45. Comparison of the Effects of Nicotine on Catecholamine Release from Isolated Adrenal Glands of Dogs and Monkeys .......................................................... 21 -t6. Comparison of the Cardiostimulatory Effects of Nicotine in Dogs and Monkeys ............. 21 47. Comparatix e Studies of Hepatic Nicotine Metabolizing Enzyme Activities in Monkeys and Dogs 22 18. Lack of Effect of Chronic Nicotine Administration on Fatty Acid Distribution in the Liver, Testis, and Adipose Tissue of Male Fisher-344 Rats ....................................... 22 -I9. Effects of Cb-ronic Nicotine Adn.inistration arrd Age in Male Fischer-344 Rats .............. 22 50. Hepatic Function after Acute or Subchronic Nicotine Administration in Untreated Mice and Mice Treated with Hepatotoxic Chemicals ............................................. 23 51. Cadmium and Nickel-Common Characteristics of Lettuce Leaf and Tobacco Cigarette Smoke .. 23 52. Biological Disposition of Nicotine: Mechanisms of Protection Against the Acute Toxicity of Nicotine .................................................................. 23 Abs( Excr Sum I n tro( absor t iun i chem 5ecti( r,cloe hePn . contri T tru m c timuki inhali Incon ent eft lipid n marizF tall\'sl tornar\ the cus of toba quantit rettes t hese tc %vithou i n mea: 'i l l not Fo presun stituen ( 2.5.6): carbon isoprer propioi B. Dist Dv tine fo subject leaves ~ ~•arying notablv tributio a.ailabl monkev having toleranc 2 TIMN 0115748 T200239

Section I Absorption. Distribution, Metabolism, Excretion. Toxi(cPlogy Surnnlarx, Abstracts (52) A. Absorption ......................................................... ... ; 1. Effect of Cigarette. Cigar. and Pipe Smoking on Nicotine Excretion. The Influence of fnhalin, t; 2. Distribution of Nicotine in the Rat ......................................... . ...... r; 3. Tissue Distribution of ('H) Nicotine in Dogs and Rhesus ,vtonkeys ..................... . t; 4. Distribution of Nicotine in the Central Nervous Svstem ...................... . ...... - ;. Age Dependent Changes in Nicotine Distribution in the Brain of the Mouse . ......... . . . . . . - 6. Lethal Brain Concentrations of Nicotine in Mice of Different Ages ............... . ........ 8 ;.Accumulation of Nicotine in Pancreatic Islets and Calcitonin-Producing Cells in Mice and Chicks Demonstrated by Micro- and Whole-Body Autoradiography ......................... . . . . 8 8. Catechol- and Indolamines in some Endocrine Cell Systems. An Autoradiographical. His- tochemical and Radioimmunological Study ......................... . . . . . . . . . . . . . . . . t~ 9. Dependence of Nicotine-C" Distribution and Movements Upon pH in Frog Sartorius Muscle .... q 10. Uptake and Distribution of Nicotine-"C in Frog Rectus Abdominis Muscle ................. q 11. Placental Transfer and Distribution of Nicotine in the Pregnant Rhesus Monkey ............. q B. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 10 12. Protein Conformation in Biomembranes: Optical Rotation and Absorption of Membrane Sus- pensions ................................................................... 10 13. Circular Dichroism and Absorption Studies on Biomembranes .......................... 10 14. Corrections for Optical Rotation Data on Biomembranes ............................... 10 C. , tetabolism ................................................................... 10 15. Urinarv Excretion of Conjugate Forms of 1-(3-Pyridyl) Ethanol After Administration of 3- Acetylpyridine .............................................................. 10 16. Studies on the Separation of Acidic Metabolites of Nicotine by Gas Chromatography ......... 11 17. Metabolism of (±)-Cotinine-2-"C in the Rat ........................................ 11 18. The Fate and Distribution of 1-(3-Pyridyl) Ethanol Methiodide in Relation to the Toxicih• of 1-(3- P%'ridyl) Ethanol and 3-Acetylpyridine ............................................. 11 19. Studies on the Excretion of 5-(3-Pyridyl)-Tetrahydrofuranone-2 and its Intermediary Role in the Lietabolism of Nicotine ....................................................... 12 20. Additional Routes in the Metabolism of Nicotine to 3-Pvridvlacetate. The Metabolism of Dihvdrometanicotine ......................................................... 12 21. Pvridvl Acids from the Mammalian Metabolism of Trans-Metanicotine ................... 13 22. Synthesis of Trans-4-(3-Pyridyl)-3-Butenoic Acid, A Metanicotine Metabolite .............. 13 23. Structure o[ Dibromoticonine, A Bromination Product of Nicotine ....................... i 3 24. N-3-Pyridylacetylglycine as a Nicotine Metabolite ................................... 13 25. The Metabolism of Nicotine and Cotinine bv a Mouse Liver Preparation .................. 14 26. Metabolism in Vitro of "C-Nicotine in Livers of Foetal, Newborn and Young Mice .......... 1-1 TIMN 0115747 T200238 1

Absorption. Distribution. Metabolism. Excretion. Toxicologv keys in the third trimester. Nicotine was exchanged rapidlv across the placenta between mother and fetus when it was administered into the maternal circulation in a dose of 1 mg per kg of body weight. The concentration of nicotine in the fetal circulation surpassed the maternal level rapidly, reaching maxi- mum in 16 min and remaining at a higher con- centration for over two hours following the in- jection. Disappearance of nicotine from the fetal circulation was slower than that from the maternal circulation. The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high; that in placenta and skeletal muscles was low. The amount of nicotine contained in a fetus, however, was onlv a small fraction of the total dose administered to the mother. Other support: U. S. Public Health Service. B. Distribution 12. Protein conformation in biomembranes: Optical rotation and absorption of membrane suspensions Dan W. Urry Division of Molecular Biophysics, Laboratory of Molecular Biology. t'niversity of Alabama Medical Center. Birmingham, Alabama Biochimica et Biphysica .4cta 265:115-168, 1972 An analytical review of the above subject is presented. Other support: Mental Health Board of Alabama. 13. Circular dichroism and absorption studies on biomembranes Dan W. Urry, and M. M. Long Laboratorv of Molecular Biophysics. University of Alabama .1.fedical Center, Birmingham, Alabama Methods in Membrane Biology, Volume 1, edited by Edward D. Korn. Plenum Press, pp 105-141, 1974 A discussion of the techniques and results of the measurement of circular dichroism (the difference in absorbance measurement between left and right circular polarized light) and the optical absorption of a number of synthetic macromolecules and bio- membranes is presented. Most of the illustrations are derived from studies by the senior author and his colleagues. Other support: Mental Health Board of Alabama. and the U. S. Public Health Service. 14. Corrections for optical rotation data on biomembranes Dan W. Urry Laboratory of Molecular Biophysics, University of Alabama Medical Center, Birmingham, Alabama Methods in Enzymology: Biomembranes 32:220-233, 1974. Edited by Colowick and Kaplan, Academic Press, Inc. Corrections to be applied to measurements of the circular dichroism patterns of biomembranes so as to ensure their greater accuracy are presented. Other support: Mental Health Board of Alabama. C. Metabolism 15. Urinary excretion of conjugate forms of 1-(3- pyridyl) ethanol after administration of 3- acetylpyridine Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and C. Norman Lukhard Department of Pharmacology, Medical College of Virginia. Richmond, Virginia Journal of Biological Chemistry 241:1878-1881, 1966 Oral administration of 3-acetylpyridine (a me- tabolite of nicotine) to the dog led to the excretion of 1-(3-pyridyl)ethanol in two acidic conjugate forms which were fractionated with the aid of ion ex- change resins. The more acidic of the conjugate forms had the properties of a sulfate ester in comparison with synthetic material. Acidic hydrolysis of the sulfate ester fraction led to the release of (-)-1-(3-pyri- dyl)ethanol. Acidic hydrolysis of the other conjugate form, which had the properties of a glucuronide, led to the release of 1-3(3-pyridyl)ethanol which after chromatographic purification was approximately 95% pure on the basis of extinction coefficient and which was approximately 60% in the levorotatory form on the basis of optical rotation. The pyridyle- thanol separated after the hydrolysis of the glu- curonide contained, in addition, two other Koenig- positive materials. By the chromatographic behavior TIMN 0115756 T200247 of tat giL cy e. [)t: Co An 16. me Her %to DeF Virs t'ir_ gas met met y-(3 acic vest a C, Aqu met p roc colu com of c in ( and sent The butx affoi the i OthE and 17. Paolc and I Depa ['irgi. Jo u rn rneta 10

Absorption. Distribution. Metabolism. Excretion. Toxicolog%- and its metabolites into mice foetuses and placentae. abs 15. In addition. also see three papers comprising basic studies on the circular dichroism and optical absorption of synthetic polypeotides on a variety of biomembranes, since these mav ultimatelv become pertinent in understanding distribution of both en- dogenous and exogenous substances (12. 13, 14). C. Metabolism The complex metabolism of nicotine has been extensivelv and precisely studied by the isolation and characterization of intermediate compounds. etc. Such knowledge is not only important to an understanding of the pathways of biotransformation of nicotine, but also makes possible the further study of any biological activities that are inherent in the transformation products. Studies contributing di- rectlv or indirectlv to knowledge concerning the metabolism of nicotine appearing in this subsec- tion include the following: Urinary excretion of conjugate forms of 1-(3-pridyl)ethanol after adminis- tration of 3-acetvlpyridine (15); Studies on the sepa- ration of acidic metabolites of nicotine by gas chro- matographv (16): Metabolism of (±)-cotinine-2-14C in the rat (17): The fate and distribution of 1-(3- pvridvl)ethanol methiodide in relation to the toxic- itv of 1-(3-pvridy1)ethanol and 3-acetylpyridine (18); Studies on the excretion of 5-(3-pyridyl)-tetrahydro- furanone-2 and its intermediarv role in the me- tabolism of nicotine (19): Additional routes in fhe metabolism of nicotine to 3-pvridylacetate: The metabolism of dihvdrometanicotine (20): Pyridvl acids from the mammalian metabolism of trans- metanicotine (21): Svnthesis of trans-4-(3)-pyridvl)- 3-butenoic acid. a metanicotine metabolite (22); Structure of dibromoticonine, a bromination product of nicotine (23): :\'-3-Pvridvlacetvlglycine as a nico- tine metabolite (24): The metabolism of nicotine is carried out most notablv bv the liver. an activity that is shared in lesser degree bv kidnev and lung, but apparently not b%- brain. Of two studies, summaries of which have been placed in this subsection, one deals with the metabolism of nicotine and cotinine by a mouse liver preparation (?5), the other with metabolfsrn in vitro of "C-nicotine in livers of foetal, newborn and young mice (26). A number of compounds are known to cause enz~Itne induction which may accelerate their own metabolism andlor that of other drugs. In mice. treatment with nicotine for up to 17 days in moder- ate dosage does not appear to significantly affect its metabolism by liver, and in toxic dosage depresses it (27). However, pretreatment with nicotine causes an elevation in the metabolism of ethylmorphine. nor- codeine and aniline by liver microsomes, possibly bv inducing enzyme svnthesis (28). Phenobarbital is a well-documented inducer of manv microsomal enzymes including those causing enhancement of liver metabolism of nicotine both in vitro and in vivo (29). Stimulation of nuclear protein synthesis in rat liver after phenobarbital administration has been the subject of another studv (30), followed by a compari- son on effects of phenobarbital and nicotine on nuclear protein synthesis in rat liver (31). In contrast to the stimulating effect of phenobarbital, pilocar- pine. DPEA. SKF 525-A and imipramine. all have been shown to have inhibitorv effects on the me- tabolism of nicotine bv microsomes obtained from the livers of rabbits (32). For studies on induction of aryl hydrocarbon hvdroxvlase by cigarette smoke see Section II, abs 9. 10. Along a different vein, the possibilitv that tobacco (cigarette) smoke mav contain constituents capable of binding with trace metals in the body, in particular metalloenzyznes thereby inhibiting their metabolic functions has been explored. Tobacco smoke condensate was found to have binding activ- ity for copper, zinc, cadmium, iron and lead (33. 34). Subfractions of tobacco smoke condensate were prepared, certain of which showed inhibitory activ- ity in vitro on galactose oxidase and catalase (me- talloenzymes) but not on glucose oxidase (a gly- coprotein containing flavin-adenine dinucleotide and no metals) (35); certain fractions were also found to inhibit tvrosinase (36). It is suggested that the presence of potent inhibitors of tyrosinase (and other metalloenzymes) in tobacco smoke, if absorbed by an organism, could affect the essential trace metal metabolism, thus impairing vital metal-requiring cellular processes, such as oxidative phosphorvla- tion. lipid metabolism, connective tissue synthesis and repair, and microsomal mixed function oxy- aertases. A smaller amount of cepper-c^~.^.-~p!exina f material was also found in the smoke from lettuce- leaf cigarettes (37). This material inhibited the oxygen uptake of rat liver slices, presumably by TIMN 0115750 T200241 intc~ sm(- aver ope; smo lke, Am l una the tam. rem, carh tion i nca is a perfi and of tF (40) (41). D. E 1 and u nde lism nific cotir (42). terer mu0 and( heim- E. T ( fects are tc port gathe C findii resist tine t 4

4 , t; f, f; 7 8 8 8 9 9 t t > 3 3 3 - .r .-i- a -.-_ s. Section I Absorption, Distribution, Metabolism, gxcretion, Toxicolo9y tiunlnlarV fntrr,cjuction-Clasicall\, knowledge concerning ,,l,:urption. tissue distribution. metabolism, excre- ti„ii and tdxicolog} has become basic to the studv of hemicals to which man becomes exposed. In this tir, tion, some of the contributions to such knowl- ,.r{;e achieved by grantees under this Project have hr•r n assembled: cross-reference is made to related ()ntributions appearing in other Sections. \. .-lbsorption The lesser pharmacodynamic effects resulting trnnl cigar and pipe smoking compared to cigarette ;mnking have been attributed to differences in inhallna practices between these modes of smoking. In connec:tion with studies on quantitativelv differ- rnt effects of cigar, pipe and cigarette smoking on lipid metabolism and catecholamine excretion, sum- marized in Section III (Abs 184). it was experimen- tall% shown (1) that on smoking cigarettes in the cus- tomary manner of inhaling, and cigars and pipes in the customary manner of not inhaling (equal weights nf tobacco being consumed). about three times the quantit\ of nicotine was excreted (urine) with ciga- rettes than with cigars or pipes. However, when t hese tobacco forms were smoked, with inhaling and k% ithout inhaling, there was no significant difference i ii mean nicotine excretion when all were inhaled or All not inhaled. For studies on respiratory tract retention (and t)rr",cnned absorption) of other tobacco smoke con- .tituNnts, see Section VII: ammonia (8); acetaldehyde ( 2.;.fi): acetone (2.8): acetonitrile (2): acrolein (7); i,rrhnn monoxide (2): formaldehyde (7); furan (10); :.nprene (2.9): methanol (9); particulate matter (2); ;)n~pionaldehvde (7): toluene (2.11); benzene (11). li Distribution Dvnamicc changes in tissue distribution of nico- tine following its administration have been the suhject of numerous investigations. Nicotine rapidly 'aves the blood to become more concentrated to ~ arving degrees in the body organs and tissues, most notablv in brain, adrenals, liver and kidneys. Dis- tribution data in relation to time have been made ,rvailable for the rat (2) and the dog and rhesus nlnnkey (3'„ the comparative data in the lattcr study having additional interest in view of the higher tolerance of the monkey for nicotine. More detailed studies have been conducted on the distribution of administered "C-nic:otine in sev- eral tissues. In the central nervous system of mice. the highest level of activity was observed in the arev matter (lower in the white matter): the nuclei of the diencephalon and the medulla obiongata and the cellular lavers of the hippocampus also concentrated activity (4). Study of change with age in mice of nicotine distribution in the brain (3 vs. 12 vs. 26 dav old animals) showed shifting patterns (5). This studv (5) followed on an earlier one (6) in which sig- nificant age dependent differences between lethal brain and blood concentrations of nicotine and latent period before death occurred were found. For additional studies on brain area nicotine levels in relation to behavioral effects see Section IV. abs 54. 55: also. for studies on nicotine distribution and movements in brain slices from monkev_ s and rats see the same section. abs 69. 70. There also appears to be a specific accumulation of administered nicotine in the pancreatic islets and in the parafollicular cells of the thyroid in mice. as well as in the ultimobranchial glands of chicks (7). Since biogenic amines are operative in these en- docrine organs (the ultimobranchial glands produce calcitonin), it is suggested that nicotine can share common transport and/or storage mechanisms with biogenic amines in the cells, and that an effect of nicotine on hormone storage and/or release may take place via an interference with aminergic mech- anisms in the cells. Attention may be called here to a autoradiographical. histochemical and radioimmu- nological study of catechol- and indolamines in some endocrine cell svstems (8). In frog sartorius muscle (9) and frog rectus abdominis muscle (10), uptake and efflux of nicotine has been shown to be dependent upon the cellular pH gradient. Placental transfer of nicotine in the rhesus monkey occurs rapidly, the concentration in the fetal circulation surpassing that in the maternal circula- tion (11). The concentration of nicotine in adrenal glands, heart, kidneys, stomach wall, and spleen of fetuses was high: that in placenta and skeletal muscles was low. The amount contained in a fetus. however, was only a small fraction of the total dose administered to the mother. Also see Section VI for an additional stuiiv on the passage of 1a1--nicCtir:c TIMN 0115749 T200240 3

Absorption. Distribution, Metabolism, Excretion. Toxicology medulla spinalis and medulla oblongata, while on the other hand. radioactivity was very low in the forebrain structures. High radioactive concentra- tions were also seen in the hippocampal formation, lobus olfactorius and cerebellum. Again in the 12- day old mouse the highest levels of radioactivity were seen in the medulla and lobus olfactorius, but in this case the radioactivitv in the forebrain struc- tures was comparatively higher than in the 3-day old mouse. A higher radioactivity was also seen in the hippocampal formation. However. in the CNS of the 26-day old mouse a quite different pattern of dis- tribution was seen. Here the forebrain structures had the highest level of radioactivity and the medulla the lowest. At this age too, the radioactivity was high in the hippocampal formation. Other support: The Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope work). 6. Lethal brain concentrations of nicotine in mice of different ages Torbjorn Stalhandske, and Premysl Slanina (Carl G. Schmiterlow) Department of Pharmacology, Royal Veterinary College. Stockholm. Sweden Acta Pharmacologica et Toxicologica 28:233-240. 1970 Albino mice aged 3 days, 12 days and 35 days, were injected intraperitoneally with lethal doses of "C-nicotine; mice aged 3 and 35 days were given 23 mg/kg and ones aged 12 days 5.4 mg/kg, the doses corresponding to 2-fold LD5o dose for each age. At death the concentrations of nicotine in the brain and blood were measured. Significant age dependent differences between lethal brain and blood con- centrations and latent period before death occurred were found. The brains of mice aged 12 days were found to have the lowest (2.10 ± 0.15 µg/g wet weight) and the brains of mice aged 35 days the highest (8.35 ± 1.02 µg/g) nicotine concentration at death; in mice aged 3 days the concentration was 4.87 - 0.66 µg/g. The lethal concentration of nic- otine in the blood was highest in mice aged 3 days (8.75 ± 1.11 µglg wet weight) and lowest in mice age 12 days (2.11 ± 0.33 µg/g); in mice aged 35 days the concentration was 4.47 ± 0.47 µg/g. Brain/blood ratios increased with age. The latent period before death occurred was shortest in mice aged 12 days (98 ± 8 sec) and longest in mice age 3 days (375 ± 25 sec); in mice aged 35 days in was 157 - 17 sec. The significance of these conditions for age dependent differences in tolerance to nicotine is discussed. Other support: Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 7. Accumulation of nicotine in pancreatic islets and calcitonin-producing cells in mice and chicks demonstrated by micro- and whole-body autoradiography Premvsl Slanina, and Hans Tjalve (Carl G. Schmiterlow) Department of Pharmacology. Royal Veterinar} College. Stockholm, Sweden Journal of Endocrinology 58:21-30. 1973 By autoradiographic methods, nicotine was shown to be specifically accumulated in the pan- creatic islets in mice. The results also indicated a high accumulation of nicotine in the parafollicular cells of the thyroid in mice and an accumulation was also shown in the ultimobranchial glands in chicks. Like the parafollicular cells of the thyroid in mam- mals, the ultimobranchial glands of birds are known to produce calcitonin. Metabolic studies with nic- otine in vitro and autoradiographic studies with the main nicotine-metabolite cotinine, indicated an ac- cumulation of unchanged nicotine (not metabolites) in the cells. The results are discussed in view of the fact that biogenic amines have been shown to be operative in these endocrine organs. It is suggested that nicotine can share common transport and/or storage mech- anisms with biogenic amines in the cells. An effect of nicotine on hormone storage andlor release may take place via an interference with aminergic mech- anisms in the cells. Other support: Swedish Tobacco Company, and 'Ragnar and Torsten Soderbergs Stiftelse'. 8. Catechol- and indolamines in some endocrine cell systems. An autoradiographical, histochemical and radioimmunological study Hans Tjalve (Carl G. Schmiterlow) Department of Toxicology, University of Uppsala, and the Department of Pharmacology, Royal Veterinary CnIlege .Stnr.kholm. Sweden Acta Physiologica Scandinavica. Supplementum 360:1-122, 1971 A study is reported focusing on extraneuronal localizations and functions of catechol- and in- 9. m Gi Dt 1'1 Jo 16 fin 26 TIMN 0115754 T200245 8

,s Y ;s \V rt re- of ;A, 21. Pyridyl acids from the mammalian -metabolism of trans-metanicotine Roger H., .1,4eacham. Jr.. C. T. Sprouse. Edward R. Bou•man. and Herbert McKennis. Jr. .~fedical College of Virginia. Richmond, Virginia Federation Proceedings 32:511, 1973 The natural occurrence of metanicotine (3-(4- methN'lamino-l-butenyl)pyridine) and dihvdrometa- nicotine has given each of these alkaloids a possible role in the metabolic degradation of the pyrroli- dine ring of nicotine. For current studies on the metabolism of inetanicotine, trans-benzoylmetanico- tine, from the Pinner-Etard reaction of benzoyl chloride and nicotine, was freed of the cis isomer by frational crystallization. Trans-metanicotine from acidic hvdrolysis of the benzoyl compound was administered (15 mg/kg) orally to male dogs. The pyridyl acids in the urine were concentrated with the aid of Dowex 50 (H') and Dowex 21K (OH-), and then converted to methyl esters. Gas chromatogra- phy (30% SE-30, 205°) provided two major com- ponents (retention time 10 min and 25 min). The 25 min component formed a picrate, m.p. 127-128°. Data (NMR, UV, IR and mass spectral) on the free base was consistent with the methyl ester of trans-4- (3-pyridyl)-3-butenoic acid. Similar evidence led to identification of the other major components as 2- pvridylacetate, previously identified as a mamma- lian metabolite of nicotine and dihydrometanico- tine. Other support: Council for Tobacco Research- L'.S.A.. and The American Tobacco Company. 22. Synthesis of trans-4-(3-pyridyl}3-butenoic acid, a metanicotine metabolite C. T. Sprouse. and Herbert McKennis, Jr. Department of Pharmacology, Medical College of l"irginia. Richmond, Virginia 1'irginia Journal of Science 24:220, 1973 in the metabolism of cis and trans isomers of metanicotine in the dog, a number of pyridyl car- boxylic acids are formed. Physical and chemical data suggested that one of the metabolic acids was trans- 4-(3-pyridyl)-3-butenoic acid, which is subsequently metabolized to 3-pyridylacetic acid. To facilitate fLrtl:er studies, and confirmation of structure, a svnthesis of trans-4-(3-pvridyl)-3-butenoic acid was sought. 1-Methoxy-2-(3-pyridyl) ethylene, prepared from pyridine-3-carboxyaldehvde and methox}-me- thylenetriphenyl phosphorane, was reacted w' ith carbomethoxymethylenetriphenvl-phosphorane to form methyl trans-4-(3-pvridvl)-2)butenoate. Upon treatment with aqueous base, for hydrolvsis and isomerization, this ester afforded trans--t-(3-pyrid%•1)- 3-butenoic acid in good yield. The synthetic product was identical in all respects to the metabolite pre- viously isolated from dog urine. Other support: Council for Tobacco Research- U.S.A., and the American Tobacco Compan\. 23. Structure of dibromoticonine, a bromination product of nicotine Herbert McKennis, Jr., Edward R. Bowman, Louis D. Quin. and Ronald C. Dennev Department of Pharmacology, Virginia Commonwealth University, Richmond, Virginia, and the Gross Chemical Laboratory, Duke University, Durham. North Carolina Journal of the Chemical Society; Perkin Transactions I: Organic and Bio-organic Chemistry 1:2046-2049. 1973 The fate of the pyrrolidine ring of nicotine on its oxidative bromination to dibromoticonine has been re-examined. From chemical and spectral (including "C n.m.r.) properties dibromoticonine is now es- tablished as 3,4-dibromo-5-hydroxy-l-methvl-5-( 3- pyridyl)-0'-pyrrolin-2-one. The bromine atoms can be selectively replaced by hydrogen under proper reductive conditions to give the isomeric monobro- moticonines. Other support: American Tobacco Company, and the Council for Tobacco Research-U.S.A. 24. N-3-pyridylacetylglycine as a nicotine metabolite Edward R. Bowman. Ravmond S. L. Chang, C. T. Sprouse, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia, Richmond, Virginia Abstract, 27th Tobacco Chemists' Research Conference, Winston-Salem, North Carolina, October 3-5. 1973, p32 Occurrence of metanicotine as a possible bac- terial metabolite of nicotine (Wenusch) and as a tobacco smoke constituent led to a study of mamma- lian metabolism of metanicotine, in which 3-pvr- idylacetate was identified as a metabolite. Although TIMN 0115759 T200250 13

Absorption. Distribution. Metabolism. Excretion. Toxicology ternan- ammonium compounds, showed no great tendency to pass the blood-brain barrier. In rats, after i.p. administration. and mice, after i.v. administra- tion, the radioactivity of (+,-)-N-methyl-''`C-3-(1- hvdroxvethvl) pvridinium iodide was eliminated largely (78-90%) by way of the urine within 24 hr. This excreted radioactivity was predominantly in the form of the administered cation. The signs of acute toxicity from large doses of (+.-)-1-methyl-3-(1-hvdroxyethvl) pvridinium io- dide differed from the previouslv cvell-established similarity of those arising from 3-acetylpyridine and (+,-)-1-(3-pyridyl)ethanol; and the levorotatory form of 1-(3-pyridyl)ethanol appeared to produce gross toxic effects similar to those from the racemic alcohol. Other support: U. S. Public Health Service, the Council for Tobacco Research-U.S.A., the Ameri- can Tobacco Company, and the Swedish Tobacco Company. 19. Studies on the excretion of 5-(3-pyridyl)- tetrahydrofuranone-2 and its intermediary role in the metabolism of nicotine Edward R. Bowman (Herbert McKennis, Jr.) Department of Pharmacology, Medical College of Virginia. Richmond. Virginia Virginia Journal of Science 19:115-121, 1968 After the administration of 5-(3-pyridyl)tetrahy- drofuranone-2 to the rat, the urine contains five or more Koenig-positive compounds. Two of these, metabolites of the administered compound, were isolated by means of gas chromatography and identified (in derivative form) as y-(3-pyridyl)--~- hydroxvbutyric acid and 3-pyridylacetic acid. The pattern of excretion of the Koenig-positive com- pounds resembles in part that obtained from the administration of nicotine or cotinine and provides additional evidence for the intermediary role of -y(3- pyridyl)--y-hydroxybutyric acid, or the correspond- ing lactone 5-(3-pyridyl)tetrahydrofuranone-2, in the formation of 3-pyridylacetic acid from nicotine. In the rat 5-(3-pyridyl)tetrahydrofuranone-2 was vir- tuallv devoid of the CNS depressant properties of y- butyrolactone. In common with cotinine and other metabolites of nicotine, the acute toxicity of 5-(3- pyridvl)tetrahydrofuranone-2 was considerably less than that of the parent nicotine. This studv was the subject in part of a preliminary report appearing in Federation Proceedings 26(2):616, 1967. Other support: Council for Tobacco Research-USA, and the American Tobacco Company. 20. Additional routes in the metabolism of nicotine to 3-pyridylacetate. The metabolism of dihydrometanicotine Roger H. vieacham. Jr., Edward R. Bowman, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia. Richmond, Virginia Journal of Biological Chemistry 247:902-908, 1972 The metabolism of dihydrometanicotine was investigated in the rat and the dog. In preliminary studies, dihydrometanicotine difumarate was ad- ministered to rats. The Rf values and retention times of methyl esters prepared from acidic Koenig-posi- tive metabolites in the urine suggested the presence of 3-pyridylacetate and 4-(3-pyridvl)butyrate. Fol- lowing administration of dihydrometanicotine di- fumarate to dogs. the pattern of excretion of acidic metabolites in the urine was similar to that found in the rat. Chemical conversion of the acidic metabo- lites to their methyl esters for separation by pre- parative gas chromatography afforded methyl 3- pyridylacetate and methyl 4-(3-pyridyl)butyrate. Methyl 3-pyridylacetate was identified by melting point, elemental analysis, and the infrared spectrum of the methyl ester picrate. Methyl 4-(3-pyridyl) butyrate was identified by mass spectroscopy and by the melting point, elemental analysis, and the in- frared spectrum of the methyl ester picrate. Previous studies have provided evidence for the formation of 3-pyridylacetate from (-)nicotine via either (-)co- tinine or (-)demethylcotinine. In view of the report (de Clercq, and Truhaut, Bull Soc Chim Bio144:227, 1962) that dihydrometanicotine is a metabolite of nicotine in the rat, the present findings in the rat and dog suggest a third alternative route (via dihydro- metanicotine) to 3-pyridylacetate. This study was the subject in part of a pre- liminary report appearing in the Virginia Journal of Science 22:133, 1971. Other support: Council for Tobacco Research-USA, and the American Tobacco Company. TIMN 0115758 T200249 21. meta Roge Bo«•' Medr Feder meth nicot role dine meta tine. chlor fratio acidi, admi pvrid the ai then phy I ponei min ~ Data base x ( 3-pvi identi pyrid- lian i tine. Other U.S.A 22. S~ acid, . C. T. S Depart Virgini Virgini In metan boxyli sugge< -}-(3-p, metab, f urthet 12

Absorption. Distribution. `ietabolism. Excretion, Toxicologv prior studies provided no firm evidence for con- jugation of 3-pvridylacetate. analagous substances are well known for their ability to form conjugates in both plants and animals. Administration of synthetic N-3-pvridt•lacetvlglvcine to dogs led to urinary ex- cretion of 3-pvridvlacetate. while administration of 3-pvridvlacetate led to urinary excretion of V-3- pvridvlacetylglvcine. In light of the previous iso- lation of 3-pyridvlacetate as a nicotine metabolite, N- 3-pyridylacetylglycine itself is now implicated in the metabolism of nicotine. Amounts of the con- jugate formed and excreted are theoretically de- pendent upon competing biological factors. 25. The metabolism of nicotine and cotinine by a mouse liver preparation Torbjorn Sta' lhandske (Carl G. Schmiterlow) Department of Pharmacology, Royal Veterinary College. Stockholm, Sweden Acta Physiologica Scandinavica 78:236-248. 1970 The metabolism of nicotine and cotinine by a 10.000 x g supernatant fraction of mouse liver homogenate has been studied by using "C-labelled compounds. The metabolism of nicotine was found to be TPNH and 0, dependent and chromatographi- cal evidence for the formation of cotinine, y-(3- pvridvl)-y-oxo-N-methylbutyramide and hydroxyco- Linine are presented. Three unidentified metabolites and delayed formation of small amounts of 14COZ were also observed. The observed metabolites were also excreted in urine after intraperitoneal adminis- tration of nicotine. Chromatographical evidences revealed that cotinine is metabolized to hydroxy- cotinine, y-(3-pvridvl)-y-oxo-N-methylbutyramide and/or demethvlcotinine and one unidentified me- tabolite. Other support: the Swedish Tobacco Company. and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 26. Metabolism in vitro of 14C-nicotine in livers of foetal, newborn and young mice T. Stalhandske. P. Slanina, H. Tjalve, E. Hansson, and C. G. Schmiterlow Department of Pharmacology. Royal Veterinary College, Stocl:ho!m. Stredcn Acta Pharmacologica et Toxicologica 27:363-380, 1969 The metabolism of nicotine in the livers of fetal, young and adult mice was studied in vitro. The metabolism of nicotine in the fetal liver with the formation of cotinine was seen at the end of fetal life. Cotinine was found to be the major metabolite at all of the ages investigated. After birth the ability of the liver to metabolize nicotine increased and ap- proached the adult level at four weeks of age. The LD,o value for intraperitoneally injected nicotine increased with age in two to eight weeks old mice. A relation between the rate of metabolism in vitro and the lethal toxicity of nicotine in young mice was observed. However, despite a very low metabolism of nicotine in the livers of three day old mice, the LD5o at this age was almost of the same order as for adult mice. In three day old mice, nicotine did not induce the convulsive pattern seen in adult mice. Other support: Swedish Tobacco Company, and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 27. Effect of nicotine treatment on the metabolism of nicotine in the mouse liver in vitro Torbjorn Stalhandske. and Premvsl Slanina (Carl G. Schmiterlow) Department of Pharmacology. Royal Veterinary College. Stockholm, Sweden Acta Pharmacologica et Toxicologica 28:75-80, 1970 'aC-nicotine was incubated with a 10,000 x g supernatant fraction of liver homogenate from mice pretreated with nicotine for 3, 10 and 17 days, respectively. The rate of metabolism of nicotine was measured by the determination of the oxidative nicotine metabolite, cotinine. After intraperitoneal injections of 5 mg/kg of nicotine three times daily for three days, a 50% significant decrease in the me- tabolism of nicotine was observed. A 71% sig- nificant decrease in hepatic glycogen was also seen. Giving nicotine 27.8 and 24.5 mg/kg/24 hours in drinking water for 10 and 17 days respectively, did not significantly change the metabolism of nicotine or the hepatic glycogen levels. Other support: Swedish Tobacco Company, and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). TIMN 0115760 T200251 28. foil niu R. IDep .% fec. Tox 19%( mg; pvrc adrr reac retu trati 3 cc mor choi c hro in t elev, code incrE we6 How dav. activ amin An i syntl treat: indic meta acute conv suggt drug that i ncrei possil T repor 28(2): 14

Absorption. Distribution. Metabolism. Excretion. Toxicology the nuclear fraction on DEAE-cellulose columns indicated that there was a 5-fold stimulation in the specific activity of one of the protein peaks from the nuclear protein fraction of the Pb-treated rats. These data indicate that Pb administration enhances the synthesis of various protein fractions in hepatic cells and suggest that these events may be involved in the enzyme induction produced by this drug. Other support: U. S. Public Health Service. 31. Comparison on effects of phenobarbital and nicotine on nuclear protein synthesis in rat liver Raymond W. Ruddon, and Cynthia H. Rainey Departments of Pharmacology and Oral Biology, University of Michigan Medical and Dental Schools, Ann Arbor. Michigan FEBS Letters 14:170-174, 1971 In order to determine whether there might be a relationship between nuclear protein synthesis and elevated enzyme activity, nicotine was administered to rats in the drinking water at a dose (5 mg/kg/day for 7 days) previously observed to increase hepatic microsomal enzyme activity. There was an increase in the incorporation of labeled leucine Snto the total non-histone nuclear protein fraction after a 40 min- ute pulse in the nicotine-treated rats. Doses of nicotine of this magnitude do not produce an increased uptake of labeled amino acid into the acid soluble pool. Electrophoretic analysis of hepatic nuclear proteins showed no difference between the absorbancy profile of the proteins extracted from nicotine-treated and control animals. The radioactiv- itv contained in the major peaks was generally higher in the nicotine-treated samples, in agreement with the higher specific activity observed in the total nuclear protein extract from the nicotine-treated rats. However, when the ratio of the cpm-treated/ cpm-control was plotted from the radioactivity con- tained in the acrylamide gels, there was an apparent selectivity to the labeling pattern. A comparison of relative migrations of labeled nuclear protein fractions whose synthesis is stimu- lated by either phenobarbital or nicotine administra- tion is included. On the basis of the available evidence, the authors propose a schematic model for the control of gene readout in mammalian cells. Other support: U. S. Public Health Service. 32. Potent inhibitory action of pilocarpine on hepatic drug metabolism Akira Tsujimoto, Toshihiro Dohi. and Sekizo Kojima Department of Pharmacotogv. Hiroshima University School of Dentistry. Hiroshima. Japan Japanese Journal of Pharmacology 22:736-739. 1972. Pilocarpine is shown to have an inhibitorv effect on the metabolism of nicotine bv microsomes ob- tained from the livers of rabbits. Pilocarpine was also found to have an inhibitory effect on the metabolism of hexobarbital and of aniline. Other findings in- dicated that pilocarpine may interact with the mi- crosomal cytochrome p-450 with a greater affinitv than that of nicotine. In the same preparation, DPEA (2.4-dichloro-6- phenylphenoxyethylamine hydrobromide). SKF 525- A (2-diethylaminoethyl-2,2.diphenylvalerate hydro- chloride), and imipramine were shown to be inhibitors of nicotine metabolism. 33. Copper-binding activity of tobacco smoke condensate Leslie W. Michael, Edward E. Menden, and Harold G. Petering Department of Environmental Health, College of Medicine. University of Cincinnati, Cincinnati, Ohio Environmental Science and Technology 5:249-251, 1971 The distribution of copper between a buffered aqueous solution of the metal ion and an organic solution of tobacco smoke condensate (Tsc) in 4- methyl-2-pentanone (MIBK) was studied. The ana- lytical parameters, metal ion and hydrogen ion concentrations, were investigated. Metal ion concen- trations were determined by atomic absorption spec- trophotometry. TSC was collected with the use of a Mason Mark III Smoker and acetone traps main- tained at 0°C. This material was transferred to MIBK by evaporation at reduced pressure. A 10-ml aliquot of an aqueous solution 0.1 M in 2-amino-2-(hy- droxymethyl)-1,3-propanediol (TRIS) 0.01 M in CuClZ and pH 5.6 to 6.1 was equilibrated with an equal volume of the MIBK solution of TSC, which contained the equivalent of five cigarettes. Analysis of an aliquot from the organic phase after equilibration of the two phases yielded a maximum value of 300 µg TIMN 0115762 34• tob+ \'in, Pete Dep 11ec. Ent rest exc Cu ing and sis : tron cop 42 f rac p rot f rac beh; T he c:a d t binc rnet rett iron OtH 35. smo Vinc Depc .tifed A rc1- pres tabo nun- sate T200253 16

he -al in he ral 28. Alteration of enzyme activity in rat liver following the acute and chronic administration of nicotine R. W. Ruddon, and A. M. Cohen Department of Pharmacology. University of Michigan ttedical School, Ann Arbor. Michigan Toxicologt' and Applied Pharmacology 16:613-625, 1970 The intraperitoneal injection of nicotine (2 or 4 mg/kg) to rats produced an increase in tryptophan pvrrolase activity in liver within 2 hours after drug administrati.on. The elevation in enzyme activity reached a peak at 4 hours after drug injection and returned toward control levels by 6 hours. Adminis- tration of 4 mg of nicotinelkg to rats 4 times a day for 3 consecutive days produced an elevation of ethyl- morphine and norcodeine metabolism in postmito- chondrial supernatant fractions from liver. The chronic administration of nicotine (5.9 mglkg/day) in the drinking water for 7 days produced an elevation in the metabolism of ethylmorphine, nor- codeine. and aniline by liver microsomes. This increased enzyme activity was maintained for 5 weeks when nicotine was continued at that dose. However. if the dose was lowered below 4.4 mg/kg/ day, the increased activity was not maintained. The activities of tryptophan pyrrolase and tyrosine trans- aminase were not altered by chronic drug treatment. An increase in the capacity of liver microsomes to scnthesize protein was observed when nicotine treatment was continued for 4 weeks. These data indicate (1) that an elevation of microsomal drug- metabolizing enzymes can be achieved after the acute administration of nicotine, but only when convulsive doses are given several times a day, suggesting that the increased activity after acute drug treatment may be a stress-related event; and (2) that the chronic administration of nicotine can increase the activity of drug metabolizing enzymes, possibly by inducing enzyme synthesis. This study was the subject of a preliminary report appearing in Federation Proceedings 28(2):418, 1969. 29. Effects of increased liver metabolism of nicotine on its uptake, elimination and toxicity in mice Torbjorn Stalhandske (Carl G. Schmiterlow) Department of Pharmacology. Royal Veterinary College. Stockholm. Sweden Acta Physiologica Scandinavica 80:222-234. 1970 Phenobarbital is a well documented inducer of many microsomal enzymes. In the present stud,,•. male albino mice were injected intraperitoneally or intravenously with 14C-labeled nicotine. Both un- treated and phenobarbital pretreated mice were used. The concentrations of nicotine and metaboli- cally formed cotinine were determined in the brain, liver and blood 1, 2.5, 5, 10, 20 and 60 minutes after injection. It was found that phenobarbital treated mice exhibited an enhanced liver metabolism of nicotine both in vitro and in vivo. The increased liver metabolism caused a significant decrease of nicotine concentration in the brain onlv when nic- otine was given intraperitoneallv. Phenobarbital pre- treatment elevated the intraperitoneal LD;,, value 2-3 times and also increased tolerance to repeated sub- lethal doses of nicotine. No change in the in- travenous LD, was observed. Other support: Swedish Tobacco Company, and the Knut and Alice Wallenbergs Stiftelse (for general equipment for isotope research work). 30. Stimulation of nuclear protein synthesis in rat liver after phenobarbital administration Raymond W. Ruddon, and Cynthia H. Rainev Departments of Pharmacology and Oral Biology, University of Michigan Medical and Dental Schools, Ann Arbor, Michigan Biochemical and Biophysical Research Communications 40:152-160, 1970 When a 40 min pulse of leucine-'H was ad- ministered to rats 2 hrs after 75 mg/kg of phenobarbi- tal (Pb), there was a 70% increase above control in labeling of microsomal protein and of a nuclear protein fraction extracted with 1 M NaCl. When a 40 min pulse of labeled leucine was given 24 hrs after a single injection of Pb (75 mg/kg), there was a 42% stimulation in the specific activity of microsomal protein but an 87% enhancement, compared to control, in the nuclear protein fraction. Separation of TIMN 0115761 T200252 15

_cse wn ce. 'ter- its s it an ,or- bly I is nal of ivo rat the ari- on •ast :ar- ave ne- om i of see hat mts , in leir ccC' 4). ,re iv- ie- iv- .de rnd the her .. an etal ing vl a- esis Ixv- ing ice- the by interac:tiun with a metalloenzyme system. The bio- I,,,ical significance of these findings must await ;muke exposure studies in animals. but a new d%encle for exploration appears to have been opened. The effects of various fractions of whole tobacco srnoke extracts on the metabolism of rat heart and li%er mitochondria have also been investigated (38). :ymong findings. a fraction extractable with ether uncoupled oxidative phosphorylation and inhibited the transport of pyruvate, a-ketoglutarate and glu- tarnate into mitochondria. Other components which remained in the aqueous phase (mainly cyanide and carbon monoxide) inhibited mitochondrial respira- tion. Observations on rat lung mitochondria are also included. Although unrelated to tobacco, attention is called here to studies on "Xvlitol metabolism in perfused rat liver: interactions with gluconeogenesis and ketogenesis" (39), and polarographic behavior of thiamine. oxythiamine and thiamine phosphates (40) and histidine and related imidazole derivatives (41). D. Excretion For findings on urinary excretion of nicotine ,ind certain of its metabolites, abstracts collected under the subsections on Absorption and Metabo- lism (above) may be consulted. Additionally, sig- nificant salivar_v excretion of nicotine, largely as cotininet in the dog and monkey has been reported I4''). There also occurs concentration of adminis- tered nicotine in the glandular portion of the gastric mucosa and in the stomach contents of mice, rats and cats. with both unchanged nicotine and cotinine being found (43). E. Toxicology Observations on toxic or potentially toxic ef- fects of tobacco smoke or certain of its constituents are to be found throughout the Sections of this Re- port (possim). A few studies have, however, been ;athered here under this rubric. Considerable interest has been aroused by the finding that the rhesus monkey is markedly more resistant to pharmacologic and toxic effects of nico- tine than is the dog, and possible reasons for this have been explored. Findings have been made suggesting that the difference in the pharmacologi- cal responses to nicotine of dogs and monkeVs. except that of respiration. may result in part from the less potent effect of nicotine on catecholamine release from adrenal glands in monkeys than in dogs (44, 45). as well as a lesser amount of norepinephrine in the monkey heart for release (46). Microsomal nicotine-metabolizing enzyme activities in the liver of monkey were found to be significantly greater than those in the liver of dogs (47). However, from a lethal effect standpoint, differences in tissue dis- tribution of administered nicotine between the two species (lower in brain and higher in skeletal muscle of the monkey) may partially explain the lesser sensitivity to nicotine of the monkey as compared to the dog (3). Two studies have examined effects of chronic (2 and 22 months) administration of nicotine in a "smoking" dose in male rats. In one of these, no effect was found on fatty acid distribution in the liver, testis, and adipose tissue as compared to controls (48). In the other study (49), no significant changes in hematologic values were found as com- pared to controls, and there was no effect on the Ca2+-dependent myosin ATPase activity or on the lactic dehydrogenase activity and isoenzyme pattern from predominantly fast (gastrocnemius), slow (so- leus) or cardiac muscles: MgZ'-dependent ATPase activity from soleus muscle was considerably de- pressed after 22 months of nicotine treatment. Tu- mor incidence did not differ significantly between nicotine-treated and control animals. but the in- cidence of Leydig cell hyperplasia was higher in the treated rat. A study of possible interactions of nicotine with the known hepatotoxic agents carbon tetrachloride, chloroform, and 1,1,1-trichloroethane in mice was also conducted (50). Findings were negative. Nico- tine administered alone did not affect liver function. nor did it modify the cholestatic effect 2-naphthvli- socyanate. Concern has been expressed as to possible toxic effects of cadmium (e.g. in production of emphy- sema) and nickel (carcinogenesis) present in to- bacco. As regards tobacco substitutes, a study has found significantly higher Cd and Ni content of lettuce cigarettes in comparison with tobacco ciga- rettes (51). In the direction of protecting against the acute toxicity of nicotine, is a study demonstrating TIMN 0115751 T200242 5

S at z t dolamines. An extensive bibliography is also in- cluded. Othersupport: the Swedish Medical Research Coun- c il. the Knut and Alice «'allenbergs Stiftelse, and The Ragnar and Torsten Soderbergs Stiftelse. 9. Dependence of nicotine-C14 distribution and movements upon pH in frog sartorius muscle George B. Weiss Department of Pharmacology. Medical College of l7qainia. Richmond. Virginia lournal of Pharmacology and Experimental Therapeutics 160:13 5-147. 1968 The uptake and efflux of inethyl-C'{-labeled nicotine in frog sartorius muscle were measured at pH 8.40 and pH 7.40. At both pH levels nicotine-C'a uptake is about 90% of maximum in 10 minutes. The total tissue space occupied after 1-hour incubation is 2.722 = 0.155 times the extracellular concentration of nicotine at pH 8.40 and 1.003 ± 0.089 times at pH 7.4. Lowering the extracellular pH initiates a sus- tained increase in the efflux rate of nicotine-C'}, whereas raising the pH decreases efflux. Addition of 2.50 mM nonradioactive nicotine induces a transient increase in the rate of loss of nicotine-C" which is larger at pH 8.40 than at pH 7.40. Nornicotine (10.0 m%f) initiates a similar but much smaller transient increase in nicotine-C" efflux and also a sustained increased efflux, whereas cotinine, a nicotine me- tabolite, has no effect on nicotine-C'" uptake or efflux. Intracellular pH estimations derived from C14- labeled 5,5-dimethvl 1-2,4-oxazolidinedione tissue spaces indicate that nicotine is distributed across the cell membrane in accordance with the pH gradient. The transient increase in nicotine release may rep- resent a displacement of bound nicotine from sites in the cell membrane, whereas alterations in extracellu- lar pH are rapidly followed by shifts in intracellular nicotine-C" that appear to be due to an altered backflux of nicotine-C14 into the muscle cell. This study was the subject in part of a pre- Iiminary report appearing in Federation Proceedings 26:397, 1967. 10. Uptake and distribution of nicotine-"C in frog rectus abdominis muscle G. B. Weiss Departments of Pharmacology. .lledical College of Virginia. Virginia Commonwealth Universitc. Richmond. Virginia and The University of Texas Southwestern Medical School, Dallas. Texas Archives Internationales de PharmacodYnamie et de Therapie 195:99-108, 1972 The effects of variation of extracellular pH as well as of intracellular pH changes induced bv procaine or iodoacetic acid were determined in isolated frog rectus abdominis muscles. Intracellular pH was estimated with14C-labeled 5.5-dimethyl-2,4- oxazolidinedione. The uptake of nicotine was de- pendent upon the cellular pH gradient. Raising extracellular pH or addition of iodoacetic acid in- creased the pH gradient and the tissue uptake of methyl-"C-labeled nicotine. Lowering extracellular pH or addition of procaine decreased the pH gra- dient and nicotine-14C uptake. Efflux of nicotine-"C was increased by a decreased pH gradient and inhibited by an increase in extracellular pH. Appar- ent changes in efflux rate induced by extracellular pH changes can be attributed to an altered nicotine- "C reuptake. The uptake and movements of nic- otine-"C in frog rectus abdominis muscle are quali- itatively similar to those previously observed in the less sensitive frog sartorius muscle. 11. Placental transfer and distribution of nicotine in the pregnant rhesus monkey Kotaro Suzuki. Terusada Horiguchi, Arsenio C. Comas- Urrutia, Eberhard Muller-Heubach. Hisayo 0. Morishima, and Karlis Adamsons Department of Obstetrics and Gynecology. Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts; the Department of Obstetrics and Gynecology and the Department of Anesthesiology. College of Physicians and Surgeons, Columbia University, and the Department of Obstetrics and Gynecology and the Department of Pharmacology, The Mount Sinai School of Medicine of the City University of New York, New York American Journal of Obstetrics and Gynecology 119:253-262, 1974 Placental transfer and distribution of nicotine in fetal tissues were determined in 10 pregnant mon- TIMN 0115755 T200246 9

Absorption. Distribution. Metabolism. Excretion. Toxicology that agents which prevent the cardiovascular effects of nicotine reduce the uptake of nicotine by brain and thereb~~ decrease the intensitv of its actions (52) A. Absorption 1. Effect of cigarette, cigar, and pipe smoking on nicotine excretion. The influence of inhaling Alfred Kershbaum. Samuel Bellet. Masami Hiraba_vashi. Leonard I. Feinberg, and Ralph Eilberg Division of Cardiology. Philadelphia General Hospital. Philadelphia, Pa. Archives of Interna! Medicine 120:311-314, 1967 A comparative study of urinary nicotine ex- cretion with cigarette, cigar. and pipe smoking was made, using a gas chromatographic method to de- termine the nicotine content of the urine. In 29 normal male smokers, smoking cigarettes in the customary manner of inhaling, and cigars and pipes in the customary manner of not inhaling, about three times the quantity of nicotine was excreted with cigarettes than with cigars or pipes. There was no significant difference in nicotine excretion between cigar and pipe smoking. Each subject smoked four cigarettes (4 gm tobacco), a 4- gm segment of cigar, or 4 gm of pipe tobacco per hour for four hours. In each of ten subjects, ciga- rettes, cigars and pipes were smoked, both with inhaling and without inhaling; there was no sig- nificant difference in mean nicotine excretion with the three forms of smoking when all were inhaled or all not inhaled. In anesthetized dogs, equivalent amounts of cigarette. cigar. and pipe tobacco smoke were ad- ministered intratracheally with inhalation kept con- stant. Nicotine excretion was similar with each tobacco form. It is evident from these findings that the greater nicotine excretion with cigarette smoking is a result of the tendency to inhale cigarettes and not to inhale cigars and pipes. The differences in nicotine ex- cretion are most probably a consequence of similar differences in nicotine absorption. Other support: National Institutes of Health, and the Cnuncil for Tobacco Research-USA. 2. Distribution of nicotine in the rat C. C. Hug, Jr. Department of Pharmacology, Universitr oiMichigan Medical School. Ann Arbor. Michigan Pharmacologist 12:220. 1970 Radioactive nicotine (100 µg/kg) was adminis- tered intravenously to rats which were decapitated between 2 and 60 minutes after injection. As mea- sured by a method specific for unchanged nicotine. nicotine concentrations in plasma were 102 and 12 ng/ml at 2 and 60 minutes, respectivelv. Plasma time-concentration curves had two components. k = 0.5 and 0.02 min-'. The early, more rapid component was associated with the entry of nicotine into tissues, the slower component with elimination of nicotine. Certain brain areas, adrenals and liver maintained a 3 to 5-fold concentration gradient over plasma between 2 and 60 minutes. Nicotine con- centrations in lung, heart, intestinal wall, spleen, thvmus and skeletal muscle were about 2 times greater than those in plasma, and in kidney were from 6 to 15 times greater. Nicotine uptake by fat was slow and did not exceed plasma concentration. Mecamylamine (1 mg/kg intravenously, 20 minutes before nicotine) prevented the behavioral effects of nicotine and reduced nicotine concentrations in brain, adrenals and plasma, and increased nicotine concentration in skeletal muscle. Mecamvlamine also reduced nicotine uptake by cerebral cortical slices. 3. Tissue Distribution of ['H] nicotine in dogs and rhesus monkeys Akira Tsujimoto, Toshikatsu Nakashima. Shiro Tanino. Toshihiro Dohi. and Yutaka Kurogochi Department of Pharmacology, Hiroshima University School of Dentistry, Hiroshima; Department of Pharmacology, Nara Medical School, Kashihara. Nara, Japan Toxicology and Applied Pharmacology 32:21-31. 1975 The distribution of ['H] nicotine among dif- ferent tissues was examined in unanesthetized dogs and monkeys following iv injection of small dose. 100 µg/kg. Nicotine was rapidly distributed throughout tissues. Five mi,iutes after icrjci,tiofi, adrenal medulla and cerebral cortex contained high concentrations of nicotine (961 and 505 ng/g for TIMN 0115752 T200243 do ~4 and tis5i fnr tex. hotl fere tipe( 11101 Skei ron( t hos he ( niro ,~ iti\ do;. and relat almc marl werf spec rou g cantl adre; 4. D sVstE C. G. Appt Depcr StocA .-lnnr. Art i c I i ntra initia This tabol to 1 simil trave l grey whitE ;f tl;E a ten 6

Labeling . .......................... .......... . li, t;ro%%-th Chambers for Large Scale Production of Plants of Unusual Isotopic. Composition ...... 4h ~~ I:SR and ENDOR Studies of Free Radicals Generated in Combustion of Tobacco Smoke ....... 41) 421)c~uterium Isotope Effects in Nicotia tabacum .......................... 4_ dification of Plant Tissue Co l M t i i i Hi h " o n a n ca ng g 4;. (;vtnlog Levels of C ....... . 1- 44 Effrc ts of Carbon-13 on the Structure and Morphology of Pollens ................. 4- ; ; (:ulture of "C-Substituted Plants of Interest in Pharmacognos'.- and Pharmacnlop. . . . . . . 48 a- 42 . . 42 n -} 2 45 -t 5 45 n . . 45 .. 45 46 TIMN 0115772 T200263 2;

of some of the latter and by analogy to the me- tabolism of phenyl-1,2-ethanediol. the formation of a glucuronide of (3-pyridyl)-1.2-ethanediol is sug- gested. Other support: U. S. Public Health Service, the COuncil for Tobacco Research-U.S.A., and the American Tobacco Company. 16. Studies on the separation of acidic metabolites of nicotine by gas chromatography Herbert McKennis. Jr., Edward R. Bowman, and mohammad Saeed Dar Department of Pharmacology. Medical College of vireinia. Richmond, Virginia t'irvinia Journal of Science 18:13-18, 1967 As part of an investigation of procedures for the gas chromatographic determination of mammalian metabolites of nicotine, a synthetic mixture of the methyl esters of nicotinic acid, 3-pyridylacetic acid, v-(3-pvridvl)butyric acid. -y-(3-pyridyl)--y-oxobutyric acid. and 5-(3-pyridvl)tetrahvdrofuranone-2 was in- k-estigated and found to give adequate separation on a Carbowax 20M column (5.6% on Anakrom ABS). Aqueous solutions of synthetic (±)-y-(3-pyridyl)-y- methvlaminobutyric acid, prepared by an improved procedure, were converted by the apparatus and column conditions to cotinine as determined by comparison with the response of equimolar amounts of cotinine in a flame ionization detector. Cotinine, in contrast to methyl y-3(3-pyridyl)-Y-oxobutyrate and 5-(3-pyridyl)tetrahydrofuranone-2, provided es- sentially no signal to the electron-capture detector. The ease with which -y-(3-pyridyl)-y-methylamino- butyric acid can be converted to cotinine appears to afford a basis for the quantitative determination of the methvlamino acid. Other support: Council for Tobacco Research-USA. ,jnd the American Tobacco Company. 17. Metabolism of (±)-cotinine-2"C in the rat Paolo L. Ivtorselli, Helen H. Ong, Edward R. Bowman, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia, Richmond. Virginia Journal of Medicinal Chemistry 10:1033-1036, 1967 A route to the synthesis of a number of nicotine rnetabolites bearing a'*C label adjacent to the pvridine ring is described. This synthesis, which starts with the condensation of ethyl nicotinate-7-"C and diethyl succinate, provides -y-(3-pyridvl)-y-oxo- butyric acid, -y-(3-pyridyl)hvdroxvbutyric acid, y-(3- pyridyl)-y-methylaminobutyric acid. cotinine, and demethvlcotinine, as well as the two alkaloids nicotine and nornicotine. After administration of (±)-cotinine-2-'aC to the rat, the urine was examined chromatographically for radioactive and Koenig- positive (pyridine) substances. The general pattern of excretion of the radioactive Koenig-positive sub- stances resembled those previously encountered in some other species and paralleled that found earlier with nonisotopic material. The radioactivity of ad- ministered (±)-cotinine was eliminated with a high degree of efficiency (90-97% of the administered dose), predominantly by way of the urine. Virtuallv no radioactivity was encountered in expired air. suggesting little or no conversion to nicotinic acid. Demethylcotinine and -y-(3-pyridyl)-y-oxo-N-me- thylbutyramide were identified in the urine by isotopic dilution. Similar experiments where carrier nicotine was employed failed to provide evidence for the reversibilitv of the metabolic reaction nic- otine --+ cotinine. Other support: the Council for Tobacco Research- U.S.A., and the American Tobacco Company. 18. The fate and distribution of 1-(3- pyridyl)ethanol methiodide in relation to the toxicity of 1-(3-pyridyl)ethanol and 3- acetylpyridine John P. Bederka, Jr., Eskil Hansson, Edward R. Bowman, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia. Richmond, Virginia, U.S.A.. and the Royal Veterinary College, Stockholm, Sweden Biochemical Pharmacology 16:1-10, 1967 (+,-)-N-Methyl-"C-3-(1-hydroxyethyl) pyridin- ium iodide, prepared from methyl-"C iodide and (+,-)-1-(3-pyridyl)ethanol, was administered i.v. to mice. Sequential wholebody autoradiograms showed a rapid elimination of the radioactivity of (+,-)-N-methyl-"C-3-(1-hydroxyethyl) pyridinium iodide from the animals. The compound, in common with the established behavior of many other qua- TIMN 0115757 T200248 11

lis- ted iea- ne. 12 •ma k= ent nto I of ve r )ver o n- •en, nes 'ere fat ion. ites ; of in =ine tine ;cal A Q. 3 dif- ze d nall ited ion, tigh for dogs. 1163 and 310 ng/g for monkevs. respectivelv) and tissue/serum concentration ratios for respective tissues were 13.7 and 7.2 for dogs. and 20.7 and 5.5 for monkeys. Concentrations in spleen. adrenal cor tex. kidne}•. and pancreas were relatively high in hoth species. There were significant regional dif- ferences in the concentration in the CNS of both species. Concentrations in various areas of CNS in monkevs were markedlv lower than those in dogs. Skeletal muscle concentrations and tissue/serum c oncentrations ratios in monkeys were almost twice those in dogs. Lower brain content in monkevs mav be due to the high affinity of skeletal muscle for nicotine. and mav partially explain the lesser sen- ;itivitv to nicotine of the monkey as compared to t',e doc'. Thirtv minutes after injection, kidnev, gastric and intestinal mucosa. and salivarv glands had relativelv high concentrations in both species and almost all tissue concentrations in monkeys were markedly higher than those in dogs. Lowest levels were found in adipose tissue (10 to 38 ng/g) in both species. Tissue concentrations of nicotine were roughly proportional to dose. Pentobarbital signifi- cantlv reduced nicotine concentrations in CNS and adrenal medulla in dogs. 4. Distribution of nicotine in the central nervous svstem C. G. Schmiterlow, E. Hansson. G. Andersson. L. -E. Appelgren. and P. C. Hoffmann nopartment of Pharmacology. Royal Veterinary College. Stockholm. Sweden .lnnals of the .\'eir York Academy of Sciences 142. Article 1:2-14. 1967 Whole-body autoradiograms of mice injected intravenouslv with (-)-nicotine-methyl-'}C show an initial accumulation of radioactivitv_ in the CNS. This high concentration of nicotine and/or its me- tabolites in the brain disappears within 30 minutes to 1 hour. Autoradiograms of cat brain show a similar accumulation of radioactivity following in- travenous injection of 14C-nicotine. The highest level of activity is observed in the grey matter, whereas the radioactivity is lower in the white matter. It can also be observed that the nuclei of the diencephalon and the medulla oblonQata have a tendency to concentrate radioactivity. Chromato- graphic investigations of extracts from c.at hrain show that the major part of the radioactivitv durino the first 30 minutes after the injection is still due to nicotine. titicroradiography of freeze-dried sectinns showed a concentration of radioactivity in ner. e ( ells after injection of 'H-labeled nicotine. Especiall\ high levels were observed in the cellular lavers of the hippocampus. Chromatographic investigations of extracts from brains of mice at various times after injection show that the mouse brain mainlv contains unchanged nicotine during the first 20 minutes. but later con- tains mainlv metabolites. Cotinine was the major metabolite found in brain. liver. and kidnev. The metabolism of nicotine has also been in- vestigated in tissue slices of various organs of the mouse by means of 14C-labeled nicotine. Of those tissues studied, the liver, kidney, and lung were found to metabolize nicotine. while the brain did not. Other support: Swedish Tobacco Company. 5. Age dependent changes in nicotine distribution in the brain of the mouse Torbjorn Stalhandske, and Premysl Slanina (Carl G. Schmiterlow) Department of Pharmacology, Royal Veterinary College. Stockholm, Sweden Acta Pharmacologica et Toxicologica 31:341-352. 1972 Mice of different ages, from newborn to 26-day old, were injected intraperitoneally with 1 mg/kg of 14C-nicotine and the nicotine concentrations in the blood and brains were determined 10 minutes after the injection. In 3-day old and 12-day old mice the nicotine concentrations in the blood and brains were also determined 1, 2.5, 5, 20 and 60 minutes after the injection. In addition, the nicotine distribution in 3- day old, 12-day old and 26-day old mice was studied by whole-body autoradiography. The nicotine con- centrations in the brain at 10 minutes increased slightly during the first 5 days after birth, but increased rapidly from that time up to the age of 12 days; thereafter, in older mice, a decrease in the concentration was observed. The brain-to-blood ra- tios steadily increased from 1.3 at birth un to 5.5 at 26 days. Autoradiographic studies in the 3-day old mouse showed high levels of radioactivity in the TIMN 0115753 T200244 7

Absorption. Distribution. Metabolism. Excretion. Totiicologv 47. Comparative studies of hepatic nicotine metabolizing enzyme activities in monkeys and dogs Toshihiro Dohi. Sekizo Kojima. and Akira Tsujimoto Department of Pharmacolog' v. Hiroshima Cniversitv School of Dentistrr. Hiroshima. Japan Iapanese Journal of PharmacologY 23:748-751. 1973 Microsomal nicotine metabolizing enzyme ac- tivities in the liver of monkevs were found to be significantly greater than those in the liver of dogs. The Michaelis constants (Km) for nicotine, obtained theoretically using the least squares method, were much lower in monkeys (7 3 x 10-{M) than in dogs (14.0 x 10-aM). The contents of cytochrome P-450 and cvtochrome b; in monkey liver rnicrosomes were about 2 and 1.5 times higher respectively than those in dogs. Additional findings are reported that sug- gest that the greater amount of microsomal P-450 in the livers of monkevs may contribute to the higher enzyme activities as compared to dogs. which may partially result in the quantitative differences in responses to nicotine between the two species. 48. Lack of effect of chronic nicotine administration on fatty acid distribution in the liver, testis, and adipose tissue of male Fischer- 344 rats R. G. Brindis. B. J. Petersen. J. H. Thompson, and R. B. Alfin-Slater Departments of Public Health and of Pharmacology and Experimental Therapeutics. University of California School of.\tedicine. Los Angeles. California Journal of Lipid Research 14:688-691, 1973 A comparison is made of the percentage com- positions of major fatty acids in liver and testis phospholipids. liver and abdominal adipose tissue trigh•cerides. and liver sterol ester in male Fischer- 344 rats administered a physiological saline control or a"smoking" dose of nicotine (1000 µg base/kg/ dav. subcutaneously) for 2 or 22 months. Results indicate that there is no major trend, or significant difference. between nicotine- or saline-treated rats with respect to major fatty acid distribution. Some differences in fath• acid distribution in the various lipid fractions were found between young and old :ats. 49. Effects of chronic nicotine administration and age in male Fischer-344 rats Jeremy H. Thompson. F. Dennis Irwin. Shigeto Kanematsu. Krikor Seravdarian. and M. Suh Departments of Pharmacology and Experimental Therapeutics. Pathology. Anatoml. and PhYsioluar. C'niversitY of California School of Medicine. Los Angeles. California Toxicologr and Applied Pharmacologr 26:606-620. 1973 VO The chronic effects of nicotine were studied in male Fischer-344 rats over periods of 2 or 22 months, Nicotine (1000 µg base/mlJkg/da%,) was given sub- cutaneously in 6% gelatin. whereas control rats received 0.85 g/100 ml wiv NaCl in 6°o gelatin. Nicotine-treated rats achieved and maintained a significantly lower (p < 0.01) weight gain than control animals. and the weight of several tissues in both control and nicotine-treated groups showed a change with age. There was no significant change in hemoglobin, or hematocrit, or mean corpuscular volume. hemoglobin, and hemoglobin concentra- tion, or in red and white blood cell counts between control and nicotine-treated rats. Similarly. the dif- ferential white cell count did not differ between two groups; however, an increasing percentage of neu- trophil polymorphonuclear leucocytes and a de- creasing percentage of lymphocytes was shown for both groups with age. Platelet number and mor- phology were not altered. Several neoplasms de- veloped in the animals injected for 22 months. Two tumors, an adenocarcinoma of the lung and chromo- phobe adenoma developed in the 6 old control rats (33%), and 9 tumors (3 instances of pheochromocy- toma, 4 cases of epidermoid carcinoma of the skin. I rat with leukemia and 1 animal with fibrosarcoma) developed in 8 of the old treated rats (29%). The incidence of Leydig cell hyperplasia differed sig- nificantly with lesions developing in 66% of the old control rats compared to 89% of the old treated rats (p < 0.05). It is possible that the physiologic age of the nicotine-treated rats was significantly greater than the controls, and thus the animals were at greater risk for the development of this lesion. Nicotine administration had no effect on the Ca'-• dependent myosin ATPase activity or on the lactic dehydrogenase activity and isozyme pattern from predominantly fast (gastrocnemius), slow (soleus) or cardiac muscles. Mgz--dependent ATPase activitr• from soleus muscle myofibrils was considerablY depressed after 22 months of nicotine treatment, n l ~: ( )t' bet anc dn( Srg cia Ou the rrt e in€ c.ac cig w h the Ott 22 TIMN 0115768 T200259

~ G. Cvto,enetic s-Effec.t of Chemical Carcinogens-Relation to Natural Histor% . . . . . . . . . . .......... 3 ( ~ 1-. E;ar\c t\-pes of Rats from Strains of Different Susceptibility to \tammarv Cancer Incluction .... . 3 18. Chromosomes of Mammarv Carcinomas Induced hv 3-Methvicholanthrene in Rats .......... 3y iq. Chances in Chromosomes of Bone Marrow After Intravenous Injections of 7.12-Dimethv1- henz(a)Anthracene and Related Compounds ............................. . ..... . . . 3y H. In Vitro-\uc:leolar Chan.oes ............................................. . ......... 39 20. Nucleolar Aging in Tetraht•mena During the Cultural Growth Cycle .............. . ..... . 3y [. Carcino~en-Effect of Aflatoxin B on Human Peripheral Blood Lymphocyte ........ . ....... . .. 40 21. Effect of Aflatoxin B, Upon Phvtohemagglutinin-Transformed Human Lvmphocytes ..........3r1. Carcinogenesis-Colon-Chemical Induction .. . ....................................... 4; 22. \.\'2.7-Fluorem•lenehisacetamide (2.7-F.A.A.)-Induced Rat Bowel Cancer ......... . .... . . .3t K. Carcinogenesis-Inhibition/Enhancement-PAH Metabolism ............................ . . t; 23. Induction of Increased Benzpyrene Hvdroxvlase Activity by Flavones and Related Compounds ..3; 24. Induction of Increased Benzpyrene H_%,drox,,-lase Activitv_ by 2-Phenvlbenzothiazoles and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 25. Induction of Increased Benzpvrene Hydroxylase Activity in Pulmonary Tissue In Vitro ....... 4 ~ 2E;. Inhibition of the Carcinogenic Action of 7.12-Dimethvlbenz(a)anthracene b~~ Beta-naphthofla- vone .....................................................................42 27. Inhibition of the Carcinogenic Action of Benzo(a)pyrene by Flavones .................... 42 28. Phenothiazine Inhibition of Intestinal and Urinary Bladder Tumors Induced in Rats bv Bracken Fern ..................................................................... 42 29. Enz%Tnatic Reactions and Carcinogenesis .......................................... 43 30. Dietarv Modification of Intestinal and Pulmonarv ArvI Hvdrocarbon Hvdroxvlase Activitv .... 43 :31. Exo'enous Factors Affecting Polvcvlic Hydrocarbon Hvdroxvlase Activity ................. 43 32. Antiviral-Anticancer Activity of Tobacco Leaf and the Smoke Condensate ................. 43 L. Coc arcinogenesis-Cigarette Smoke-Bioassa\ .......................................... 44 33. Brief Communication: Cocarcinogenic Agents in Tobacco Carcinogenesis ................. 44 :34. Cocarcinogenic and Tumor-Promoting Agena in Tobacco Carcinogenesis ................. 44 :35. The Role of Alpha-Emitting Isotopes in the Effects of Cigarette Smoking .................. 44 \I. Carcinogenesis-Cell Proliferation in Man ............................. . .............. 45 36. Smoking and the '~4itotic Index of Oral Mucosa ..................................... {S N. Carcinogenesis-Mechanisms ...................................................... 45 37. Characterization of the Cvtochrome Oxidase Inhibitor Found in The Gas Phase of Tobacco Smoke .................................................................... 15 38. Assav of a Volatile Inhibitor in Tobacco Smoke Using Bacterial Cytochrome Oxidase ........ 45 ~ 39. Investigations on the Formation of the Microbial Electron Transport System as Effected B,, .............................................................. Tobacco Smoke 46 26 TIMN 0115771 T200262

•ct .b- so :m n- 11- itv 71 •ed ic 4- a- -n I - c- a in- :IBK uot hy- Cl, ual ied an I of µg I copper per cigarette when fresh, nonfractionated -,L; was used. This simple. rapid extraction tech- rnique has been used to determine variations in the hemical activity of tobacco smoke condensate. e Tther support: L`. S. Public Health Service. 34. Isolation of metal binding fractions from tobacco smoke condensate v,nc:ent N. Finelli. Edward E. Menden. and Harold G. Petering oepartment of Environmental Health, College of Medicine. C'nirersitr of Cincinnati, Cincinnati. Ohio Fn ironmental Science and Technology 6:740-742, 1972 Tobacco smoke condensate (TSC) from nonfilter research cigarettes was fractionated on a weak cation exchange column [carboxymethyl cellulose in the Cu (II) form], yielding three fractions: noncomplex- ing substances. protonated copper-binding ligands, and nonprotonated copper-binding ligands. Analy- sis for copper was done by atomi.. absorption spec- trometrv and showed the amount of complexed copper in the protonated ligand fraction to be 271 ± 42 µg/cigarette and in the nonprotonated ligand fractions to be 720 ± 59 µg/cigarette. Several known protonated and nonprotonated ligands were also fractionated on the cation exchanger, and their hwhavior was compared to that of the Tsc fractions. The cation exchanger was also used in the zinc, tadmium. iron (III), and lead forms to determine the binding activity of whole TSC solutions toward these metals. Results, expressed in µ mol of inetal/ciga- nettt~. were copper, 14.6; zinc, 12.8; cadmium, 8.3; iron. 0.5: and lead. 0.5. tJther support: U. S. Public Health Service. 35. Effects of metal-binding fractions of tobacco smoke on in vitro activity of enzymes ', incent N. Finelli. and Harold G. Petering UFportment of Environmental Health, College of Vedicine. University of Cincinnati, Cincinnati, Ohio Archi: es of Environmental Health 25:97-100, 1972 To investigate the role of metal-binding agents present in the environment on trace metal me- tabolism, the authors successfully isolated from the numerous constituents of tobacco smoke conden- sate, two fractions: protonated ligands (II-FZ) and nonprotonated ligands (II-F,). Both of these frac- tions, along with a copper containing fraction (I-F.). have been tested for inhibitory activity toward catalase, galactose oxidase and glucose oxidase. Galactose oxidase and catalase are metalloenzymes and glucose oxidase is a glycoprotein containing flavin-adenine dinucleotide and no metals. Both fractions, II-F, and II-F,, strongly inhibited galactose oxidase, a copper-containing enzyme, while they did not affect glucose oxidase. The protonated ligands (II-FZ) and their copper complexes (I-F,) inhibited catalase but the nonprotonated ligands (II- F,) had no effect on this enzyme. Other support: U. S. Public Health Service. 36. Potent in vitro inhibitors of tyrosinase: Metal- binding fractions of tobacco smoke condensate Vincent N. Finelli, Peter M. Eller, Patricia J. Montague, and Harold G. Petering Department of Environmental Health. College of Medicine, University of Cincinnati, Cincinnati, Ohio Toxicology and Applied Pharmacology 27:415-421. 1974 A fraction of tobacco smoke condensate (TSC) containing copper-binding agents was subjected to a classical functional group extraction, yielding basic, acidic, phenolic and neutral subfractions. Two of these fractions (the acidic and the phenolic) contain potent in vitro inhibitors of mushroom tyrosinase. The mechanism of inhibition was studied in com- parison with known copper-binding agents. The concentration of active inhibitors in the TSC frac- tions has been estimated using the Easson-Stedman method and is of the same order of magnitude as their titratable acidity. This procedure is therefore applicable in the determination of concentrations of enzyme inhibitors in unidentified mixtures of com- pounds. The TSC fractions exhibited noncompeti- tive inhibition with K; values of 1.3 x 10-'M (acidic fraction) and 9.3 x 10-BM (phenolic fraction), similar in magnitude to K; values found for the most potent of the inhibitors studied. The presence in tobacco smoke of potent in- hibitors of tyrosinase and other metalloenzymes is noted to strengthen the authors' hypothesis that TIMN 0115763 T200254 17

-ied in thf. •spirationn ry chains n monox effect. In ~ isolated to their zt and the islocators re normal ially high a, which Another was that iration of =her with ular Ca2- ance over lung mi- f tobacco t to one from one ! 3 rate of ruvate + ;ible link d abnor- is being \%-litul also caused an increased reduction of hound NAD as determined analytically and by inc reased, pk-ridine nucleotide fluorescence mea- ;ured bv surface fluorometry. The latter technique <huwed that half-maximum reduction was obtained with 6.4 m%t xylitol. suggesting that xylitol was motabc~lized principally to D-x}•lulose via the low K,,, vtt,solic NAD-linked xvlitol dehydrogenase. [n the perfused liver xylitol was converted Tndink to Oucose. after phosphorylation of D-xy- huins«, to D-x}'lulose-5-P and metabolism via the pt-ntn,N phosphate pathway. Lactate uptake and , nn\EIrsinn to glucose were strongly inhibited by .k litnl. This effect was interpreted as resulting from the inc reased cx-tosolic NADH:NAD ratio which ( .,usO'cl p}.ruvate levels to fall. An observed decrease .,f a( etvl cnenzyme A levels may also contribute to ii:nini>hed flux through pyruvate carboxvlase. Xyli- !,)I uptake was less affected by the presence of I.,( tate. presumably because of the lower mid-po- tt-ntial of NAD-xylitol dehydrogenase (-236 mvolts) ; 11mpared with that of lactate dehydrogenase (-216 ;;ivnlts). Ketone body production from endogenous acids was inhibited equally by lactate and .~ litul. The production of ketone bodies from added I c ac:id was marginally inhibited by xylitol, sug- ;~~s in- the absence of a specific antiketogenic effect . t'le isolated liver. Yvlitol metabolism was not ,i:s"( iated with any increased rate of respiration, er: h,w. ing that reoxidation of NADH produced by ngenesis \\litol dehvdrogenase caused an inhibition of the ).sakura tri( ic:id cx-cle. Total adenine nucleotide levels ii:nini,hed slightly during xylitol metabolism, but 0ht, :\TP:ADP ratio was not appreciably changed. 1971 )th,,r ,upport: U. S. Public Health Service, the n with perfused ved as a tems as uvate, a- and tri- hondrial f the p- less af- d in the n in the itochon- ni-ni,in Di<ibetes Assoctation, and the American ! i,,irt :\,scx iation. 40. %tolecular struc lures and catalytic activity. I. (:atalytic polarography of thiamine, oxythiamine and thiamine phosphates Si-Mundur Gudbjarnason ).t,nrtment ol Aledicine. Wayne State University School \teelicine. Detroit. Ntichigan r?+,,( himica et Biophysica Acta 148:22-36. 1967 The biochemical mechanism of the coenzyme thiamine pyrophosphate or cocarboxylase has been Iuggested on the basis of studies of the reaction mechanism of thiamine. It is assumed that thiamine and cocarboxvlase have the same reaction mecha- nism. although thiamine cannot replace cocarboxvl- ase in the enzymatic reaction. The purpose of this report is to describe differences in the catal\tic polarographic activities of thiamine and coc:arbo\~ 1- ase. The polarographic data indicate that thiamine and cocarboxylase may differ both in reaction rnech- anisms and reaction rates. The catalytic polarographic activities of thia- mine, oxythiamine, thiamine monophosphate and thiamine pyrophosphate were studied during the intramolecular rearrangement of these substances in a basic solution. The halflife of the polarographic maxima were 6 min for thiamine, 19 min for thiamine pyrophosphate, 35 min for thiamine mono- phosphate and 188 min for oxythiamine. The thia- mine phosphates appear to be more stable in the tricyclic dihydrothiachromine form. whereas thia- mine is converted more rapidly to the yellow thiol form. The polarographic waves of cocarboxvlase rep- resent the active protons of carbon 2 from the thiazole ring and the amino group of the pyrimidine ring. The polarographic wave of thiamine corre- sponds to the hydrogen of carbon 2. Reaction of pyruvate with thiamine or cocarboxylase results in the formation of a second polarographic wave or increase in existing wave corresponding to the amino group. The possible role of the amino group of the pyrimidine ring in the binding of the carbonyl dipole of pyruvate is discussed. Other support: U. S. Public Health Service. the Michigan Heart Association. the Council for To- bacco Research-U.S.A.. and the Detroit General Hospital Research Corporation. D. Excretion 41. Molecular structures and catalytic activity. II. Proton reactivity and catalytic polarography of histidine and related imidazole derivatives Sigmundur Gudbjarnason Departments of Medicine and Biochemistry. CVavne State University. School of Medicine. Detroit. .llichigan Biochimica et Biophysica Acta 177:303-313. 1969 The catalytic polarographic activity of histidine. imidazole. imidazole acetic ecid hictaminP. I- TIMN 0115765 T200256 19

Section II (.arcinogenest<s ~ . ttnt~tr~ lbstracts (45) \ itrf, (:ulture Techniques ........... . ........................................ . . . .~ 1 ;; ('uItur«' ; liItlrit.1IL:e of Vitamin A and 3.7-Dimeth},l-2. 6-Octadienal (Citral) on the Effect of Benzo(a) pVrene ,,,I Hamster Trachea in Organ Culture ........................................... . 3 1 \l,jintenance of Normal. Metaplastic, and Dysplastic States of Adult Human Bronchial Mucosa in ( )r,an Culture .............................................................. 3 2 T,)\ic it~ of Benzo(a)pyrene and Air Pollution Composite for Adult Human Bronchial `tucosa in ( )r,,m Culture .............................................................. :3 2 ( iilturr~ ; I'I,,(ental Transfer of 3.4-Benzpyrene and Fetal Benzpyrene Hvdroxylase Induction .......... 33 ( .ultur~~ ; 1,11 tate Deh},drogenase Activity of Human Cells Infected with Adenovirus . ............... 33 f \iwrimental Carcinogenesis ................................................... .. 34 t, Chemical Carcinogenesis in Syrian Hamsters ....................................... 34 - Study of Smoke Condensate of Cigarette Tobacco as a Possible Bladder Carcinogenic Agent in the Copenhagen Rat ............................................................. 3-t Fiii(i«,mioloo\•-Genetics-Human .................................................. 34 .,t. \lortalitv in Smoking Discordant Vlonozygotic and Dizygotic Twins. A Study on the Swedish Tk~-in Registry ............................................... ....... ....... 34 •.1t•tdh0lism-Arvl Hvdrocarbon Hydroxylase (AHH)-Human Mechanisms ... . ............... 35 'a lnduction of Aryi Hydrocarbon Hydroxylase in Human Alveolar Macrophages by Cigarette 5itioking .................... ........................................... 35 !n fnduction of Aryl Hydrocarbon Hydroxylase in Human Lymphocytes and Pulmonary Alveolar \1<+crophages-A Comparison .................................................. 35 \1,1;iholism-\ucleic Acid-Chemical Carcinogenesis ................................... 36 t \u(leolar Pathoingy Produced by Acridine Orange and Proflavine ....................... 36 ' Inhihition )I DNA-Dependent RNA Polvmerase by 4-Nitroquinoline-N-Oxide in Isolated Nu- I(~i ...... .............................................................. 36 ; s h:ffects of -t-Nitruquinoline-N-Oxide on RNA Synthesis ............................... 36 14. lnteraction of 4-Nitroquinoline 1-Oxide with Deoxyribonucleic Acid and Synthetic Polydeoxvri- honucleotides .............................................................. 37 t ~ .-\ Proposed Model of the Interaction of 4-Nitroquinoline 1-Oxide with DNA ............... 37 ( anc:er Treatment-Experimental Tumors ............................................. 38 16 [nfluence of Five Anticancer Drugs on the Induction and Growth of Experimental MammarCancers: Comparison with Ovariectomv ............................................ 38 TIMN 0115770 T200261 25

Absorption. Distribution. Metabolism. Excretion. Toxicology tobacco smoke and other pyrolytic products. if absorbed by an organism, could affect the essential trace metal metabolism, thus impairing vital metal- requiring cellular processes, such as oxidative phos- phorylation, lipid metabolism, connective tissue synthesis and repair. and microsomal mixed func- tion oxidase. Other support: U. S. Public Health Service. 37. Isolation of metal-binding agents from lettuce cigarette smoke and their effect on OZ-uptake of liver slices Victor J. Elia. Lalitha Murthy, and Harold G. Petering Kettering Laboratory. Department of Environmental Health, College of Medicine, University of Cincinnati. Cincinnati. Ohio Environmental Letters 5:7-16, 1973 Lettuce leaf cigarettes, a tobacco substitute, were smoked under standardized conditions and the smoke condensate (LSC) was collected. The LSC, amounting to 7.3 mg/cigarette, was fractionated on a carboxymethylcellulose column in the Cu[II] form, yielding copper-complexing fractions (60% of the total weight) and a noncomplexing fraction (40% of the total). These values are compared to results for tobacco smoke condensate, which yields about five times as much total solids, of which 25% is in copper-complexing fractions. The ligands from the LSC were found to be inhibitors of OZ uptake by rat liver slices, a metalloenzyme requiring system. Thus, the ligands may have adverse effects on cellular energetics. Other support: U. S. Public Health Service. 38. Effect of tobacco smoke extracts on mitochondrial respiration and anion transport Kathryn F. LaNoue, and John R. Williamson Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania Presented at the workshop conference on Tobacco and Health, Newport Beach, California, May 7-9, 1972 The effects of various fractions of whole tobacco smoke extracts have been investigated on the me- tabolism of rat heart and liver mitochondria. Crude fractionation of the whole smoke indicated that a portion extractable with ether uncoupled oxidative phosphorylation and inhibited the transport of py- ruvate, a-ketoglutarate and glutamate into mito- chondria. chondria. Other components which remained in the aqueous phase inhibited mitochondrial respiration Difference spectroscopy of the respiratonr chains cytochromes identified cyanide and carbon mon,,x. ide as being mainly responsible for this effect. ln further studies, rat lung mitochondria were isolated. purified and characterized with respect to their cytochrome and pyridine nucleotide content and the presence of the various substrate anion translocators identified in liver mitochondria. These were normai with respect to the liver except for an unusually high activity of a-glycerophosphate oxidation. which needed µM amounts of CaZ- for activation. Another unusual feature of the lung mitochondria was that addition of 40-300 CaZ- inhibited the respiration of NAD-linked substrates. These data, together with other evidence suggest that the intracellular Ca:- concentration may have a regulatory influence over phospholipid synthesis. When added to lung mi. tochondria, an aliquot of ether extract of tobacco smoke (dissolved in ethanol) equivalent to one hundredth of the total smoke production from one cigarette caused 50% inhibition of the state 3 rate o' respiration with glutamate + malate, pyruvate - malate and succinate as substrates. A possible lini between the effects of tobacco smoke and abnor. malities of the Caz- balance in the lung is being explored. 39. Xylitol metabolism in perfused rat liver: Interactions with gluconeogenesis and ketogenesis Andrea Jakob. John R. Williamson, and Toshio Asakura Johnson Research Foundation, University of Pennsylvania, Philadelphia. Pennsylvania Journal of Biological Chemistry 246:7623-7631. 1971 Xylitol metabolism and its interaction wit lactate metabolism has been investigated in perfusec livers from fasted rats. Xylitol (5mM) served as e strong reductant for cytosolic NAD systems & shown by increased ratios of lactate to pyruvate, m glycerophosphate to dihydroxyacetone-P, and tri ose phosphates to 3-P-glycerate. The mitochondria NAD system, as monitored by changes of the ~ hydroxybutyrate to acetoacetate ratio, was less ai- fected, particularly when xylitol was added in t~ presence of lactate, suggesting a limitation in thr rate of transport of NADH from cytosol to mitochoo- TIMN 0115764 T2U0255 dri.~ hou incT sur, sho wi t 1 rnet c"-t( mai 1u1c, pen con xvli the cau of a dim tol lact tent c;orr tnt•c `att, xt'i' olei gest in t assc sho xt•I citr din the ()tf, Am He, 40. Cat. and Sigr Dep of N Bior thiz sug mei 18

Absorption. Distribution. Metabolism. Excretion. To\icologt methvl histidine and t-meth%-l histidine was studied in cobaltous ammoniacal medium. The effect of pH. concentration and temperature upon the electrode potential and catalytic current of the polarographic waves of histidine were exam- ined. as well as the effect of pH upon the catalytic polarographic activity of imidazole. The results indicate that the catalytic polaro- graphic waves of histidine and the other imidazole derivatives represent the reduction of protons of protonated amino and imidazole groups. The elec- tron to proton transfer is catal~~zed br complex formation of the protonated group with Co''. The catal%-tic electron transfer is limited to a specific electrode potential or energy level and the polarographic waves illustrate the specific energy levels for protons of specific pronated groups in these cobalt complexes, at which the protons can accept electrons. Other support: U. S. Public Health Service. and the Michigan Heart Association. 42. Excretion of nicotine and its metabolites in dog and monkey saliva Akira Tsujimoto, Sekizo Kojima. Masahiro Ikeda. and Toshihiro Dohi Department of Pharmacology. Hiroshima University School ot Dentistry. Hiroshima. Japan Toxico,'ogr and Applied Pharmacology 22:365-374. 1972 Salivary and plasma levels of unchanged nic- otine and total radioactivities (nicotine and its metabolites) were determined sequentially at short time intervals following intravenous injection of'H- labeled nicotine (100 µg/kg) to dogs and rhesus monkevs. Immediately after the drug injection, high concentrations of nicotine appeared in parotid and submandibular saliva induced by pilocarpine in- fusion or bv auriculotemporal nerve stimulation. Excretory patterns of nicotine and total radioactivi- ties in both glands were almost similar but were significantly different between the two species. Nic- otine was excreted more readily in dog saliva than monkey. The salivary: plasma concentration ratios for nicotine were higher than those for total ra- dioactivities in both species. Nicotine seemed to be 1 more readilv excreted in saliva than its metabolites. The concentrations of nicotine and total radioactivi- ties in saliva were independent of variation in salivarv flow rate and were not affected bv the method of stimulation. The chromatographic pat- terns of parotid and submandibular saliva resembled those of plasma in the same species. Chromato- graphic studies showed that most of the adminis- tered nicotine appeared in plasma and saliva in the form of cotinine 5-60 minutes after the injection.'H. Nicotine: total 'H-radioactivity concentration ratios ('H-nicotine: total 'H) of both saliva and plasma in monkeys were lower than those in dogs. The times at which 'H-nicotine: total 'H ratio became 0.5 in plasma and saliva were very short (2-9 minutes); in addition. those in monkeys were short relative to dogs. 43. Gastric excretion of C"-nicotine G. Andersson, E. Hansson, and C. G. Schmiterlow Department of Pharmacology. Roi•aI Veterinarr College. Stockholm. Sweden Experientia 21:211-213. 1965 (-)-Nicotine-methyl-C'was given i.v. to mice, rats and cats which were sacrificed at 5, 15 and 30 min and 1 and 4 hr after the injection. Whole body autoradiograms show that, in addition to other sites, nicotine and/or its metabolites become concentrated in the gastric mucosa and stomach contents. From stomachs cut open and wiped free of content. it appears that radioactivity occurs only in the glandu- lar portion. Studies on mice and rats of the amount of radioactivity excreted in the stomach gave rather wide variations, the values obtained varying from 1.8 to 10% of the amount initiallv administered. To study this, it was arranged to perfuse the stomach of rats in situ with solutions buffered to pHs ranging from 1 to 9. Labeled nicotine was given i.v. and the amount of activity appearing in the perfusate was determined. Results showed that the highest ex- cretion (in 30 min) occurred at pH 1 (mean value 4.4%) and that excretion diminished as the pH increased (down to 0.6% at pH 9). Chromatographic analysis of the perfusate showed that after 30 min 65% of the radioactivity repcebented unchanged nicotine, the remainder be- ing present in the only metabolite that could be detected, namely cotinine. TIMN 0115766 T200257 . dra% nic( that stor lea,~ ston oth. E. 44. nic( adr A. 1 DeF, tich Eur bol epi froi nic sen as I rat( eve res ver ticm1 of su ca ex le~- le. 45 cal gli A. De Sc Eu ch 20

-: _=_7m~• Carcinogenesis activity of cellular extracts and an isozyrne pattern compatible with malignancy. The types of cells infected included: an established human lung epi- thelium and 9 primary fibroblastic cell strains. 2 derived from human lung and 7 from human fore- skin. %tonolaver cultures of the cells growing in svnthetic medium containing 20°1o inactivated fetal calf serum were inoculated with 20 TCD 50's of type 12 virus. At frequent intervals up to 14 days and at more prolonged intervals up to 3 months. the cell lines were studied morphologicallv and the total LDH activitv of the cells and the media determined bv the method of Standjord et al. Isoz}Ttles were separated by disc electrophoresis and quantitated bv microdensitometrv. Definite morphologic changes were seen in infected cells. LDH changes varied with the type of cell, but did not follow the pattern described by Latner et al. B. Experimental Carcinogenesis 6. Chemical carcinogenesis in syrian hamsters F. Homburger Bio-Research Institute. Cambridge. Massachusetts Progress in Experimental Tumor Research 16:152-175. 1972 (Karger. Basel) New work since 1966, when the last reviews of the field were made. is reviewed in the present paper and some hitherto unpublished material from the author's laboratorv is described. Other support: U. S. Public Health Service, the Council for Tobacco Research-U. S. A., the Virginia and D. K. Ludwig Foundation, the Fannie E. Rippel Foundation, Bio-Research Consultants, Inc. 7. Study of smoke condensate of cigarette tobacco as a possible bladder carcinogenic agent in the Copenhagen rat Wm. B. Deichmann and W. E. MacDonald Research and Teaching Center of Toxicology, University of Wiami. School of Medicine, Coral Gables, Florida Progress report to the AMA-ERF In recent years there have been reports linking the excessive smoking of cigarettes with cancer of the urinarv bladder. To provide additional informa- 34 tion on this subject. a study was initiated with a total of 580 (male and female) Copenhagen rats. fed dietary supplements of cigarette smoke condensate. Some rats were fed an additional supplement of phenobarbital (a hepatic microsomal stimulant) or parathion (a hepatic microsomal depressant). (This feeding experiment is still in progress.) After 24 months of feeding the individual diets, our observations indicate: (a) a somewhat reduced gain in body weight of all experimental male and female rats. and (b) the survival rate of the male control rats was 80%, while that of the experimental groups ranged from 57 to 70%. At 24 months. the survival rate of the female control rats was 64°%, while that of the experimental groups ranged from 47 to 60%. Gross examination of the urinary bladders of the animals that died or that were killed in extremis showed tumors as follows: in 2 rats fed the control diet, in 9 rats fed the maximal tolerable dose (MTD) of the condensate, in 5 rats fed 50% of the MTD, in 7 rats fed the MTD plus phenobarbital, in 9 rats fed 50% of the MTD plus phenobarbital, in 3 rats fed the MTD plus parathion, in 11 rats fed 50% of the MTD plus parathion, in 3 rats fed the control diet plus phenobarbital, and in 3 rats fed the control diet plus parathion. There were 100 rats in the control group and 60 in each of the experimental groups. Microscopic examination of the tumors and bladder contents (which in many instances included calcium oxalate, calcium or magnesium phosphate crystals, and amorphous clumps) will determine the significance of ingested cigarette smoke condensate as an agent supporting the production of bladder tumors in the Copenhagen rat. C. Epidemiology-Genetics-Human 8. Mortality in smoking discordant monozygotic and dizygotic twins. A study on the Swedish twin registry Lars Friberg, Rune Cederlof. Torbjorn Lundman. and Hans Olsson Departments of Environmental Hygiene, Karolinska Institute and National Institute of Public Health, and the Medical Department, Seraphimer Hospital, Karolinska Institute, Stockholm Archives of Environmental Health 21:508-513. 1970 Among 706 male, dizygotic, smoking discor- dant twin pairs born in 1901 to 1925, 13 deaths TIMN 0115778 T200269

)n and u, lied in onths. n sub- A rats elatin, ned a i than ;ues in wed a nge in .scular :entra- •hveen ~le dif- m two f neu- a de- .vn for mor- 's de- Two rats tocy- .in. 1 ,)ma) The I sig- aeold •d rats ,ige of .reater ~re at esion. Ca=-- lactic from •us) or :tivity 3rably ment, ti~ it\ from cardiac and gastrocnemius i,as unchanged. ,t;pl,r,rt: Los --xn°eles Countv Heart Associa- u Hepatic function after acute or subchronic ni(otine administration in untreated mice and m i(e treated with hepatotoxic chemicals ; ,-tl~t and G. L. Plaa !,?,nt (ie Pharmacologie. Faculte de Medecine ;- iN %lontreal. Quebec. Canada lntt-rnationales de Pharmacodrnamie et de 223:1 )2-141. 1976 \1,!e rni(e treated with nicotine hydrochloride ;. .,, utek 15 InS1kg i.p.) or subchronically (5 mg/ ; i.pdailv ~ for 3weeks: 25 mg/liter in drinking •}, 7 f,,r 2-3 months) showed no evidence of ;,;ti, (k;function. as measured by serum glu- -p% ru\ ic transaminase or serum alkaline phos- ~.,t,,>t' M-tiVities. Neither acute nor subchronic : i::itni;tration modified the hepatotoxic response to "t,~t t hepatotoxin (carbon tetrachloride)• nor that >; potent hepatotoxins chloroform or 1.1, 1- i: ~noethane. nor was the cholestatic effect of 2- .,:;''ith~ li;ocvanate modified. :o. Cadmium and nickel-common characteristics ,if lettuce leaf and tobacco cigarette smoke r! E;i,,. Edward E. Menden. and Harold G. P4-tt,ring , b16(,rntorr. Department of Environmental ~ ~"?!ree of Aledicine. Universitt• of Cincinnati. ',t;. i)hio ,tul Letters 4:31 7-32-4. 1973 ttu I,•af cigarettes. a tobacco substitute. have ; unri to cont mi significant levels of Cd. Ni l.rn .~ !ottuce 1 itl,,uette contains 1.39 µg of Cd s u, ,)f \i pei ~ igarette. These levels indicate ,ntlv higher t',i and Ni content of lettuce in compdrison with tobacco cigarettes. ro,ult, show that only 5% of the Cd and none of V ,ppeared in the mainstream smoke (LSC). By t,,! analvsis of LSC, ash and butt portions remain- ;, after smoking. it was estimated that 80% of the •,dmium and 10% of the nickel left the smoked i~.;arette portion as part of the sidestream smoke• ~ hich is inhalable by individuals in the vicinity of the smoker. Uther support: U. S. Public Health Service. 52. Biological disposition of nicotine: mechanisms of protection against the acute toxicity of nicotine Carl C. Hug, Jr. Department of Pharmacologr. L'nii•ersih ot 11it hi,~nn Medical School. -tnn Arbor. Michi;an .-lbstract reprinted from the Third Research Conterenc~ on Tobacco and Health. .\'enport Beach. Calitornia. \ta% ;-9. 1972 Nicotine (0.1 mg/kg) injected in 3 to 4 seconds into the tail veins of conscious rats produced con- vulsions within one minute. The animals ,vere sacrificed at one minute and the tissue concentra- tions of unchanged radioactive nicotine were de- termined. Pretreatment with saline altered neither the incidence of convulsions nor the brain levels of nicotine. Pretreatment with mecamvlamine. hex- amethonium• non-radioactive nicotine. tolazoline or phenoxybenzamine significantly reduced both the incidence of convulsions and the brain levels of nicotine. Pentobarbital prevented the convulsions but did not alter the tissue concentrations of nico- tine. The uptake of radioactive antipyrine bx tissues was used as an index of their blood flow. Nicotine (0.1 mglkg, injected intravenouslv simultaneousl\ with antipyrene) increased the one-minute levels of antipyrene-"C in various areas of rat brain by more than two fold. Pretreatment with either mecamvla- mine or nicotine completely prevented the effects of nicotine on the uptake of antipyrene by brain. The accumulation of radioactive nicotine (3 µ\l to 3 mM) by rat cerebral cortical slices incubated in an oxygenated Krebs-Ringer medium was saturable and was inhibited by a variety of metabolic inhibi- tors. Preincubation of the slices with mecamvla- mine or non-radioactive nicotine significantlv reduced the accumulation of radioactive nicotine. Mecamvlamine also reduced the uptake of nicotine by homogenates of rat brain. Hexamethonium had no consistent effect on the accumulation of nicotine by rat brain tissue in vitro. We conclude that agents which prevent the cardiovascular effects of nicotine reduce the uptake of nicotine by brain and thereby decrease the inten- sity of its actions. This conclusion is in accord with the hypothesis originally stated by Laurence and Stacey (Briti,sh Journnl of Pharmncolo;_y 8:62. 1953) We also suggest that mecamylamine may act within brain tissue to alter the uptake of nicotine. TIMN 0115769 T200260 23

~- ~-- 4.~.--r Carcinogenesis E. Metabolism-Nucleic Acid-Chemical Carcinogenesis 11. Nucleolar pathology produced by acridine orange and proflavine R. C. Reynolds and P. O'B. Montgomery, Jr. Department of Pathology. University of Texas Southwestern Medical School, and The Laboratories for Cell Research Woodlawn Hospital, Dallas. Texas .9merican Journal of Pathology 51:323-339, 1967 The two acridine derivatives, acridine orange and proflavine, have been found to produce nuclear and nucleolar changes similar, but not identical, to those produced by actinomycin D and 4-nitroquino- line N-oxide. The nuclear changes produced by the acridine compounds include (1) nucleolar exhaus- tion. manifested by a decrease in the size of the nucleoli; (2) reorganization of the nucleolar granules to produce unusual structures at the margins of the nucleoli which have been called light and dark nucleolar "caps;" and (3) an increase in nuclear size. It is suggested that these nuclear lesions result from the inhibition of nucleic acid synthesis which the ac- ridine and other related compounds are known to produce. Other support: The Damon Runyan Fund. 12. Inhibition of DNA-dependent RNA polymerase by 4-nitroquinoline N-oxide in isolated nuclei J. S. Paul. R. C. Reynolds, and P. O'B. Montgomery, Jr. University oi Texas. Southwestern Medical School, Department of Pathology, and Laboratories for Cell Research. Woodlawn HospitaJ, Dallas, Texas .Vature 215:749-750. 1967 The activity of DNA-dependent RNA polymer- ase (E.C.2.7.7.6) was assayed on intact isolated nuclei from Chang liver cells grown in spinner cultures. Each of the RNA precursors (ATP, GTP, UTP. and CTP) were added to the assay mixture. The carbon-14 label was introduced in labelled ATP or labelled CTP. The incorporation of '°C into acid insoluble RNA in the controls required the presence of all four precursors as has previously been shown. Incorporation of '4C into the acid insoluble fraction (RNA) was inhibited by low temperatures. RNase. DNase and actinomvcin D as has been shown bv other investigators. Inhibition was also caused br the deletion o1` one or more of the ribonucleotide precursors. An unexpected finding was the lowered de- pendence of groups treated with 4-nitroquinoline N- oxide (4-NQO) on the presence of all four precursors. Control groups in assay mixture. with ATP. UTP or CTP omitted, showed smaller contents of RNA polymerase than groups pretreated with 4-NQO when assayed with one nucleotide omitted. Base sequence studies are in progress. The lower dependence of RNA polymerase ac- tivity in 4-NQO treated nuclei may be caused by increased utilization of nuclear RNA as a ribosetri- phosphate pool or to the partial substitution of another ribonucleotide in the assav medium for the one that is absent from the assay medium. The second alternative is believed to be the correct one. Other support: Damon Runyon Memorial Fund, the U. S. National Aeronautics and Space Administra- tion. and the U. S. Public Health Service. 13. Effects of 4-nitroquinoline-N-oxide on RNA synthesis J. S. Paul, R. C. Reynolds, and P. O'B. Montgomery, Jr. Department of Pathology, University of Texas Southwestern Medical School, Dallas, Texas, and Laboratories for Cell Research, Woodlawn Hospital. Dallas, Texas Cancer Research 29:558-570, 1969 4-Nitroquinoline-N-oxide, a potent mutagenic, carcinogenic, and carcinostatic agent, has been studied with respect to its effects on RNA synthesis using sucrose gradients, methylated albumin Kie- selguhr columns, and the electron microscope. An analysis of polyribosomal amino acid incorporation in tissue culture systems exposed to 4-nitroquino- line-N-oxide demonstrated a marked inhibition of the incorporation of amino acids by polyribosomal structures; the inhibition of amino acid incorpora- tion was apparent within one hour after exposure to 4-nitroquinoline-N-oxide. A few hours after the TIMN 0115780 i 36 T200271

~tabolites dioactivi_ iation in d b%- the phic pat- esembled hromato- adminis- !va in the ction. 'H- on ratios dasma in . times at e 0.5 in iutes); in lative to w The authors state that no conclusions can be !ra,%,n irom these findings as to the part played by ;1,cotine in causing stomach ulcers but that the fact ;h,,t it is e\creted via the gastric mucosa into the ;c,,mac:h contents in substantial amounts, fulfills at ,,,,st a prerequisite for the action of nicotine on the •t,1tnach %vall. t tthor suppnrt: the Sivedish Tobacco Companv. E. Toxicology 44. Comparison of pharmacological responses to nicotine and release of catecholamines from the adrenals in dogs and monkeys 1. Tsulimoto. T. Nishikaxva. T. Dohi, and S. Kojima ;),•uurtment oi Pharmacology. Hiroshima Universitt• ;, or Dentistr}-. Hiroshima. Japan ;,-,,prun Journal of Pharmacology 26:236-242. 1974 Comparative studies were made on the meta- College, hnlic. and physiological responses to nicotine and ,~pinephrine and on the release of catecholamines rmm the adrenal glands of dogs and monkeys by i; cotine. Dogs were approximately 10 times more to mice, ensitive to nicotine than monkeys to responses such and 30 ,; h\-pergl%•cemia, hyperlipidemia, increase in respi- ,le body r,,t„rv rate. heart rate and pressor responses. How- .er sites. the metabolic. pressor and tachycardiac mtrated rvspnnses of the two species to epinephrine were s. From ~er% similar quantitatively. The plasma concentra- 'rent, it !iO+ns of catecholamine in the vena cava of dogs and landu- :lonkeys were significantly increased by i.v. doses J 5o and 400 µglkg of nicotine, respectively. It is int of -w,,ested that the differences in the pharmacologi- rather , ,1 1 responses to nicotine of dogs and monkeys, from 'xcPpt that of respiration, may result in part from the ~d. To t.sti potent effect of nicotine in catecholamine re- ~ach of '':+>r in monkeys than in dogs. anging ind the te was est ex- : value he pH rfusate ctivitv ier be- ild be 45. Comparison of the effects of nicotine on catecholamine release from isolated adrenal glands of dogs and monkeys A. Tsujimoto, and T. Nishikawa iJ-partment of Pharmacology, Hiroshima University ~( huul of Dentistry. Hiroshima, Japan F~jrupean Journal of Pharmacology 29:316-319. 1974 The potencies of the effect of nicotine on cate- cholamine release from isolated perfused adrenal glands of dogs and monkeys and the concentrations of catecholamines in these glands were determined. Both nicotine and acetvlcholine had much less effect on catecholamine release in monkevs than in dogs. The epinephrine concentration in the adrenal glands of monkeys was significantly less than that in dogs while the norepinephrine concentrations in the two species were approximately equal. The lower con- centration of epinephrine in the adrenal glands of monkeys seems to be only a minor factor in causing less release of catecholamines by nicotine in mon- keys than in dogs. 46. Comparison of the cardiostimulatory effects of nicotine in dogs and monkeys Takashige Nishikawa. and Akira Tsujimoto Department of Pharmacology, Hiroshima L'niversitr School of Dentistrv. Hiroshima. Japan Journal of Pharmacy and Pharmacologr 27:716-; 18. 1975 Earlier studies by the authors and others have shown that dogs are 5-10 times more sensitive than rhesus monkeys to the effects of nicotine on certain metabolic and physiological responses. and that nicotine also causes greater release of catecholamine from the adrenal glands in vivo in dogs than in monkeys. In the present study, the effects of nicotine on the ventricular tension development in isolated perfused hearts of dogs and monkeys and on cate- cholamine release from and content of these hearts were determined. Data are presented suggesting that the differ- ence in the cardiostimulatory response to nicotine of dogs and monkeys is due to the different amounts of noradrenaline released in the two species, it being also noteworthy that there is less noradrenaline in monkey heart to be released. It is also suggested that there may be similar differences between dogs and monkeys at the terminal adrenergic nerve fibers of other organs or at the sympathetic ganglia. TIMN 0115767 T200258 21

'.rop the inal in- '_1H I is be- ates en- ~mi- tion has ears it of the ons. ~ of iati- bv a iarv V is ates ater l' ;ted tem inal :ght or I of the the bv t~ 1101 ind p\ ro,alloi and the fact that these potent ,rc inugens did not show promoter actic•itN in the t,,~~l,~rd tt~o stage system. Invaluable contributions heen made. to our know•ledge• of structure i,,,u tion relationship in c:arcinogenesis and to an f111i1er,t.lndinQ of cocarcinogenesis. The particular ,•,lii,, „t the work cited has been the demonstration Il,,t tht• ctivity of cigarette smoke is the resultant of rries of active agents. reinforcing agents and ,nhihitorv agents. Considerable light is cast on the .',,.rr\<ition that fractions of tobacco smoke con- i,•,;:,,te (annot be shown to have an activity com- ,,,r,,hi«, tn whole smoking condensate. Smokers were found (35) to excrete five times ~:,iiro Pn-210 and two times more Ph-210 as non ,,,u,h,.rs and it was concluded that daily normal ,,,t,,),e of these substances was increased accordinglN• t hn,u,h smoking. \1 c:arcino;enesis-Cell Proliferation in Xtan Pr,~liferative activity in the oral mucosa of man u;t n.,is stimulated by smoking as evidenced by a _h mitotic index. Ex-smokers had less. These findings may be construed as relating to fu n tion of cigarette smoke in the induction of oral .,n, er. .-\ proliferating epithelium is an advanta- u: ,ubstrate for the action of carcinogens. Al- 'irn,,telv. proliferation may be considered an ex- r~.i, n nf promoting activity. The demonstration of of this phenomenon is of considerable t:(;r( inoGenesis-Mechanisms Three papers (37-39) detail the inhibition of •.tfl, hrmme oxidase and consequently of oxidative ;.h,,;phonlation bv fresh tobacco smoke. In the rt i ul,,te phase. benzo(a)pyrene was said to inhibit h,rmation and function of the membrane-bound trim transport svstem. In the gas phase, a toxic hii,itor of cvtochrome oxide was identified, proba- !,i% ( kanide. The postulation here was that inhibition of wn,hi( . glvcolysis might select for the mutant cells „ith ~,Iycolvtic pathways characteristic of tumor. 1'ht~re seems to be little reason, however, to believe that this is the way malignant teansformation is induced by tobacco smoke. O. Carcinng-enesis-Isotopic Lahelin, A series of papers (40-45) described the in- troduction of carbon-13 and other stable isotopes into tobacco plants. Changes in cell morpholo',,\ were noted as w•ell as in structure and mnrphnln,~ of pollens. A. In Vitro Culture Techniques Organ Culture 1. Influence of vitamin A and 3.7-dimethvl-2.6- octadienal (citral) on the effect of benzo(a )pvrene on hamster trachea in organ culture T. Timothy Crocker and Lora L. Sanders Cancer Research Institute and Department of MedicinP. L'niversitr of California. San Francisco Medica! Center. San Francisco, California Cancer Research 30:1312-1318. 1970 Benzo(a)pyrene (BaP). 10.5 µg/mL produced cellular pleomorphic or squamous metaplastic epi- thelia and shrinking of cartilage matrix in sucklin~ hamster trachea in organ culture. The squamous state appeared earlier if citral, a presumptive vitamin A antagonist. was present with BaP than if BaP alone was present in culture medium tit,hile citral alone produced abnormally differentiated epithelia with- out clear-cut squamous metaplasia. DNA synthesis preparatory to cell division, indicated bv autoradio- graphic evidence of incorporation of tritiated thy- midine in nuclei, was increased in some pleomor- phic and metaplastic states produced bv BaP alone or by BaP plus citral, but citral did not produce significant effects on proliferative activitv. Vitamin A, 10 or 20 i.u./ml. produced shrinkage of cartilage matrix but sustained well-differentiated columnar epithelia with normal low degrees of replicative activity during 15 days of cultivation. When vitamin A was added to media containing BaP, columnar differentiation of epithelia was preserved. Prolifera- tion induced bv BaP was also inhibited bv vitamin A in some instances and more effectively bv 20 than by 10 i.u. Dissolution of cartilage matrix was greater in the presence of BaP and vitamin A. The present in vitro studv demonstrates that both vitamin A and fiaP act directly on the respiratory epithelium in a competitive fashion and on cartilage with an ad- TIMN 0115775 T200266 31

;ur i am , nonsmokers or less exposed part- ,, ,t,,inst 34 among smokers or more exposed. In 4 ,; ,rre,ponding monozygotic pairs the figures 14 against 9. The excess mortality among male ,()tic smokers was not associated with any l„ ific cause of death. Four cases of lung cancer ,:,,,,n, males occurred only in smokers. Accidents ,,,d ,uicides seemed to be associated with smoking, t,,, rtin~ the hypothesis regarding differences in type between smokers and nonsmokers. ()n!% tirne will show whether trends found are The data suggest. however, that part of the r, ,,t,>r mortality in smokers is not due to smoking ,,,,r ;t, but to factors associated with smoking. e tthpr support: Swedish %fedical Research Council. I). %tetabolism-AryI Hydrocarbon Hydroxvlase ~ oHHt-Human Mechanisms 9. Induction of aryl hydrocarbon hydroxylase in human aleveolar macrophages by cigarette smoking F: T CantrNll. G. A. Warr, D. L. Busbee. and R. R. %tartin I), partment ot Biology, M. D. Anderson Hospital and Turnor Institute. and the Department of :bfedicine, Nu~ ;"r College of Medicine. Houston. Texas l.urna/ of Clinical Investigation 52:1881-1884. 1973 Pulmonarv alveolar macrophages were obtained ;n,nr healthc % olunteers by saline pulmonary lavage, ,tnd an-l hvdrocarbon hydroxylase was measured in !i o c ells. Enzvme activity was low in cells from five „on,mokers with a mean of 0.008 ± 0.004 U/106 ~. Cells obtained from nine cigarette smokers ('11,tained higher enzvme levels, with a mean of n ott3 = 0.024 U/10fi cells. A former cigarette smoker A_i• la\acled on five occasions. Enzyme activity ;,rino two lavages four months apart were 0.010 i; (i n 009 C;/10° cells, respectively. One week after ,;•,0Lino was resumed, the enzyme activity rose ,:i;htlv to 0.013. and reached 0.041 U1108 cells by ,nk• month. Upon cessation of smoking, the enzyme ,( ti% itv returned to control levels by the next lavage, t++o months later. These data indicate that aryl h}•drocarbon hvdroxvlase may be induced in pul- monarv alveolar macrophages of subjects chroni- callv exposed to cigarette smoke. Further studies are said to be needed to determine whether any cor- relation of aryl hydrocarbon hydroxvlase and cancer exists, particularly in man. To find such a relation- ship would be a step toward identification of high- risk individuals. Other support: National Tuberculosis and Respira- torv Disease Association: the National Aeronautics and Space Administration; and the U. S. Public Health Service. 10. Induction of aryl hydrocarbon hydroxylase in human lymphocytes and pulmonary alveolar macrophages-A comparison Elrov Cantrell. David Busbee, Glenn 51'arr, and Russell Martin Department of Biology. M. D. Anderson Hospital and Tumor Institute and Baylor College of Medicine. Houston, Texas Life Sciences 13:1649-1654, 1973 Exposure of animals to cigarette smoke causes an increase in the levels of aryl hydrocarbon hv- droxylase (AHH) in various tissues. The innate capacity for enzyme induction is genetically de- termined but the extent of induction and AHH levels in various tissues may vary. In the present study. AHH levels in human pulmonary alveolar macro- phages (PAMs) were determined and AHH induc- ibility of cultured lymphocytes from corresponding volunteers was also determined. The inducibilitv bv 3-methylcholanthrene of cultured lymphocytes was similar in both smokers and nonsmokers. ranging from 0.2-4.2 fold induction. AHH levels of non- smokers PAMs was 0-0.020 units and in smokers was 0.032-0.253 units. The correlation of AHH activity in PAMs with lymphocyte inducibility was significant in both nonsmokers and smokers. The regression of AHH in PAMs as compared with AHH inducibility in lymphocytes was 8.5 fold higher in smokers than nonsmokers, reflecting the induction of AHH in PAMs by smoking. Other support: National Tuberculosis and Respira- tory Disease Association, the National Aeronautics and Space Administration, and the U.S. Public Health Service. TIMN 0115779 T200270 35

Carcinogenesis dikive effect. The organ cu.ture method thus appears applicable to the studv of the mechanisms of action of vitamin A and a car.--inogenic polvcyclic hv- drocarbon on tracheobronchial tissues. Other support: Council for Tobacco Research- U. S. A.. and the U. S. Public Health Service. 2. Maintenance of norma l, metaplastic, and dysplastic states of adult human bronchial mucosa in organ culture Thomas V. O'Donnell. T. Ti.noth_v Crocker, and Lora L. Nunes - Carciioc'nscular Research Institute (T.t'.O./ and Cancer RPsearrh Institute and Department of Medicine /T.T.C,.L.L. N.]. School of Medicine. ['niversitY of California. Snn Francisco. C3lifornia Cancer Research 33:78-87. 1973 This work was done to learn whether normal and diseased adult humau respiratory mucosa could be maintained in organ culture. to describe the stability of epithelial le:>ions. when encountered, and to compare the naturally occurring human lesions with epithelial abnormalities that have been produced in vivo and in vitro by carcinogenic hvdrocarbons, cigarette smoke condensate, and air pollutants. - Bronchial mucosa was obtained from 10 patients undergoing lo'Lectomy (8 for squamous bronchogenic carcinoma; and from 13 patients ex- amined by bronchoscopv. Randomly chosen muco- sal patches were incubatod with tritiated thyznidine and fixed as fresh samples, while others were labeled and fixed after 5 to 15 days of maintenance in organ culture. Histolo.;ical and autoradiographic observations indicated t zat some mucosal patches bore epithelia with mc)re than one histological state. Of 43 fresh mucos:- l samples, 26 had viable epithelium. in which 29 histological states were identified. Five were pathological, with squamous metaplasia in two instances and with localized dysplasia sufficiently marked to be considered as possible carcinoma in sbu in three instances. Of 133 cultured exFlants. 106 bore viable epi- thelium in which 110 histological states were ideqtificd. When initially present, columnar epi- thelium was retained in -3bout 70% of explants; this retention occurred more often if explants were from whole bronchi rather :han from biopsies. Nine cultured explants bore dysplastic or squamous meta- plastic epithelium: these were from patients vielding such lesions in fresh samples. Dvsplasias ,vere as highly cellular and replicative after maintenance in culture as they were before incubation, suggesting continuity of replication among pleomorphic cells, consistent with potentially preneoplastic lesions. Squamous metaplasia,s were poorly sustained. Swelling and sloughing of abnormal cells were consistant with failure of the system to sustain this abnormal state or with early phases in the reversal of squamous metaplasia in organ culture. Other support: Council for Tobacco Research -U. S. A.: U. S. Public Health Service. 3. Toxicity of benzo(a)pyrene and air pollution composite for adult human bronchial mucosa in organ culture T. Timothy Crocker, Thomas V. O'Donnell. and Lora L. Nunes Cancer Research Institute. Department of Medicine [T.TCL.L.M] and Cardiovascular Research Institute (T.['.O'D.]. School of Nfedicine. Lrniversitt' of California, San Francisco. California Cancer Research 33:88-93. 1973 Bronchial mucosal patches from seven adult human subjects undergoing lobotomy and from five subjects during examination by bronchoscopy were maintained in organ culture for 5 to 11 davs. Benzo(a)pyrene (BaP), 15 µg/ml, or an air pollution composite (AP), 700 µg]ml. was present in culture media during incubation of treated explants. Some control mucosal samples were fixed at time zero, and others were maintained for 5 to 11 davs on media containing only 0.4% acetone as vehicle for the test materials. All samples were incubated for 30 min with tritiated thymidine before fixation, and each tissue piece was examined by histological and autoradiographic methods. Fresh mucosal samples did not have uniformly normal epithelia because of underlying disease or injury during handling. Some mucosal pieces were in more than one state of epithelial differentiation because mucosal lesions were often smaller than the mucosal piece prepared for culture. A range of abnormalities was therefore observed during maintenance on control culture media. Exposure to BaP and AP produced suffi- ciently similar changes to permit the results to be pooled for comparison with control cultures. In- stances of each state of epithelial differentiation in 'I'IIVIIN] 0 1157'76 . ; 1: I t t I 32 T200267

Carcinogenesis of reduction of the nitroquinolines to the corre- sponding hydroxvaminoquinoline does not always parallel carcinogenicity. A model of complex formation between deoxv- guanosine in DNA and carcinogenic 4-NQO's and 4- hvdrotyaminoquinoline 1-oxides was constructed based upon molecular orbital results and steric factors. which allowed the prediction of the qualita- tive carcinogenicity of 26 derivatives of 4-NQO. Carcinogenicity may result from complex formation of the 4-nitro derivatives and/or from the interaction of the -I-hydroxyaminoquinoline 1-oxides with DNA. Other support: U.S. Public Health Service, the Damon Runvon Memorial Fund, the National Aero- nautics and Space Administration, and the South- western Medical Foundation. F. Cancer Treatment-Experimental Tumors 16. Influence of five anticancer drugs on the induction and growth of experimental mammary cancers: Comparison with ovariectomy E. Douglas Rees and Alan Ross Universitr of Kentucky College of Medicine. Lexington. Kentuckr• Cancer 21:1029-10-11. 1968 The effects of cyclophosphamide, thiotepa. methotrexate. vinblastine. and 5-fluorouracil on mammary carcinomas induced in female rats by 3- methvlcholanthrene were assessed. In one factorial experiment a group of rats received drugs con- currentlv with 3-methylcholanthrene; a second (de- lay) group received drugs in the postcarcinogen period during which cancers became palpable. Mor- talitv was less and drug effectiveness greater in the delay group. Methotrexate and 5-fluorouracil sig- nificantly prolonged time of tumor appearance but combination of the two showed a negative interac- tion. In a second experiment simultaneous adminis- tration of drugs and 3-methylcholanthrene was shown to increase mortality, especially in older rats. Drugs delayed tumor appearance but elicited more tumors than expected in older (relatively resistant) rats. Some rats receiving drugs (but not 3-methyl- cholanthrene) developed mammary carcinomas. In a studv of the effect on established carcinomas of cvclophosphanlide. 5-fluorouracil. 3-methylcholan- threne and ovariectomv. ovariectomy elicited more rapid regressions in more tumors. Drug treatment prior to ovariectomy did not affect hormone de- pendency. Other support: U.S. Public Health Service and the American Cancer Society. G. Cytogenetics-Effect of Chemical Carcinogenesis-Relation to Natural History 17. Karyotypes of rats from strains of different susceptibility to mammary cancer induction E. Douglas Rees, Amy Eversole Shuck. loseph C. Christian. and Joe R. Pugh Departments of Medicine and Pharmacology. L'niversih_ of Kentucky, Lexington, Kentucky Cancer Research 28:823-830, 1968 The female Sprague-Dawley rat is quite vu1- nerable to the induction of mammarv_ carcinomas by 3-methylcholanthrene and by 7. 12-dimethvl• benz(a)anthracene; a majority of the carcinomas induced are sex hormone dependent. The Long- Evans rat is relativelv resistant to mammary carci- noma induction but is more susceptible to leukemia. Karyologic studies were performed on cells from rats of both strains. No difference was noted in the X or Y chromosome. :[n the Sprague-Dawley. both members of the #3 pair were subterminal, whereas the mem- bers were generally heteromorphic (one subterminal and one terminal) in cells from Long-Evans rats. In inbred Long-Evans rats in particular the #12 chro- mosome was interesting in that one of the members frequently had. larger upper arms than the other, and, not infrequently, one of the homologs had large satellites on prominent upper arms. Study of diploid cells of both strains indicated a tendency, especially after administration of 3-methylcholanthrene or 7.12-dimethylbenz(a)anthracene. for a chromosome of subterminal morphology to be missing and a terminal chromosome to be gained, apparently through loss of upper arms of the former. Karyotypes of inbred Fischer, Marshall, and Osborne-Mendel rats were also established. The present data do not indicate a correlation between karyotype and sus- ceptibility to mammary cancer induction. TIMN 0115782 I I 38 T200273 --- ---~.~.u

:ng as in ing ei Ils. [1S. ed. ere his iof rch I L. ia. ,,.~~itrnl and treated cultures were distributed in the ratios I"(, in control:°o in treated): co- 36:15: metaplastic or dvsplastic. 8:?: ab- unclassified. 31:22: regenerative. 11:13: i,,1. 14:;0. BaP and AP produced toxic destruction f,III cell tvpes or. as a less marked effect. sup- ,r,,ssion of DNA synthesis and distortion of mor- ;,hnlo;ical states of columnar but not of regenerative I,ithelia. Pleomorphic replicative lesions were not i„lured in adult human epithelium although such I,-~inns were present elsewhere in untreated mucosal ,,inn1,Ic>s and have been produced by similar con- ,entrations of BaP in suckling rodent tracheas in iin,mn culture. Toxicity due to AP cannot be identi- iiecl with either a single component of AP nor any 1,,irtiCular cell type. Toxicitv of BaP for adult human respiratorv mucosa is interpreted as evidence that mic rosomal mixed-function oxidases are present ,[nd active in metabolic conversion of BaP. Adult human and suckling rodent respiratory epithelia are vuceptible to biological effects of BaP in the organ ( ulture svstem, but toxic effects were more common in the human mucosal samples examined in this st udt-. Other support: U. S. Public Health Service. Tissue Culture 4. Placental transfer of 3,4-benzpyrene and fetal benzpyrene hydroxylase induction I. V. Princiotto. M. Rubin. D. Abramson, and E. 1. /..itmiski 1)ot>nrtment of Phi•siology and Biophysics, Schools of \ttdirine and Dentistrn% Georgetown UniversitY. lt rr,hinl-ton. D. C. ('npublished Report to the AMA-ERF In vitro studies conducted with sheets of term human amnion, chorion laeve, and intact chorioam- nion. indicate that benzo(aJpyrene is transferred ,IC ross these tissues in either direction probably by simple diffusion. A small fraction of the hvdrocar- hon was metabolized in these tissues. The water soluble metabolites only appeared in the transmem- brane compartment. In addition to all the reported metabolites. we consistently found a previously unreported fluorescent compound identified as 9- t01" " banzpviceue. Chorion laeve-hvdroxvlase ac:tivitv was greater than amnion tissue activity, in placental tissues obtained from both smoking and nonsmok- ing mothers. In control experiments with visking sheets. no hvdroxvlated metabolites were produced. The major benzo(aJpyrene metabolite. 3-OH henzp\ - rene- did not readily cross the tissue preparation.s. nor did any other oxvgenated products. Homogenates of livers and placental tissue, collected from animals sacrificed on the 20th da% ()f gestation. incubated with benzo(a]pvrene produced 9-hydroxv_ benzo(aJpyrene in addition to other me- tabolites. This metabolite was not isolated from male liver homogenates. Prior administration of 3\iC stimulated arvi hvdroxvlase activitv and increased the vield of metabolites but did not alter the relative amounts of each product. At the time these findin;s were submitted for publication. there was some reluctance for acception of our results and the editor of Biochem Pharmacol requested stronger proof of identity of the 9-OH BP metabolite, which we unfortunatelv were unable to provide. (one reviewer suggested that our finding tvas an artifact). Wa- terfall. J. F. and P. Sims. Biochem /(1972) 128:265-277. then described this phenol metabolite and showed that it arises from decomposition of benzo(a]pyrene 9,10-oxide. Our finding, that there is a difference in the metabolism of the hydrocarbon in males and females was reported by several other investigators before we were ready for publication. Cell Culture 5. Lactate dehydrogenase activity of human cells infected with adenovirus /. F. Foley, B. Th. Aftonomos. and Nt. Heidrick Department of Internal Medicine and Epplet• Cancer Institute, University of Nebraska College of Medicine. Omaha, Nebraska Clinical Research 15:425. 1967 Considerable cancer investigation has been fo- cused on the enz-vtne lactate dehydrogenase (LDH). since increased glycogenolysis is a feature of tumors and LDH is a key enzyme in the process. Tumors have been found to produce increased amounts of LDH and have a distinctive isozyme pattern. Since several adenoviruses are known to be oncogenic in hamsters, we endeavored to follow the influences of adenovirus type 12 infection on the LDH of human cell lines. Latner et al. (1QFi41 have previrnislv reported that adenovirus type 12 infection of mon- key kidney cells induced an increase in the LDH TIMN 0115777 T200268 33

;.: AW40-AW es _ Carcinogenesis second half. In late decelerated growth phase. the nucleoli enter stage 4. In this stage. only a few large nucleoli are present and these are apparently inac- tive in ribosome production. In stationary phase. where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle. the granular component dominating stage I nucleoli and the fibrillar. stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated actinomycin D-cul- tures. fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase. The authors' conclu- sions suggest that nucleoli age during population growth and that this aging can be accelerated by treating the cultures with actinomycin D. Other support: The Cancer Research Co-ordinating Committee of the Universitv of California and the U.S. Public Health Service. 1. Carcinogen-Effect of Aflatoxin B on Human Peripheral Blood Lymphocyte 21. Effect of aflatoxin B, upon phytohemagglutinin-transformed human lymphocytes Herbert Savel. Ben Forsyth. Warren Schaeffer, and Thomas Cardella Departments of Internal Medicine and Medical .tficrobiologv. Universit}• of Vermont College of .~fedicine. Burlington. Vermont Proceedings of the Society for Experimental Biology and Medicine 134:1112-1115. 1970 The present study indicates that the extremely potent carcinogen aflatoxin B, is able to inhibit the stimulated uptake of tritiated thymidine by phy- tohemagglutinin in human peripheral blood lym- phocytes. and to decrease the stimulation induced by potent antigens-in this case, tuberculin and mumps. The data suggest that the human peripheral blood lvmphocytes are as sensitive to the effects of aflatoxin B, as are the human embryonic liver cells, and that aflatoxin B, appears to block a pathway common to both antigenic and phytohemagglutinin stimulation. Studies are in progress to determine whether there are any specific morphological al- terations in the aflatoxin B,-exposed l}•mphocvtes. Studies on the possible s_vstemic immunosuppres- sant effects of aflatoxin B, in animals appears to be warranted by the present observations. Other support: U.S. Public Health Service. J. Carcinogenesis-Colon-Chemical Induction 22. N, N' 2,7-Fluorenylenebisacetamide (2,7- F.A.A.)-Induced rat bowel cancer Jeremy H. Thompson Department of Pharmacology and Experimental Therapeutics, U.C.L.A. School of tifedicine. Los Angeles. California Irish Journal of Medical Sciences. 7th Series 2:565-583, 1969 Gastrointestinal serotonin levels. and the in- cidence of toxicitv and cancer were studied in 12 control and 36 carcinogen-treated male Buffalo rats. Animals received Purina rat chow with a tryptophan content of 0.22%; treated rats received 0.025g% (i%,1! w) N.N'-2-7 -Fluorenvlenebisacetamide (2.7-F.A.A.) added to the chow for 8 months. Body weight gain was normal in control rats but greatly reduced with 2,7-F.A.A. feeding. Two control and 8 treated rats died of chronic bronchopneumonia; no tumours were found. Two control and 3 treated rats were examined histologically after 3 months; no tumours were found. The experiment was terminated at 8 months as the treated rats were emaciated. Serotonin was determined spectrophotofluorometrically after complexing with o-phthaldialdehyde in the mucosa of stomach fundus, pyloric antrum. upper duode- num, small bowel, ascending colon and descending colon from all rats, and in all tumours and adjacent microscopically normal mucosa in treated rats. De- tailed histological studies were also performed. No tumours were found in control rats and apart from mild chronic bronchopneumonia, histological ex- amination was normal. The major pathological find- ings in the treated rats were atrophy of most body tissues and organs, severe chronic bronchopneu- monia with bronchiectasis, and numerous cancers. Atrophy was particularly prominent in the stomach, small bowel, kidney, suprarenal, bladder, testes. TIMN 0115784 I i I I I I 40 T200275

1 , i I hedrt, spleen, pancreas. and liver. Increased gas- ,rc~intestinal mucin production was usual. Two rats had an adenoma in the heart and suprarenal gland, rpspectively: liver atrophy was followed by cirrho- ;is. Cancers occurred as follows (n=25): skin 4%. oastric rumen 12°'0, pyloric antrum 12%. upper duodenum e%. small bowel 56%, ascending colon descending colon 8%, primary hepatoma 72%, i:holangiocarcfnoma 48%, reticulum cell sarcoma t2J,,. and lymphosarcoma 4%. Serotonin levels %vere. in general. significantly increased in non- malignant mucosa of treated rats compared to con- ,rols (P < 0.05-0.001), and significantly reduced in , ancer mucosa when compared to non-malignant mucosa from the same rat and controls (P < 0.05- 0.o011. It is suggested that a fall in gastrointestinal mucosal serotonin may be related to malignant degeneration in the bowel. Other support: California Institute for Cancer Re- search and the American Cancer Society. K. Carcinogenesis-Inhibition Enhancement-PAH Metabolism 23. Induction of increased benzpyrene hvdroxylase activity by flavones and related compounds Lee W. Wattenberg, Mary Anne Page, and J. Lionel Leong Department o,f Pathology, University of Minnesota ~ledical School, Minneapolis. Minnesota Cancer Research 28:934-937, 1968 A study of relationships between the structure of flavones, flavanones, and chalcones and their capacitv to induce increased 3,4-benzpyrene hy- droxvlase activity in the liver and lung of the rat was carried out. Appropriate halogenation increases in- ducing activity. In the compounds studied, hydrox- ~ lation reduces inducing capacity, but the corres- ponding methoxy compounds are active. Two nat- urallv occurring polymethoxy flavones, tangeretin and nobiletin. have been found to be active inducers of benzpyrene hydroxylase activity. 24. Induction of increased benzpyrene hvdroxylase activity by 2-phenvlbenzothiazoles and related compounds Lee W. Wattenberg, yfarv Anne Page, and J. Lionel Leong Department of Pathology. University of Minnesota Medical School, :Winneapolis. Minnesota Cancer Research .28:2539-2544.1968 A study of the relationships between structure of 2-phenylbenzothiazoles and related compounds and their capacity to induce increased benzpyrene hydroxylase activity in the liver and lung of the rat has been carried out. Thirty-three compounds have been studied. It has been found that introduction of an appropriate halogen into the 4'-phenyl position approximately doubles inducing activity compared to unsubstituted 2-phenylbenzothiazole. Other sub- stitutions and modifications of the molecule result in either lesser increases in inducing activity or, in some instances, reduction or total loss of inducing activity. Most compounds show similar inducing effects on both lung and liver. Some, however, do not. Of the more potent inducers, the compound showing this selective effect to the greatest degree is 2-(4'-cyanophenyl)-benzothiazole, which has more than double the inducing effect on lung as on liver. Other support: U. S. Public Health Service. 25. Induction of increased benzypyrene hydroxylase activity in pulmonary tissue in vitro Lee W. Wattenberg, J. Lionel Leong, and Arthur R. Galbraith Department of Pathology, University of Minnesota School of Medicine. Minneapolis, Minnesota Proceedings of the Society for Experimental Biology and Medicine 127:467-469, 1968 A technique was developed for obtaining in- duction of increased benzypyrene hydroxylase activ- ity in short-term cultures of rat pulmonary tissue. Several phenothiazines, polycyclic hydrocarbons, and other compounds which induce increased benz- pyrene hydroxylase activity in vivo when adminis- tered to rats will likewise induce an increased activity of this enzyme system in vitro when added to the culture system. Other support: U. S. Public Health Service. TIMN 0115785 T200276 41

a n- ore ent d e- the ty ul- tas vl- ias lg- c i- ia. its .Y ,rs ,n- a s 18. Chromosomes of mammary carcinomas induced,by 3-methylcholanthrene in rats ; ti%lajumdar and E. Douglas Rees .),.partrnents of.Wedicine and Pharmacology. C.'niversity ~,,ntuck). Lexington. Kentucky ; arnni uf HereditY 61:231-236. 1970 Chromosome preparations of cells from mam- marv carcinomas induced by 3-methylcholanthrene ,,l Sprague-Dawlev rats were studied by direct and h% short-term culture techniques. The primary stem- line in all the carcinomas was that of a cell with a normal appearing karvotvpe. Although chromosome ,{berrations were found. the type of aberration and the chromosome(s) affected varied from cell to cell in the same carcinoma. Marker chromosomes were rarelY seen. Hoivever, chromosomes in the no. 11-13 group. which have a centromere in the submedian rNgion. were of interest, since 27 percent of cells %vith a diploid chromosome number that were studied in detail lacked at least one of the chromo- ;omes in this group-apparently due to loss of short ,irms. The karvotype of the mammarv carcinomas ~, ere less deviated from normal than the karvotvpes Of most transplanted rat tumors that have been reported. This is in accord with the well-differen- tiated histologic and metabolic characteristics of these mammary carcinomas. Other support: Agricultural Research Service, U.S. Department of Agriculture. 19. Changes in chromosomes of bone marrow after intravenous injections of 7,12- dimethvlbenz(a)anthracene and related compounds E. Douglas Rees, S. K. %tajumdar, and Amy Shuck :)-oartment oi.lfedicine and Pharmacology, University &rntucki .tiledical Center, Lexington. Kentucky eedings of the :`'ationa/ Academy of Sciences 66.1_28-1235.1970 %Iultiple intravenous injections of an emulsion nntaining 7,12-dimethylbenz(a)anthracene (DMBA) t)r 7.8.12-trimethvlbenz(a)anthracene (TMBA) in- duce a high incidence of leukemia in rats. The pres- ent investigation focused on a study of changes in chromosomes of bone marrow following single or multiple administration of these and related conl- pounds. Twenty-four hours after a single injection. about half of the metaphase cells in the marro« have chromosomes with breaks. Although hreaks were inflicted on chromosomes of various sizes ind morphology, these aberrations were nonrandom in that members of nos. 1 and 2 chromosome pairs were involved to a greater extent than expected on the ba- sis of their size and number. Distinctive karvotypic: abnormalities involving the no. 2 chromnznme were observed in half of the leukemic rats. «here,3s these abnormalities were not observed in nonleuke- mic. DMBA-treated rats. Benzo(a)pvrene and ben- zo(e)pvrene, polycvclic aromatic hvdrocarbons which did not induce leukemia, produced fewer breaks in the no. 2 (and other) chromosomes than did DMBA or TMBA. Other support; Agricultural Research Service. U.S. Department of Agriculture, Eastern Utilization Re- search and Development Division. H. In Vitro-Nucleolar Changes 20. Nucleolar aging in Tetrahvmena during the cultural growth cycle Birgit Satir and Ellen Roter Dirksen (P. Satir) Department of Physiology-Anatomy, Universitr of California, Berkeley. California, and the Cancer Research Institute. University of California. San Francisco. California Journal of Cell Biology 48:143-154. 1971 Nucleolar morphology was studied by electron microscopy in control and actinomvcin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the TIMN 0115783 T200274 39

Carcinogenesis interac:tions. direct binding of 4-NQO to DNA pro- ~-ides a suO_Cgested mode of action. F. Cancer Treatment-Experimenta/ Tumors The effect of five anticancer drugs (16) on the induction and growth of inethylcholanthrene in- duced breast cancer in female rats was examined. Drugs and carcinogen were given concurrentl\• in one group and after development of tumors in another group. Combinations of drugs were also used and all drug effects were compared with ovariectomv. G. CvtoQenetics-Effect of Chemical Carcinogens- Relation to ,1'atural Historv Three papers (17-19) are devoted to the relation of karyotvpes of rats to susceptibility to tumor induction, with no evidence of anv relationship between the two being reported. Additional studies were performed to determine the effect of selected carcinogenic PAH on chromosome patterns during tumor induction and progression. H. In ['itro-Nucleolar Changes The purpose of this study (20) was to determine the sequence of aging changes in nucleolar mor- phology of cells from Tetrahvmena pyriformis. Evi- dence is adduced that nucleoli age during pop- ulation growth and that aging is accelerated by Actinomvcin D. I. Carcinogen-Effect of Aflatoxin B on Human Peri pheral Blood Lymphocyte In this study (21) aflatoxin B was shown to inhibit the uptake of tritiated thymidine by phy- tohema~lutinin or antigen stimulated human pe- ripheral blood lymphocytes. It is suggested that aflatoxin B blocks a pathway common to both types of stimulation. A possible immunosuppressive ac- tion of aflatoxin B is deemed worthy of investiga- tion. J. Ccrcinogenesis--Colon----Chemiccl Inducticn A known carcinogen N,N'2.7-Fluorenylenebisa- cetamide, was used (22) to induce bowel cancer in the rat. Tumor induction was associated with a drop in serotonin levels. This observation has led the investigator to suggest that a fall in gastrointestinal mucosal serotonin may be related to cancer in- duction in the bowel. K. Carcinogenesis-Inhibition/Enhancement-P.-tH .'lletabolism This series of nine papers (23 through 31) is directed toward establishing the relationship be- tween induction of AHH bv hN•drocarbons. the rates and kinetics of induction. the interference or en- hancement of induction by a wide %,arietv of chemi- cal agents and the correlation between AHH action and ultimate tumor induction. The investigator has worked in this field for approximately fifteen vears and data suggest that stimulation or enhancement of AHH activity has a protective effect against the induction of cancer by carcinogenic hydrocarbons. The recognition that AHH is one of a group of mixed-function oxidase systems provides wide lati- \ 1 tude in stimulating the formation of this enz%-me b~- a I t varietv of innocuous ubiquitous agents. hi This observation is at odds with the preliminan data of others who suggest that AHH activitv is positively correlated with susceptibility to, and rates of. tumor induction-the more enzyme. the greater the effect of the carcinogen. Antiviral screening (32) with an ethanol insoluable fraction of tobacco leaf and a nontumoro- genic fraction of tobacco smoke condensate sug- gested activity against encephalomyocarditis infec- tion and suppression of Erlich ascites tumor cells in mice. L. Cocarcinogenesis-Cigarette Smoke-BioassaY pt Five cigarette smoke components were tested for cocarcinogenicity using mouse skin system (33). A subsequent paper (34) extended the original study to 21 tobacco smoke components that might play a role in carcinogenesis, promoting agents. or inhibitors of benzo(a)pyrene carcinogenesis. All of these varieties of actions were observed among the tested materials. Particularly noteworthy was the enhancement of berrzo(a)pyrene carcinogenicitv b~ TIMN 0115774 pi th el in bl af W T tF ir 30 T200265

Carcinogenesis ~ Se fewer in number. However. close study of the pollen of such carbon-substituted plants revealed gross changes ip the microscopic appearance of the pol- len. Further study of the isotopically substituted pollen by means of scanning electron microscope techniques showed extensive and obvious altera- tions of morphology. The extremely regular ellipsoi- dal, trilobate appearance of the control pollen was seen in only a small percentage of the isotopically substituted pollen. Irregularity of size and shape, distortion of the longitudinal furrows and irregular- ity of the pores and the surface substructure were typical of "C pollens. Study of the pollen of morning glory, grown concurrently during one growth cycle. indicates that these effects may be of general oc- currence as a consequence of "C-substitution. Ad- ditional studies are in progress, using the techniques of pollen paleontology. Other support: Atomic Energy Commission. 45. Culture of 13C-substituted plants of interest in pharmacognosy and pharmacology R. A. Uphaus. M. 1. Blake, and J. J. Katz Department of Pharmacy, College of Pharmacy, University of Illinois, Chicago, Illinois, and Chemistry Division, Argonne National Laboratory, Argonne, Illinois Unpublished report to the AMA-ERF. The techniques of deuterium substitution for the production of drugs of pharmaceutical interest were extended to the use of the heavy, stable isotope of carbon, carbon-13. Use of this isotope in studies of biosynthetic pathways, specific labeling for NMR and ESR studies, and general appreciation of iso- tope-induced biological effects is now feasible with the use of sealed, self-sustaining growth systems to produce almost any plant of interest. In addition, the continued use of these growth chambers indicated the feasibility of producing insects and other ani- mals in situ, when the appropriate food chain is established. Extensive incorporation (above 90%) of "C into animal metabolic pathways may thus be realized. Past work (by others) on the use of in vivo site labeling for the purpose of studying the free radicals in tobacco smoke and their interaction with the substrate, could now become possible with the use of the 13C culture techniques developed here. Other support Atomic Energy Commission. TIM.N 0115792 Ca At T200283 48

Carcinogenesis 26. Inhibition of the carcinogenic action of 7,12- dimethylbenz(a)anthracene by beta- naphthoflavone L. W. Wattenberg and J. L. Leong Department of Pathology, University of Minnesota Medical School, Minneapolis. Minnesota Proceedings of the Society for Experimental Biology and Med icine 128:940-943. 1968 Beta-naphthoflavone, a potent inducer of in- I creased polycyclic hydrocarbon hydroxylase activ- ! ity. was found to inhibit the occurrence of neoplastic I lesions resulting from 7,12-dimethylbenz(a)anthra- i cene (DMBA) in experiments in which the beta, naphthoflavone was given prior to the carcinogen. Pulmonary adenoma formation in the AIHeJ strain mouse resulting from oral administration of DMBA and mammary tumor formation in the Sprague- I Dawley rat also from oral administration of the same I carcinogen were both inhibited by beta-naphthofla- I vone. Other support: U. S. Public Health Service. 27. Inhibition of the carcinogenic action of benzo (a) pyrene by flavones Lee W. Wattenberg and J. Lionel Leong Department of Pathology, University of Minnesota Medical School. Minneapolis, Minnesota Cancer Research 30:1922-1925. 1970 A study of the effect of feeding flavone inducers of increased benzo(a)pyrene (BP) hydroxylase activi- ty on pulmonary adenoma formation resulting from oral administration of BP to AIHeJ mice was carried out. Three flavones were used: R-naphthoflavone, a highly potent inducer; quercetin pentamethyl ether, intermediate in inducing potency; and rutin, a weak inducer. Administration of R-naphthoflavone re- sulted in almost total inhibition of pulmonary ade- noma formation: with quercetin pentamethyl ethe approximately 50% inhibition occurred, and wit rutin there was no inhibition. In a second investiga tion, the effect of inducing increased BP hydroxylaseJ activity in the skin of Ha/ICR mice on epidermat, neoplasia initiated by BP was studied. Topicau application of f3-naphthoflavone resulted in slightly ~ I more than 50°io inhibition of skin tumor formation. Other support: U. S. Public Health Service. 28. Phenothiazine inhibition of intestinal and urinary bladder tumors induced in rats by bracken fern A. M. Pamukcu. Lee W. Wattenberg, J. %f. Price. and George T. Bryan 1 Department of Pathological Anatomy. College of Veterinary Medicine. University of Ankara. Ankara. Turkey; Department of Pathology. University of Minnesota Medical School, Minneapolis, Minnesota: Scientific Divisions, Abbott Laboratories. North Chicago. Illinois: and Division of Clinical Oncology. UniversitY oi Wisconsin Medical School. Madison. Wisconsin Journal of the National Cancer Institute 47:155-159, 1971 Bracken fern (Pteris aquilina), mixed with grain (1:3 by weight), was fed to 2 groups of male and female albino rats that also received 2 mg thiamine hydrochloride subcutaneously once weekly. In Group 1, fed t:his diet, 19 of 21 rats surviving more than 6 months developed intestinal neoplasms (ad- enomatous polyps or adenocarcinomas) and the same number developed urinary bladder tumors (papillomas or transitional cell carcinomas). In Group 2, of 28 rats fed the bracken fern diet, to which phenothiazine (2 mglg diet) was added, 11 had intestinal tumors and 10 had bladder tumors. The average survival of the rats in both groups was 11 months. Nine rats fed a grain diet containing no bracken fern developed neither intestinal nor blad- der tumors. Rats receiving the phenothiazine- bracken fern diet had persistently increased levels of benzo(a)pyrene (BP) hydroxylase activity in the mucosa of the small intestine, liver, and kidney, whereas rats fed the bracken fern diet (Group 1) showed normal or only slightly elevated levels of this enzyme. The magnitude of induction. relative to untreated control rats, was highest in the kidney and similar for the mucosa of the small intestine and liver. BP hydroxylase activity of the small intestine progressively decreased in all groups of animals. These data are consistent with the possibility that phenothiazine-stimulated microsomal metabolism of the bracken fern partially inhibits the genesis of intestinal and bladder neoplasms. However, the possibilities of a direct chemical interaction between carcinogen and phenothiazine at some point before the active site, or of a competitive inhibition of the TIMN 0115786 c I I 1 I f I , 42 T200277

Section II Carcinogenesi<s ~tlfillll,fC\ t.,,,,_Eeports of research related to carcino- ,l l,e se,re;ated into sev-era] areas on the t ro ,t,,arc.h ' (loals. .-\ variety of experimental ,,,,d approaches were used. ('ultc+re Techniques ;; (:ulture t i„• ;nfluence of polvcx,lic aromatic hydrocar- I.~F{I „n in vitro organ cultures of hamsters „as studied to determine the mode of ,f P.-\H on respiratory epithelium and pos- ,, ,i ;,n le( tk-e effect of Vitamin A on carcinoge- The studies indicate that both PAH and % ,ct directlv and competitively on epi- i,,,; Or,a n culture was shown to be a useful tool <tuciies. These reports (1-3) contribute to ! r,,,1 ,,rt,,i of organ culture research now under i,,~,-~ti;ation in man% • laboratories. ( ,rltirre i.inu, sheets of human chorion and amnion, dnd transport of benzo(a)pyrene and its i lit« . n-ere studied. A previously unreported :,mn l. tt-OH benzo(a)pyrene was identified }u-11 studies were also pursued in homoge- 4 li~ors and placental tissues of animals +,,,f on the 20th dav of gestation. The above ;,M',i n,etabolite was again identified. otft~( t of adenovirus type 12 on human cell .••,,• t,\.jnlined (5) \rith particular reference to hl induction of lactic dehydrogenase n ;,rofile that might resemble the pattern ~wli tor tumors. Although there were mor- :! ,,' , hang(l, i n the infected cells, the LDH •.,re not th-;v described previously as an t)attern , (,,npmtible with malignancy. Carcinogenesis \ r~ ir~\~ 15) reported on work related to the use Sman hamster for studying chemical car- ~~ lil,,dder carcinogenesis in rats exposed to ciga- -!tl• ,mnku condensate by fecdings was investiguted (7). The effect of concomitant treatment with phe- nobarbital. an hepatic microsomal enz\-me inducer, as well as parathion, an hepatic microsomal enzyme inhibitor, was also examined. Earlv incomplete re- sults suggest a dose-related tumor response with potentiation by phenobarbital and inhibition b\ parathion. C. Epidemiology-Genetics-Human One studv (8) using the Swedish Twin Registrv examines the mortality rates in smoking discordant monozvgotic and dizvgotic twins. The data are preliminary and firm conclusions cannot be drawn. The research had. at the time of writing of this report. failed to prove any firm basis for quantifying the relative importance of genetics vs. environment on the mortalitv of ririns. This ma\- only be achieved xrith the accumulation of a larger experience in view of the number of variables related to morbiditv and mortalitv and the multis\_•stemic effect of cigarette smoking. D. Metabolism-Ary) H<,drocarbon Ht•droxrlase (AHH)-Human Mechanisms The role of AHH in the metabolism of car- cinogenic hydrocarbons is generally acknowledged. Studies in this area (9• 10) were directed toward determining the induction rates of AHH in human alveolar macrophages and human lymphocytes. The data confirm the inducibility of enzyme by polvc}•- clic aromatic hydrocarbons with certain differences noted between lvmphocytes and macrophages as well as between smokers and nonsmokers. These papers preceded attempts to related AHH levels to lung cancer. E. ildetabolism-Nucleic.9cid-Chemical Carcinogenesis This series of papers (11-15) is devoted to the effect of the carcinogen 4-nitroquinoline N-oxide (4- NQO) on nucleic acid metabolism. The fundamental studies are concerned with correlating the potent mutagenic, carcinogenic and carcinostatic effect of 4-NQO with its effects on nucleic acid. Research on the possible mechanisms and site of artion of this compound has been conducted ultrastructurally as well, particularly for nucleolar effect. Among other TIMN 0115773 T2o0264 ~ 29

,1ddition of -t-nitroquinoline-N-oxide, increased ,jmounts of low molecular weight RNA could be ,jemonstrated by trichloracetic acid extraction or ;neth>~lated alb lre sgented c active so nthetic This increase probably Pre Y pruducts and was not derived primarily from de- ,raded RNA of higher molecular weight. Exposure of HeLa cells to 4-nitroquinoline-N-oxide for 24 huurs resulted in depressed synthesis in all RNA fractions: RNA sedimenting below 10S was less depressed than heavier RNA fractions, as evaluated bv sucrose density gradient centrifugation. The similarity between RNA synthesis in the presence of 4-nitroquinoline-N-oxide and actinomy- ( in D in low dosages is discussed. and the possible ,imilarities in the sites of action are described. Other support: The Damon Runyon Memorial Fund for Cancer Research, and the National Aeronautics and Space Administration. 14. Interaction of 4-nitroquinoline 1-oxide with deoxyribonucleic acid and synthetic polydeoxyribonucleotides 1 S. Paul and P. O'B. Montgomery, Jr. L'nirersity of Texas Southwestern Medical School, Department of Pathology. Dallas, Texas. and Laboratories for Cell Research, Woodland Hospital. Dallas. Texas 1lolec:ular Pharmacology 6:315-322, 1970 fn the present study, native calf thymus DNA and. to a lesser extent. poly dG:dC produced marked ( hanges in the absorption spectrum of 4-nitroquino- line 1-oxide (4NQ0) as determined by difference ;nectrum methods. The addition of sodium chloride from 0.5mM to 0\1 decreased the effect of native DNA on the 1 ifference spectrum of 4NQO by approximately 4- told. The interaction was partially dependent upon ionic parameters, which suggested the importance of charged sites in the interaction of 4NQO with D\A. CJrea (6Lf) abolished the effect of DNA on the difference spectrum of 4-NQO. The effect of urea may indicate some role of hydrogen bonding in the formation of the DNA-4-NQO complex or in the alteration of hydrophobic interactions resulting in disruption of the DNA-4NQO cwuplex. Strand separation (Tm) of native DNA was sig- nificantly stabilized by the addition of 4-NQO. Studies on the binding of native DNA with 4- NQO resulted in a nonlinear curve, consistent with the involvement of more than one site of interaction in the formation of the DNA-4-NQO complex. The integrity of the double-helical conformation of DNA and the presence of G-C pairs appeared to be essential for maximal complex formation. The bind- ing of 4-NQO to DNA evidently is complex and cannot be described by a single model of complex formation. Other support: U.S. Public Health Service, the Damon Runyon Memorial Fund, the National Aero- nautics and Space Administration, and the South- western Medical Foundation. 15. A proposed model of the interaction of 4- nitroquinoline 1-oxide with DNA J. S. Paul, P. O'B. Montgomery, Jr., and 1. B. Louis University of Texas Southwestern Medical School. Department of Pathology, and Laboratories for Cell Research, Woodlawn Hospital, Dallas, Texas Cancer Research 31:413-419, 1971 Extended Huckel molecular orbital computa- tions were performed on both carcinogenic and noncarcinogen.ic 4-nitroquinoline 1-oxides (4- NQO's) and related compounds. Distinct correla- tions between carcinogenicity and several molecular orbital characteristics were found. All correlations between molecular orbital properties and carcino- genicity were modified by one or more of the following structural factors: (a) the absence of a 4- nitro group, (b) the absence of a 1-oxide group, or (c) the presence of bulky substituents in position 2 or 3 of the quinoline ring. Any of the three factors resulted in either noncarcinogenicity or reduced car- cinogenicity. Conformational studies indicated that the 4-nitro group was approximately 60° out of the plane of the quinoline ring and that the addition of an electron to 4-NQO was energetically possible. 4-Hydroxyaminoquinoline 1-oxide, a metabolic reduction product of 4-NQO, has been considered a possible proximal carcinogen. The theoretical ease TIMN 01157g1 T200272 37

81. %tvocardial Blood Flow in Coronary Artery Disease: Correlation with Severity of Disease and Treadmill Exercise Response ................................................. 106 82: Mtocardial Blood Flow: Measurement b~• a Coincidence Counting System and Single Bolus of n4Rb .................................................................... 106 83. Measurement of Coronarv Flow in Ischemic Heart Disease ........................... 106 84. Recognition of Coronarv Arterc• Disease ......................................... 106 85. Coronary Blood Flow in Relation to Angina Pectoris ............................... 107 86. Interaction of the Chemoreflex and the Pulmonary Inflation Reflex in the Regulation of Coronar% ~ Circulation in Conscious Dogs ................................................ 107 87. The Action of Inhaled Cigarette Smoke on the Microcirculation, Heart Rate and Carotid Pressure of the Bat ................................................................ 107 88. Microcirculation in the Ventricle of the Dog and Turtle ............................. 108 89. The Effect of Nicotine. Propranolol. Phentolamine. and Hexamethonium on the Microcircula- tion of the Cat ............................................................ 108 go. Studies on the Coronary Microcirculation by Direct Visualization ...................... 109 91. The Coronarv Microcirculation in the Potassium Chloride Arrested Heart ................ 109 92. Effect of Nitroglycerin on Total and Regional Coronary Blood Flow in the Normal and Ischaemic Canine Mvocardium ........................................................ 109 93. Effect of Nicotine and Vasopressin on the Terminal Vascular Bed of the Heart ............ 110 94. Capillaries in Heart and Muscle of Dog and Rabbit ................................. . 110 95. Effect of Nicotine on Capillary Flow and Terminal Vascular Capacity of the Heart in Normal Dogs and in Animals with Restricted Coronary Circulation ........................... 110 96. Capacity of the Terminal Vascular Bed During Normal Growth, in Cardiomegaly, and in Cardiac Atrophy ................................................................. 111 97. Physiological Studies on Revascularization of the Dog Heart ......................... 111 98. Cholinergic Mechanisms on the Heart and Coronary Circulation ....................... 111 99. Effects of Diphenylhydantoin (Dilantin) on Peripheral and Coronary Circulation and Myocardial Contractility in the Experimental Animal ........................................ 112 100. Origin of Blood Supply to Sinoauricular and Atrioventricular Node .................... 112 Clinical Studies 101. Coronary Collateral Circulation and Myocardial Blood Flow Reserve ................... 112 102. Correlation of Computer-Quantitated Treadmill Exercise Electrocardiogram with Arteriographic Localization of Coronary Artery Disease ......................................... 113 103. Rehabilitation of the Coronary Patient .......................................... 113 104. Multistage Electrocardiographic Exercise Test: Principles and Clinical Applications ........ 114 105. Central and Peripheral Vascular Effects during Cigarette Smoking ..................... 114 106. An Analysis of the Effects of Nicotine on the Cerebral Circulation of an Isolated Perfused, in Situ Cat Brain Preparation ....................................................... 114 52 TIMN 0115796 T200287

)6 )6 )6 36 07 07 07 )8 8 )9 l9 )9 to l0 .0 2 12 12 i3 13 t-t 14 14 t07. Direct Central and Reflexly Mediated Effects of Nicotine on the Peripheral Circulation ...... 11,5 108. The Effect of Glucagon on the Coronary Circulation in Man .......................... 1 15 109. The Effect of Cardioacceleration by Right Atrial Pacing on M~~ocardial Blood Flow in Normal Human Subjects ........................................................... 116 D, Electrophysiological Studies and Studies on Contractility ................................ l lf; Fundamental Studies 110. Chronic Partial Occlusion of the Pulmonary Arterv in Cats. Change in Ventricular Action Potential Configuration during Earlv Hypertrophy ................................. 1 1e t t t. Electrophysiological Studies on Organization of Central Vasopressor Pathways ........... 116 112. The Effect of Change in Cycle Length on the Ventricular Action Potential' in Man ......... 117 113. The Pharmacological Effects of Potassium: The Occurrence of Sino-Ventricular Conduction .. 117 114. Electrophysiological Correlates of Contractile Change in Mammalian and Amphibian `tyo- cardium ................................................................. 118 115. Mechanisms of Ventricular Fibrillation .......................................... 118 116. Asynchrony of Conduction within the Canine Specialized Purkinje Fiber System .......... 118 117. T Wave Alternans: An Association with Abrupt Rate Change ......................... 1 19 118. Electrophysiologic Correlate of Exit Block ....................................... 119 119. Role of the Premature Action Potential in Contractile Potentiation: A Studt• of Paired Stimulation .............................................................. 119 120. Effects of Acetylcholine on Automaticity and Conduction in the Proximal Portion of the His- Purkinje Specialized Conduction System of the Dog ................................ 120 121. Digitalis and Vagal Stimulation during Atrial Fibrillation: Effects on Atrioventricular Con- duction and Ventricular Arrhythmias ........................................... 120 122. Effects of Cycle-Length Alteration on Canine Cardiac Action Potentials ................. 120 123. Rate-Dependent Right Precordial Q Waves: "Septal Focal Block" ...................... 120 124. Some Effects of Nicotine on Cardiac Automaticity, Conduction, and Inotropy ............. 121 125. High-Fidelity Recording of Cardiac Depolarization ................................. 121 126. The Role of Inotropic Variation in Ventricular Function during Atrial Fibrillation ......... 121 127. Exit Block ............................................................... 122 128. Electrophysiologic Studies on Wenchebach Structures below the Atrioventricular Junction . . 122 129. Exposure of the Canine Proximal AV Conducting System for Electrophysiologic Studies .... 122 130. Automaticity: Mechanisms and Electrocardiographic Consequences .................... 123 131. Mechanical and Electrical Actions of Nicotine on Hypertrophied and Failed Cat Ventricle ... 123 132 Nicotine and the Action Potential of Cat Ventricle ................................. 123 133. The Effects of Nicotine upon the Cardiac Action Potential and Contractile State ........... 124 134. Cardiac Arrhythmias Induced by Nicotine: Electrophysiological Mechanism(s) ............ 124 135. Biphasic Effect of Nicotine on Action Potential Repolarization in Electrically Driven Guinea-Pi g Atria .................................................................... 124 TIMN 0115797 T200288 53

( 5-I. Early Changes in Energy Metabolism in the Lh-ocardium Following Acute Coronary Artery Oc;c.lusion in Anesthetized Dogs .................................................. 96 55. Stimulation of Reparative Processes Following Experimental Myocardial Infarction ......... 96 i6. Acute Alterations in Energetic:s of Ischemic Heart tituscle . . . . . . . . . . . . • . . . . . . . . . . . . . . . 97 :,;. The Use of Gl% -colt•tic Metabolism in the Assessment of Hc•poxia in Human Hearts ......... 97 :,8. Metabolic Changes in Infarcted and_ Non-Infarcted Myocardium during the Postinfarcticm Period ........................................ ................ < .17 8fi ;,9- Biochemical Changes in Non-Infarcted Heart Muscle Following Mvocardial Infarction ....... 97 R% (30. Changes in Mvocardial High Energy Phosphate Levels and Left Ventricular Function after 8, Coronary Artery Occlusion in Anesthetized Dogs ... . ............................... 98 1 Kt. Storage and Metabolism of Norepinephrine after Experimental Mvocardial Infarction ........ 98 87 F'?. Changes in Norepinephrine Stores in the Canine Heart Following Experimental 1.tt•ocardial Infarction .................:............................................... 99 88 t;3. I'ptake and Subcellular Distribution of Norepinephrine in the Canine Heart Followin88 E\perimental M~~ocardial Infarction ............................................ 99 88 64. Functional Compartmentation of ATP and Creatine Phosphate in the Heart Muscle ......... 99 65. LDH-Isoenzvmes in Infarcted Heart Muscle ...................................... 100 89 66. Changes in Enzyme Pattern of Infarcted Heart Muscle during Tissue Repair .............. 100 89 • 67. Reparative Processes in Heart Muscle Following :vlvocardial Infarction ................. 101 89 68. `lvosin Srnthesis in Chronic Heart Failure ....................................... 101 89 r9. The Metabolism of the Heart in Failure ......................................... 101 70. Inhibition of Protein Svnthesis in Cardiac Hvpertrophy in Its Relation to Myocardial Failure . 101 90 ~ ; 1. Ntetabolism of the Heart in Failure ............................................. 102 90 '2. Accumulation of Norepinephrine in Rat Tissue Following Treatment with Three Beta- Adrenergic Antagonists ..................................................... 102 91 7:3. Ac:tion of a Beta Adrenergic Receptor Blocking Agent on the Positive Chronotropic Response 92 and Uptake of Norepinephrine in the Perfused Guinea-Pig Heart ...................... 102 92 74. Effects of N ic ()tine and Svmpathomimetic Amines on Potassium Intoxication ............. 103 t)3 (' (:nrnnany. Capillart•. Cerebral and Peripheral Circulation ................................ 103 9:3 I:u nclamrsntal StudiE s %:~. Fluorescence Histochemical and Ultrastructural Studies on the Coronarv Artery of the Dog .. 103 94 76. The Effects of Nicotine on Coronary Circulation of Dogs ........... . . . . . . . ........... 104 94 77. The Effect of Nicotine on the Coronary Microcirculation in the Cat Heart ................ 104 94 78. Effects of Intracoronary Infusions of Acetvlcholine and Nicotine on the Dog Heart in Vivo ... 105 ' 95 79. The Use of Positron Emitter in the Determination of Coronary Blood Flow in Man ......... 105 95 80. Measurement of Coronary Blood Flow with Radioisotopes ....... 95 105 TIMN 0115795 T200286 51

26. AdronerQic Innervation and Cocaine-Induced Potentiation of Adrenergic Responses of Aortic Strips from Young and Old Rabbits ............................................. 27. Pharmacniogic Nature of Adrenergic Innervation in Rat Aorta .............. . . . . . . . . . . . 8i 28. The Neuromuscular Mechanism of Veins ......................................... 8'j 29. An Unusual Venoconstriction Induced by Acetylcholine ............................. 88 30. Localization of Venoconstrictor Responses ........................................ 86 31. The Supersensitirity of Isolated Rabbit Atria and Aortic Strips Produced by 6-Hydroxydopa- mine .....................................................................8Fi 32. Nicotine Monomethiodide and Vascular Adrenergic Transmission ...................... 87 33. Sympathetic Mechanisms in Blood Vessels: Nerve and Muscle Relationships .............. 87 34. Pressor Effects of Four Aliphatic Aldehydes and Their Interactions with"C-Norepinephrine in an Isolated Smooth Muscle Preparation .......................................... 87 35. Dose-Dependent Sympathomimetic and Cardioinhibitory Effects of Acrolein and Formaldehyde in the Anesthetized Rat ...................................................... 88 36. Effects of Inhaled Acetaldehyde and Propionaldehyde on Blood Pressure and Heart Rate ..... 88 37. Effects of Cigarette Smoking at Rest and During Exercise ............................. 88 38. Effects of Cigarette Smoking on Hemodynamics at Rest and During Exercise. I. Normal Subjects ..................................................................89 39. Analvsis of Nicotine-Induced Vascular Reflexes in the Dog ....................... ., . .. 89 B. Cardiac Metabolism ............................................................. 89 40. Feedback Control of the Citric Acid Cycle ........................................ 89 41. Control of the Transport of Reducing Equivalents across the Mitochondrial Membrane in Perfused Rat Heart .......................................................... 90 42. Regulation of Glutamate Metabolism and Interactions with the Citric Acid Cycle in Rat Heart Llitochondria............................................................... 90 43. Feedback Interactions in the Control of Citric Acid Cycle Activity in Rat Heart Mitochondria .. 91 44. Inhibition of Citrate Synthesis by Succinvl-CoA and Other Metabolites .................. 92 ; 45. Hypoxia and Nicotine Induced Loss of Intracellular Myocardial Acid Hydrolases ........... 92 46. Free Fatty Acid Metabolism of the Human Heart at Rest .............................. 93 47. Substrate Preference and Metabolic Activity of the Aerobic and the Hypoxic Turtle Heart .... 93 ~ Acid Mobilization and I ee Fatt ki F tt d Ci S i ff f r y ng on e mo gare gar an ects o C -I8. Differences in E Catecholamine Excretion ..................................................... 94 49. Effect of Nicotine on the Mobilization of Free Fatty Acids from Adipose Tissue in Vitro ..... 94 50. Some Aspects of the Effect of Nicotine on Plasma FFA and Tissue Triglycerides ........... 94 51. Effects of Chronic Nicotine, Acute Hypoxia, and Their Interactions on Myocardial Enzymes ... 95 52. Increase in Hexosemonophosphate Shunt Activity during Tissue Repair .................. 95 53. Changes in the Activities of Lyosomal Enzymes in Infarcted Canine Heart Muscle .......... 95 50 TIlVIN 0115794 T200285

'S ,5 6 e, 7 7 7- i I 163. Direct and Reflex EffPcts of Hypothermia. Hypotension and Hypoxia on the Heart ......... 134 16.}, \qaatie•e Chronotropic Effects of McN A-343 and Nicotine in Isolated Guinea-Pig Atria: Insensitirity to Blockade by Tetrodotoxin ............................... . ........ 134 165. Cardiac Arrhythmias Observed during Maximal Treadmill Exercise Testing in Clinicallv NormalLten .............................................................. 135 E{, Atherosclerosis . . . • . - ......................................................... 135 166. Assessment of Catecholamine Function in Prevention of Experimental Arteriosclerosis by Adre- nalectomv ............................................................... 135 167. The Role of the Hepatoadrenal Axis in the Pathogenesis of Experimental Arteriosclerosis in Rabbits .................................................................. 13 6 168. Relation between Metabolism of Vitamin D and Occurrence of Calcific Arteriosclerosis in Rabbits with Cirrhosis ...................................................... 136 169. Inhibition of Nicotine-Induced Calcific Thromboarteritis in Rabbits by Adrenalectomr ...... 136 170. Effect of Chronic Nicotine Administration on Cholesterol Metabolism of Liver. Serum. Heart and Brain ................................................................... 137 171. Bioassay for Thrombopoietin Utilizing Mice in Rebound Thrombocytosis ................ 137 172. The Hemagglutination-Inhibition Assay for Thrombopoietin .......................... 138 173, Effect of Nicotine on Clot Retraction of Rat Blood Platelets ........................... 138 174. The Effect of Carbon Monoxide on Diet-Induced Atherosclerosis in Cvnomolgus Monkeys (A'facaca fascicularis) ........... • ........................................... 138 175. The Effects of Cigarette Smoking on Total Body Burden and Excretion Rates of Carbon Monoxide ...................... ......................................... 138 176. Comparison of the Cardiovascular (CV) Effects of Some Gaseous Components of Tobacco Smoke .................................................................. 139 177. Negati~•e Inotropic Action of Carbon Monoxide (CO) on the Isolated Isovolumic Heart with a Hemoglobin (Hb)-Free Perfusate ............................................... 139 178. Carbon Monoxide (CO)-Nicotine Interactions on the Isolated Perfused Isovolumic Rabbit Heart ................................................................... 140 179. Effect of Carbon Monoxide (CO) on Adenine Nucleotides in Brain and Heart ............. 140 180. Effect of Carbon Monoxide and Hypoxia on the Morphology and Metabolism of Vascular Smooth Muscle Cultures .................................................... 140 181. The Induction of Atherosclerotic Plaque-Like Mounds in Cultures of Aortic Smooth Muscle Cells ................................................................... 141 182. Smoking as a Factor in Atherosclerosis. A Review of Epidemiological, Pathological and Experimental Studies ....................................................... 141 183. Tobacco Smoking and Atherosclerotic Vascular Disease ............................. 141 184. A Comparative Study of Cigarette, Cigar and Pipe Smoking Effects on Blood Lipids. Catecholamine Excretion and Nicotine Content of the Urine .......................... 141 185. Effect of Smoking and Nicotine on the Crystallization of Cholesterol ................... 142 186. Cigarette, Cigar, and Pipe Smoking. Some Differences in,Biochemical Effects ............. 142 TIMN 0115799 T200290 55

nf is ;,;er\ation. it «ill be necessar% to observe the l), ( () sample simultaneouslv with its combustion. Thi: implles in situ generation of smoke in the ;,,trL.tion cavity itself. Development of a novel light „Ilertion scstem. together with a high intensity ,,fr,jred source. will allow instantaneous combus- „n uf tobacco particles imbedded in a quartz fiber. e,~ntintwus presentation of fresh tobacco to the nfr,+red beam is possible by rapid displacement of *ho treated fiber on a pulley takeup system. This ,,,, hnictue is similar to that sometimes used to ; r,,n;p„rt short-lived radioactive isotopes from a site .,f ,.:eneration to site of detection. Alternatively, the i, a\ kinetics of the short-lived smoke free radicals ,,,,,\ he followed by using a short burst of infrared ht. produced by a high speed optical shutter. ~nalogous experiments. using the ENDOR spec- mrt~ r. %cill be possible with the development of +r irradiation svstem for the spectrometer which %\il' alloW observations of magnetic resonance/ nuclear spin change as a function of incoming r,idiation. As in the case of ESR, but possibly even :t,o,re so. the use of partially deuterated tobacco i iuld ,,ruvide new insights concerning the chemical <i41( iN< giving rise to smoke free radicals and their behavior. t)thNr ;upport: Atomic Energy Commission. 42. Deuterium isotope effects in Nicotia tabacum .1 l'phaus. \t. 1. Blake, and J. J. Katz v•rrtmrnt ot Pharmacr. College of Pharmacy. r~,t% ot Illinois. Chicago. Illinois, and Chemistrr r.. .Ar,,oonne .\'ational Laboratory. Argonne. Illinois ln c1 n,port to the :1%tA-ERF. Substitution of deuterium for hydrogen in spe- (.apable of generating free radicals will sig- itlkantlv alter the observable electron spin reso- i:•,, e ;ESR) and electron nuclear double reasonance tra IENDOR) of these species. Establishment of -W Chemical species present in tobacco smoke ~iich gives rise to free radicals can thus be based l, ,n stud%- of compounds other than those sub- ituted u•ith "C. Tobacco was grown at various %,-1s of heavy water content in the nutrient media, t,, determine the feasibility of this material for ~nagnetic resonance studies of tobacco smoke free radicals. It appears feasible to use the growth cham- bers developed for "C growth studies for culture of tobacco at high levels of deuteration. Incorporation of deuterium into any organism results in drastic changes in growth rate, morphology, reproductive capacity and final plant size. At 50°% D,O. tobacco produces plants about one-half the size of controls: at 60% D_O, flower production is inhibited and plants may be too small for practical production of tobacco. It is thought that a compound having 50°•0 hydrogen and 50% deuterium incorporated into its tissues would be acceptable for ENDOR or ESR studies. Other support: Atomic Energy Commission. 43. Cytological modification of plant tissue containing high levels of "C E. Flaumenhaft, R. A. L'phaus• and J. J. Katz University of Akron. Akron, Ohio, and Chemistn• Division. Argonne National Laboratory, Argonne. Illinois Unpublished repo:rt to the AMA-ERF Tobacco plants grown on 13CO, were sectioned before total harvest to provide representative tissue from various parts of the plant. Homologous regions of control plants were likewise sectioned to provide samples suitable for cytological examination. Ten samples of each type of plant were then examined microscopically; the investigator did not know the isotopic origin of any of the samples provided. A 90% correlation was found between tissue derived from "normal" and the carbon-13 containing tissue. This is taken to indicate that the incorporation of carbon-13 into growing tissue may well produce hitherto unsuspected changes in cell morphology. Further studies are in progress. Other support: Atomic Energy Commission. 44. Effects of carbon-13 on the structure and morphology of pollens R. A. Uphaus, A. S. Tomb, M. I. Blake, G. T. Chubb. and J. J. Katz Department of Biology. Kansas State University, Manhattan, Kansas; Department of Pharmacy, College of Pharmacy, University of Illinois. Chicago. Illinois. and Chemistry Division, Argonne National Laboratory, Argonne, Illinois Unpublished report to the A".tA-ERF. Growth of tobacco at levels of 90% "COZ results in production of normal-appearing flowers, though TIMN 0115791 T200282 47

Cardiovascular System ,`itlllllilary .abstracts (262) \. filnu,d vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 1. Comparison of Adrenergic Mechanisms in an Elastic and a\iuscular Artery of the Rabbit .... 7; 2. Direct Method for Recording Tension Changes in the Wall of Small Blood Vessels in Vitro . .. „ 1. Distribution of Components of 'H-\oradrenaline Uptake in the Wall of the Rabbit Aorta ..... 4. The Bimodal Basis of the Contractile Response of the Rabbit Ear Arter~ to Norepinephrine and (~ther Agonists ................ . ............................................. 78 ;. Distribution of Bound Norepinephrine in the Arterial Wall ............................ : 8 ti. Effects of Nicotine. Dimethylphenvlpiperazinium and Cholinergic Blocking Agents at Adrener- ;ir Nerve Endings of the Rabbit Pulmonary Artery ......................... . . .. . .... ; g ;. The Effect of Nicotine on the Response of Vascular Smooth Muscle to Adrenergic Nerve Stimulation ............................................................... 79 H. Blockade of the Nicotine-Induced Norepinephrine Release bv Cocaine. Phenoxvbenzamine and Desipramine ............................................................... 79 ~1. Transmural and Subcellular Localization of Monoamine Oxidase and Catechol-O-Methvl Transferase in Rabbit Aorta ................................................... ; 9 t'ptake of Nicotine by the Sympathetic Nerve Terminals in the Blood Vessel .............. 80 1 1, Effect of the Chelating Agents. EDTA. 2.2'-Bipyridvl. 8-Hydroxyquinoline and Pyrophosphoric Acid. on Norepinephrine Uptake bv Rabbit Aorta ................................... 80 12. Distribution of Norepinephrine Uptake within Rabbit Aorta between Adventitia and Media ... 81 1:;. Distribution of Norepinephrine Released from Adrenergic Motor Terminals in Arterial Wall .. 81 14. The Release of H3-Norepinephrine in Arterial Strips Studied by the Technique of Superfusion and Transmural Stimulation ................................................... 81 15. Some Structural Considerations of the Reactivitv of Vascular Smooth Muscle .............. 81 1t;. Distribution Theory of Resistance of Neurogenic Vasoconstriction to Alpha-Receptor Blockade in the Rabbit ...............:.............................................. 82 17. Biphasic Constrictor Response of the Rabbit Ear Artery .............................. 82 18. Distribution of Inulin Space in the Rabbit Thoracic Aorta ............................. 82 19. Adrenergic Transmitter Release and Distribution in Blood Vessels ...................... 83 20. A Double-Labeled Frozen Section Technique for Studying Distribution of H3-Norepinephrine . 83 21. The Vascular Response to Adrenergic Transmitter in Experimental Hypertension ........... 83 22. A Superfused and Transmurallv Stimulated Artery Preparation ........................ 83 23. Comparison of Contracting Agents on the Isolated Guinea Pig Aortic Strip ................ 84 24. Reactivity to Biogenic Amines of Isolated Aortic Strips and Electrically Driven Left Atria from Guinea Pigs after Repeated Treatment with Nicotine ................................ 84 25. Bretylium Potentiation of the Contractor Responses of Isolated Rabbit Aortic Strip to Potassium and Tyramine .............................................................. 84 TININ 0115793 T200284 49

s-- Carcinogenesis water soluble. ethanol insoluble fraction of tobacco leaf and a nontumorigenic fraction of tobacco smoke condensate (TSC) have been found to have antiviral activity against encephalomyocarditis virus infec- tion in mice and several other viruses in cell cultures. Thev now report that an ethanol soluble nontumorigenic fraction of tobacco leaf also pos- sesses antiviral activitv. In addition, the nontumori- genic fractions of the leaf and TSC suppress the growth of Ehrlich ascites tumor cells in mice. L. Cocarcinogenesis-Cigarette Smoke-Bioassay 33. Brief communication: Cocarcinogenic agents in tobacco carcinogenesis B. L. Van Duuren and B. M. Goldschmidt Laboratorv of Organic Chemistry and Carcinogenesis. Institute of Environmental Medicine. New York L'nirersitr Afedical Center. New York, New York Journal of the National Cancer Institute 51:703-705, 1973 Five cigarette smoke components were tested for cocarcinogenic activity; they were repeatedly applied to mouse skin together with a low dose of benzo(a]pyrene for 52 weeks. Pronounced activity was shown by catechol and the noncarcinogenic aromatic hydrocarbons pyrene, benzo[e]pyrene, and benzo(g,h.iJperylene. Phenol had a small inhibitory effect on benzo(a]pyrene tumorigenesis. Other support: U. S. Public Health Service. 34. Cocarcinogenic and tumor-promoting agents in tobacco carcinogenesis B. L. Van Duuren and B. M. Goldschmidt Laborator' v of Organic Chemistry and Carcinogenesis. Institute of Environmental Medicine, New York L'niversitr lfedical Center. New York. New York /ournal of the .\'ational Cancer Institute 56:1237-1242. 1976 A series of 21 tobacco smoke components and related compounds were tested for cocarcinogenic activity on mouse skin. The compounds were ap- plied to mouse skin (50 female ICR/Ha Swiss mice/ group) three times weekly with a low dose (5 µg per application) of benzo[aJpyrene (B[ajP). The test com- Your.ds were ef five classes: aliphatic hydrocarbons, aromatic hydrocarbons, phenols, and long-chain acids and alcohols. The following compounds en- hanced remarkablv the carcinogenicitv of B[ajP: catechol, pvrogallol, decane, undecane. pvrene. benzo[e]pyrene and fluoranthene. The following compounds inhibited B[alP carcinogenicitv com- pletely: esculin, quercetin, squalene and oleic acid. Phenol, eugenol. resorcinal, hydroquinone, hexade- cane and limonene partially inhibited B[a]P car- cinogenicity. Six of the 21 compounds were also tested as tumor promoters in two-stage carcinogene- sis. No direct correlation existed between tumor- promoting activity and cocarcinogenic activity. The cocarcinogens pyrogallol and catechol did not show tumor-promoting activity. Decane, tetradecane, an- thralin and phorbol myristate acetate showed both types of activity. Structure-activity relationships and possible modes of action were described. Other support: U. S. Public Health Service. 35. The role of alpha-emitting isotopes in the effects of cigarette smoking Robert L. Bogner' Nuclear Science and Engineering Corporation. Pittsburgh. Pennsylvania Unpublished report submitted to the AMA-ERF (1968) Thi's study is designed to investigate the sig- nificance of polonium-210 and related radionuclides as health hazards associated with smoking. The approach has been to determine the levels of Po-210 and its precursor, Pb-210, in selected facets of the environment and in the human population. The Po-210 and Pb-210 contents of cigarette tobacco, cigarette smoke, foods, water and air have been determined. U. S. cigarettes contain an average of 0.474 picocuries (pc) per cigarette of Po-210 and 0.564 pc/cigarette of Pb-210. Results of smoking machine studies established that approximately 10% of the Po••210 as well as Pb-210 appear in the mainstream smoke. Hence, an individual smoking 20 cigarettes per day will inhale an average of 1 pc of Po-210 and the same amount of Pb-210. The natu- rally occurring Po-210 and Pb-210 levels in the atmosphere arising from the decay of radon-222 emanated from the earth's crust were shown to approach 5 x 10-3 pc/m3, and 4 x 10-Z pc/m', re- spectively. Accordingly, the intake of Po-210 and TIMN 0115788 i ( l t 44 T200279

i 1 Pb-'10 bY an individual inhaling 20 m' of air per day ,,ill be 0.1 pc and 0.8 pc per day. respectively. Therefore, it can be seen that cigarette smoking increases the normal daily intake of Po-210 by an order of magnitude and doubles the normal Pb-210 intake. These relative intakes have been confirmed by measurement of Po-210 and Pb-210 in the urine of <mokers and non-smokers where it was found that <mokers excreted more than five times more Po-210 <,od ti%-o times more Pb-210 as non-smokers. \t. Carcinogenesis-Cell Proliferation in man 36. Smoking and the mitotic index of oral mucosa Condict Moore and G. Randolf Schrodt D,partments of Surgery and Pathology, University of Lnuis% ille School of .tiiedicine, Louisville, Kentucky l 'npublished report to AMA-ERF Biopsies of grossly normal. oral mucosa from fift%--one persons with varying smoking and oral cancer histories were studied by light microscopy, and some by electron microscopy, looking for changes resulting from smoking. A significant dif- ference occurred in the mitotic index of oral mucosa hetiveen smokers and non-smokers-ex-smokers. High indices occurred in nearly half of smokers. While this study provides no direct link between high mitotic index and oral cancer, our finding is (nnsistent with other studies indicating that smok- ing predisposes to oral cancer. It also fits with other <tudies indicating benefit conferred by quitting ;moking. N. Carcinogenesis-Mechanisms 37. Characterization of the cytochrome oxidase inhibitor found in the gas phase of tobacco smoke `•I C. Fu. B. 1. Wilkinson, and D. C. White J-partment of Biochemistry, University of Kentucky ~lydi co! Center. Lexington, Kentucky r'~oceedings of the University of Kentucky Tobacco and Hoalth Research Institute Workshop Conference. C:r,nrerence Report 3:239-252, 1972 A powerful inhibitor of cytochrome oxidase has been detected in the gas phase of fresh tobacco smoke. The inhibitor found in aqueous extracts of whole smoke can be recovered almost quantitatively after filtration through a Cambridge filter. The in- hibitor can be partially trapped on activated charcoal or glass wool at -90°C; it can be transferred from aqueous solutions by a stream of nitrogen if the pH is less than 9.4, but not if the pH is greater than 12.7. The smoke inhibitor seems to effect the oxidase when it is actively transferring electrons and has little effect on the oxidized cytochrome oxidase preparation or reduced cytochrome c. During the inhibition the inhibitor greatly increases its abilitv to inhibit fresh oxidase, suggesting some toxic com- plex may be reduced. Most of the properties of the inhibitor match those of KCN which occurs in sufficient amounts in the extract to account for most of the inhibition. 38. Assay of a volatile inhibitor in tobacco smoke using bacterial cytochrome oxidase B. J. Wilkinson, M. C. Fu. and D. C. White Biochemistry Department, University of Kentuckr Medical Center, Lexington, Kentucky Proceedings o,f the University of Kentucky Tobacco and Health Research Institute Workshop Conjerence. Conference Report 3:276-294, 1972 The activity of cytochrome oxidase is essential for the generation of high energy phosphate used to drive the synthetic reactions of the cell. Total inhibition of the cytochrome oxidase by carbon monoxide or cyanide is rapidly fatal. There is much evidence that partial inhibition of the oxidative reactions that generate high energy phosphate pro- motes the neoplastic transformation. Some inhibi- tors of oxidative phosphorylation actually can be initiators of cancer. A volatile factor in the gas phase of fresh tobacco smoke has been shown to inhibit both mammalian and, in the present study, bacterial (Micrococcus denitrificans) electron transport sys- tems. The inhibition has been localized to the cytochrome oxidase or terminal end of the electron transport system. Four assays for activity of the oxidase have shown dose-response relationships to TIMN 0115789 T200280 45

T 1 136. Cardiovascular Effect of Nicotine in the Conscious Dog. Modification by Changes in Autonomic Tone ...................:............................................... 125 137. Calcium-Dependent Action Potentials Produced by Catecholamines in Guinea Pig Atrial Muscle Fibers Depolarized by Potassium .............................................. 125 138. Propranolol-Insensitive Effects of Epinephrine on Action Potential Repolarization in Electrically Driven Atria of the Guinea Pig ................................................ 126 Fundamental Studies with Reference to Nicotine and Smoking 139. The Effect of Inhalation of Cigarette Smoke on Ventricular Fibrillation Threshold in Normal Dogs and Dogs with Acute Myocardial Infarction .................................. 126 140. Hypoxia in the Genesis of Cardiac Arrht•thmias ................................... 127 141. Evaluation of Antifibrillatory Agents and Catecholamines by a Physiologic Method ........ 127 142. Neurogenic Basis for the Rise in Blood Pressure Evoked by Nicotine in the Cat ........... 127 143. A Comparative Study of Nitroglycerin and Propranolol ............................. 128 Contractilitv 144. Reduction of Liyocardial Contractilitv bv 100% Oxygen in Patients with Coronary Disease .. 128 145. Evaluation of Me•ocardial Force-Velocitv Relation in Closed-Chest Dogs ................. 128 146. %Ie•ocardial Contractility of the Intact Heart ...................................... 129 147. Nicotine and Potassium Chloride Contracture in Mammalian Ventricle .................. 129 148. Direct and Indirect Inotropic Effects of Nicotine on Cat Ventricular Muscle .............. 129 149. Effect of Nicotine on Contractility of the Intact Heart ............................... 130 150. Studies on the Auricular Stimulating Action of Nicotine ............................ 130 151. Comparative Studies of Atrial Responses Following Nicotine and Transatrial Stimulation ... 130 152. Nicotine-Induced Restoration of Action Potentials to Cardiac Tissue Depolarized by Po- tassium ................................................................. 131 153. Nicotine-Like Actions of cis-Metanicotine and trans-Metanicotine ..................... 131 Clinical Studies 15-1. Cardiovascular Function and Electrocardiographic Changes in Smokers and Non-Smokers in Response to Maximal Exercise ................................................ 131 155. Cardiac Vectors in Women Smokers and Non-Smokers .............................. 132 156. Vectorcardiographic Diagnosis of Left Ventricular Hypertrophy ....................... 132 157. A Point-Score Svstem for the ECG Diagnosis of Left Ventricular Hypertrophy ............. 132 158. Right Precordial qrS Pattern due to Left Anterior Hemiblock ......................... 133 159. Sinus Nodal Echoes: Clinical Case Report and Canine Studies ........................ 133 160. Continuous Prehospitalization Monitoring of Cardiac Rhythm ........................ 133 161. Electrophysiological Basis of Clinical Arrhythmias ................................. 133 162. Modification of the Nicotine-Induced Depolarization in Striated Muscle by Altered [H'lo, [Ca"]o, and [CI-]o ......................... I .......................... . .... 134 54 TIMN 0115798 T200289

-..W~ =• : Carcinogenesis the aqueous extracts of tobacco smoke. The dilute aqueous extracts are stable for 15 hours at 25°C. and the assav is reproducible. The assay system is sensitive enough to detect differences between the inhibitor delivered by different reference cigarettes. Other support: National Science Foundation. 39. Investigations on the formation of the microbial electron transport system as effected by tobacco smoke T. T. Lillich and D. C. White Department of Biochemistry. Universit' v of Kentuckr .1ledical Center. Lexington. Kentuckr Proceedings of the Universihv of Kentucky Tobacco and Health Research Institute Workshop Conterence. Conference Report 3:193-207. 1972 The bacteria Staphylococcus oureus can grow with glucose fermentativelv like a tumor cell or in the absence of glucose with a membrane-bound electron transport system. A bioassay system was developed using this bacterium to test smoke com- ponents for the ability to block the formation or function of the electron transport system.. The factor in the particulate phase of tobacco smoke most active in inhibition of the formation and function of the membrane-bound electron transport svstem was established to be benzo(a)pyrene (BaP). In continuation studies the BaP was shown to inhibit the formation of the respiratory system, particularly the function of the cvtochrome oxidase o which was formed but was not functional. Concomitantlv the BaP prevented increases in membrane phospho- lipid. shifted the proportions of phosphatidyl glyc- erol and cardiolipin, greatly stimulated phytoene production and inhibited synthesis of the xantho- phvilic carotenoids. Systems where mutants have been selected which control membrane function, where isolated membrane vesicles can be assaved for active amino acid transport or membranes can be fractionated chromatographically are being used to further define the mechanism of the BaP inhibition at the membrane level. This studv has been additionally described in Proceedings of the University of Kentucky Tobacco and Health Research Institute Workshop Confer- ence. Conference Report 4:617-631, 1973. Other support: U. S. Public Health Service. 46 O. Carcinogenesis-Isotopic Labeling 40. Growth chambers for large scale production of plants of unusual isotopic composition R. A. Uphaus. M. I. Blake. A. G. Kostka. and J. J. Katz Department of Pharmacy. College of Pharmacv. Unirersity of Illinois. Chicago, Illinois, and Chemistrv Division, Argonne National LaboratonY. Argonne. Illinois Unpublished report to the AMA-ERF. The controlled growth of large plants whose isotopic composition is altered by the introduction of deuterium, carbon-13 or other stable isotopes of biological importance must meet several special requirements. Conservation of costly isotope makes manditory a hermetically sealed growth svstem which must, moreover. provide conditions for long term growth which will not introduce artifacts of morphology or biochemistry into the final mature plant. Control of temperature, humidity. soil mois- ture, carbon dioxide levels, nutrient concentration and light intensity was achieved with the growth chambers developed. Carbon-13 substituted tobacco was the first plant so produced. The systems were highly automated and self-regulating to the point that in operation, only minimal human intervention was required during months of plant growth. Con- trol plants grown in carbon-12 were found to be essentially indistinguishable from normal green- house-grown tobacco of the same genetic composi- tion. Several crops of tobacco were successfully cultivated in these units, with carbon isotopic com- positions in excess of 90% carbon-13. Other support: Atomic Energy Commission. 41. ESR and ENDOR studies of free radicals generated in combustion of tobacco smoke J. R. Morris, R. A. Uphaus, and J. J. Katz Chemistry Division, Argonne National Laboratory. Argonne, Illinois Unpublished report to the AMA-ERF Work is continuing on the characterization of to- bacco smoke free radicals by means of both ESR and ENDOR measurements. To insure that even those radicals of very short lifetimes are being observed, new techniques are being evolved. Instead of spin trapping, or some procedure whereby the sraoke must be conducted to the spectrometer cavity for TIMN 0115790 T200281 t t

187. Spontaneous Occurrence of Arteriosclerosis in Rabbits with Prolonged Cirrhosis .......... 142 188. Influence of Cirrhosis on Production of Atheroarteriosclerosis and Thromboarteritis with ' Vitamin D and Dietary Cholesterol ............................................ . 142 189. Relations between Nicotine Induction of Arteriosclerotic Thromboarteritis and Nicotine- Induced Rise in Serum Free Fattv Acids in Rabbits ................................ 143 190. Relations between Metabolic Increase of Plasma Free Fattv Acids and the Occurrence of Arteriosclerotic Thromboarteritis in Rabbits ...................................... 144 F. Hemodynamics . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 191. Cardiovascular Hemodvnamics: Determination from the Distribution of Pulmonarv Blood Flow in Seated Patients .......................................................... 144 192. The Effects of Anesthesia with Pentobarbital on Hemodvnamics and Arterial Blood Gases in Splenectomized Dogs ....................................................... 144 193. Cardioe•ascular Effects of Cold Pressor Tests. 40° Head-up Tilt, and Smoking on Smokers and Nonsmokers .............................................................. 145 194. Cardiovascular Effects of Long-Term Cigarette Smoking and Nicotine Administration ...... 145 195. Effect of Mescaline on Cardiopulmonary Dynamics: Method for Determination of Right Ventricular Pressure in the Guinea Pig .......................................... 146 196. Determination of Flow in a Multicompartment Model by External Detection of Gamma Radiation ................................................................ 146 G. Epidemiological Studies ......................................................... 147 197. *Cardiovascular and Respiratory Symptoms in Relation to Tobacco Smoking. A Study on American Twins ........................................................... 147 198. Mortality in Twins in Relation to Smoking Habits and Alcohol Problems ................ 147 199. Ischaemic Heart Disease in Death Discordant Twins. A Study of 205 Male and Female Pairs . 148 200. Background of Angina Pectoris: Social and Environmental Factors in Relation to Smoking ... 148 201. Life Change Patterns Prior to Death in Ischaemic Heart Disease. A Study on Death-Discordant Twins .................................................................. 148 202. Psvcho-Social Factors in Relation to Coronarv Heart Disease and Associated Risk Factors .... 149 203. Concordance for Mortality with Special Reference to Ischaemic Heart Disease and Cerebrovascu- lar Disease. A Study on the Swedish Twin Registry ................................ 149 204. Coronary Disease in Staveley, Derbyshire with an International Comparison with Three Towns in Marion County. West Virginia .............................................. 150 205. Results and Correlations of Multistage Exercise Tests in a Group of Clinically Normal Business Executives ............................................................... 150 206. A New Diagnostic Test for Coronary Artery Disease ................................ 150 H. Physiological Action of Nicotine Derivatives ......................................... 151 207. Possible Role of Nicotine lsomethonium Ion in Physiological Responses to Nicotine ....... 151 208. Adrenergic Activity of 3-(2-Methylaminoethyl) Pyridine ............................. 151 209. Effects of 3-Pyridylacetate and Its Glycine Conjugate_on Free Fatty Acids in Rabbit Epididymal Fat Pads ................................................................. 151 56 TIMN 0115800 T200291

o. of f r, i;;,,"en bv the drug at some critical binding site. ,,i1,,,t he excluded. t>tl,Nr ;upport: U. S. Public Health Service; Ameri- ;,,n Cancer Society. Inc; and the Turkish Scientific ,n(i Tec:hnical Research Council. 29. Enzymatic reactions and carcinogenesis [,Qe W. «'attenberg ),r;artnnont o/ Pathology. University of Minnesota u! School. Minneapolis. Minnesota i!n ;rtmment and Cancer. pp 241-255. The Williams and ill.,n, Companc. Baltimore, :Vtarvland. 1972. 476 pp The primary issue to which this presentation is ,,ddressed is whether in an environment contami- nated vrith carcinogens, it is possible to enhance hinc:hemical defense mechanisms against this haz- ard. Current evidence indicates that because of i erhaps unexpected set of circumstances, it may not f r~ possible to avoid some form of decision on this m atter. Three main topics are presented: (1) items oncerning the nature and distribution of micro- .nmal enzvme systems metabolizing foreign com- Imunds which are pertinent to the subsequent por- tions of the presentation, (2) factors which affect the le~ el of activity of the polycyclic hydrocarbon hy- droxvlase svstem, (3) the capacity of increased levels (;f microsomal enzyme activity to protect against ( hemical carcinogens. Other support: U. S. Public Health Service. 30. Dietarv modification of intestinal and pulmonary aryl hydrocarbon hydroxylase activity t.ee W. Wattenberg :)A partment of Pathology, University of Minnesota t1. cGca1 School. Minnrnpolis, Minnesota nnd Appl;ed Pharmacology 23:741-748. The issue to which this presentation is ulti- :natek• addressed is the potential role of the mixed- function oxidase svstem as a survival mechanism in an environment contaminated with numerous nox- ious chemicals. Only a segment of this problem is dealt with in any detail, namely those aspects having to do with the component part of the mixed function oxidase svstem designated as arvl hvdrocarbon hv- droxylase (AHH). At the present time the range of substrates metabolized by AHH is not known. How- ever, this system does metabolize an extremelv important group of compounds, the polycyclic h%-- drocarbons, which include a large number of car- cinogens. The polycyclic hydrocarbons can be formed from combustion of almost anv organic material, e.g., wood, gasoline. coal, tobacco. and are ubiquitous in urban areas. Because of the almost inevitable exposure of urban dwellers to these com- pounds, biochemical systems metabolizing them are of importance. A factor which enhances the interest in AHH in this regard is that the activity of this system in the major portals of entry, i.e.. intestines and lung, is largely and possibly entirely the result of induction bv exogenous inducers. The implica- tion of this finding is that AHH activity will be determined bv environmental factors, and the result of such contact will control the capacity to metabo- lize this important group of compounds. Other support: U. S. Public Health Service. 31. Exogenous factors affecting polycyclic hydrocarbon hydroxylase activity Lee W. Wattenberg Department of Pathology. University of Nfinnesota Medical School. .Minneapolis, Minnesota Advances in Enzyme Regulation (G. Weber, Editor) 11:193-201. 1973. Pergamon Press. Oxford & New York This review is concerned with an aspect of control of enzyme activity by foreign compounds. Specifically, it deals with the role of exogenous inducers of increased microsomal mixed-function oxidase activity and discusses this control as a survival mechanism for organisms in an environ- ment containing diverse noxious chemicals. Other support: U. S. Public Health Service. 32. Antiviral-anticancer activity of tobacco leaf and the smoke condensate E. Furusawa, S. Furusawa, and J. Y. B. Lee Department of Pharmacology. School of Medicine. University of Hawaii, Honolulu, Hawaii Proceedings of the Western Pharmacology Society 18:322-325. 1975 Through the authors antiviral screening work in the field of natural products of higher plant origin, a TIMN 0115787 T200278 43

234. Release of Catecholamines and Dopamine-¢-Oxidase from the Adrenal Medulla ........... 161 235. Endogenous Inhibitor(s) in Adrenal Medulla of Dopamine-R-Hydroxylase ................ 161 216. Quantal Secretion from Adrenal %Iedulla: All-Or-None Release of Storage Vesicle Content .,. 161 237. Influence of Nicotine on Catecholamine `letabolism in the Rat ........................ 162 238. Effect of Smoking and Nicotine on Adrenocortical Secretion ......................... 162 239. Hypothalamic Control of Baroreceptor Reflexes ................................... 162 240. Effects of Adrenergic Agonists and Antagonists on Potassium Metabolism ............... .163 241. Molecular Interactions of Nicotine ............................................. 163 242. Sympathomimetic Amines and Heart Lactic Dehydrogenase Isoenzymes ................. 164 243. Effect of Beta Adrenergic Blocking Agents on the Noradrenaline Content of Rat Heart Before and After Noradrenaline Infusion ................................................. 164 244. Production of Severe Cardiac and Skeletal Myopathy by Interference with Hepato-Adrenal Function ................................................................ 164 K. Hematology ................ ................................................ 165 245. Effect of Tobacco Smoking on Binding of Oxygen by Hemoglobin ..................... 165 246. Linear Regression Analysis of the Heat Denaturation of Hemoglobin A. F, and S .......... 165 247. An Acute Effect of Cigarette Smoking on Platelet Function. A Possible Link between Smoking and Arterial Thrombosis ..................................................... 165 248. Effects of Nicotine on Mucopolysaccharides of Rat Blood Platelets ..................... 166 249. An Investigation of the Effects of Tobacco Smoke and Nicotine on Rat Blood Platelet Functions ................................................................ 166 ................................... 167 25G. Immunoassay and Bioassay for Thrombopoietin 251. Regulation of Thrombopoiesis ................................................ 167 252. Cigarette Smoke Induced Formation of Methemoglobin (Met-Hb) in Blood ............... 167 253. Effects of Chronic Smoking upon the Clotting Mechanism ........................... 168 Abstracts not Cited in Summary Text 254. Neuronal and Extraneuronal Uptake of Adrenergic Transmitter in the Blood Vessel ........ 168 255. Depressed Transmembrane Potentials during Experimentally Induced Ventricular Failure in Cats .................................................................... 168 256. A New Genetic Polymorphism of Human Serum: a2 Macroglobulin (AL-M) .............. 169 257. Anionic Sites on the Membrane Intercalated Particles of Human Erythrocyte Ghost Membranes. Freeze-EtchLocalization ..................................................... 169 258. Therapy of Angina Pectoris with Propranolol and Long-Acting Nitrates ................. 169 259. Continuous Prehospitalization Monitoring of Cardiac Rhythm ........................ 170 260. Hemodynamic Consequences of Atrial Fibrillation ................................. 'i70 261. A Qualitative Platelet Defect in Severe Vitamin B12 Deficiency: Response, Hyperresponse, and ThrombosisafterVitaminB12Therapy .......................................... 170 262. A Cigarette Smoking Machine Design for Cardiovascular Research ..................... 170 58 TIMN 0115802 T200293

Cardiovascular System l the offr:rt nf cnntracting agents on the isolated auinea pig aortic: strip (?3). the reactivity to biogenic iamines of isolated aortic strips (24). bretvlium po- tentiation of the contractor responses of isolated rabbit aorta to t}•ramine in Voung and old rabbits (25). adrenergic innervation and cocaine-induced potentiation of adrenergic responses of aortic strips (26). and the pharmacologic nature of adrenergic innervation in the aorta have been described (27). One paper dealt with neuromuscular mechanisms of veins (28). Some of these studies cited here describe that chelating agents inhibit norepinephrine uptake bv the aortic wall because of toxic metal-chelate compounds formed by these agents. In addition, it has been stated that adrenergic neurons can be saturated: when this is the case, labeled amines are concentrated in extraneural regions. Nicotine pre- vents the increase and accumulation of radioactive norepinephrine near the adventitia. and in the medial border. Using the pulmonary arterv. the release of tritiated norepinephrine was studied in relation to muscle contraction in the isolated rabbit main pulmonary artery. Excitation of nervous ele- ments br electric transmural stimulation causes contraction and a sharp rise in the overflow of labeled norepinephrine. There exists a fusion barrier between . the surrounding medium of the vascular =smooth muscle and the receptor organ. and the alpha-adrenergic receptors are not identical in dif- ferent vessels. Of particular interest is the influence of alpha-receptor blockade on the distribution of norepinephrine. Marked differences were found be- tween larger and smaller vessels, as far as response to sympathetic stimulation is concerned. The per- fused rabbit ear artery also shows a biphasic con- tractile response to intraluminal norepinephrine and sympathetic nerve stimulation. Much of this work on adrenergic transmitter release and distribution in blood vessels has been summarized (19). In ex- perimental hypertension. an apparent disparity ex- ists between transmitter release and response. which mav be the consequence of an alteration in the extraneuronal distribution of transmitter caused by the reactive changes in the vessel wall consequent upon the increase in arterial pressure. M arked species differences were found. as far as contractility of aortic strips is concerned (23). For example. the guinea pig aortic strip. when compared to that of the rabbit. is about 10 times less sensiti\.e to norepinephrine. and 100 times less sensiti%•e to :,, hydroxvtryptamine. Nicotine failed to contract the guinea pig aortic strip. Daily injection of nicotine to guinea pigs alters the response of aortic strips to norepinephrine and histamine (24). Apparentlt• most of the acute and chronic nicotine effects on the vascular wall are caused by release and by changes in the turnover of norepinephrine. which occur in the sympathetic nerve terminals of the aorta and the atria. The effect of nicotine on the atrial response to acetvlcholine can be attributed to an alteration by nicotine or by its metabolites in the allosteric: cod- figuration of the atrial muscarinic receptors (24), Pretreatment of rabbit aortic strips with bret}•lium potentiates the contractile response to potassium and tvramine. but not to noradrenalin. From this and other evidence. it is concluded that both bretylium and pheniprazine potentiate the action of tyramine and potassium. not by presynaptic but by postsynap- tic action. The interesting field of response of aortic strips to adrenergic innervation and potentiation by cocaine has led to the conclusion that adrenergic nerve fibers penetrate into the smooth muscle layers of the rabbit aorta. but that cocaine induced po- tentiation is not dependent on these adrenergic nerve terminals (26). In the rat there appears to be poor adrenergic innervation of the thoracic aorta (27). Research has shown that the adrenergic mech- anisms in various types of venous smooth muscle svstems are not uniform (28). Additional study is necessarv to disclose the pattern of variation in these mec han isms. Other workers have studied venoconstriction induced bv acetvlcholine and nicotine bitartrate (29). Injections of high doses of acetvlcholine di- rectly into a vein, perfused in situ. results in a rise in perfusion pressure from catecholamines released from extraneuronal stores. In contrast. nicotine even in high dosage had no effect on perfusion pressure up to 1 mg. Apparently. nicotine did not produce venoconstriction in whole vein preparations. but was effective in producing constriction in isolated venous segments (30). Chemical sympathectomy has become a helpful tool in the study of the effect of nicotine. 6-hy- droxydopamine abolished the response to nicotine. which apparently was irreversible, even after 90 minutes incubation in medium containing noradren- aline. However, reserpine-treated preparations ex- hibited recovery of the nicotine response. Chemical 60 TIA7N 0115804 T200295

Cardiovascular System (45). It is possible that the combination of hypoxia and nicotine can lead to a conversion of a sublethal I injury into a state of irreversible cell damage (45). Clbsely related to these studies are publications k%•hich\deal with the effect of nicotine and cigarette smoking on mobilization of free fatty acids and catecholamines. These studies are of particular clini- cal importance. since the release of free fatty acids following myocardial infarction has been found. by some. to result in cardiac arrhvthmias. Detailed studies on free fattv acid metabolism in the human heart at rest (46) were carried out by means of '4C palmitic acid infusion with arterial and coronary- sinus samplings. The authors believe that there may be evidence for the release of free fath• acids (FFA) into the coronare sinus. As to be expected. FFA mt•ocardial uptake was directly proportional to their arterial concentration. Thev also believe that tri.- glycerides may be utilized by the fasting human heart. The anoxic turtle heart was also used in the determination of substrate preference and metabolic activitv of the heart (47). During aerobic perfusion. the heart demonstrated active extraction and oxi- dation of free fatty acids. even in the presence of exogenous glucose. FFA metabolism -,vas similar to that seen in the mammalian heart and was adequate to meet most of the mvocardial energy requirements. Glycogen storage in turtle hearts was tenfold greater than in rat heart. and provided greater metabolic flexibility permitting the heart to function ade- quately under environmental conditions that are lethal to the mammal. One group of workers (48) found a considerable increase in free fattv acid concentrations in the serum of cigarette smokers. while cigar smokers showed only slight elevations. This difference was ascribed to the fact that cigarette smokers usually inhale (48). In vitro. nicotine fails to liberate free fatty acids (49). However, when nicotine was injected intraperitoneally and the animal was then sacrificed. subsequent incubation of the animal fat pads resulted in an increased free fatty acid release. Apparently nicotine alone has no direct lipolytic effect on rat adipose tissue, and mobiliza- tion of free fattv acids by nicotine and tobacco smoke is a result of synpathoadrenal stimulation. The effect of nicotine on increasing free fattt• acid levels in blood was absent in rats prec•iousle adrenalectomized (50). This effect. however. ap- peared to be reversible since it could be restored by the administration of corticosterone (50). Within the field of cardiac metabolism are studies on the effect of nicotine. as compared to acute hypoxia. and their interactions on myoc.ardial enzymes. From previously quoted reports. it seems likely that the combination of nicotine and hvpoxia (6 % oxygen) may be potentiating each other. It could be shown (51) that treatment of nicotine combined with hypoxia resulted in severe enzymatic changes in heart muscle. Some of the more fundamental studies on the metabolic changes in myocardial infarction have discovered changes in hexosemonophosphate shunt activity during the reparative processes following mvocardial infarction (52). %fvocardial infarction results in marked changes in activity of )ysosomal enzi-mes in both soluble and particle-bound frac- tions of heart muscle (53). In the early stages of myocardial infarction profound alterations in high- energy phosphates and lactate and alpha-glyceryl phosphate content of heart muscle were observed. In non-ischemic muscle (54) the activity of the rate- limiting enzyme phosphofructokinase increased 5 minutes after coronarv occlusion, followed bv an increase in activity of isocitrate dehvdrogenase, CPK, MDH and LDH. Of far-reaching clinical im- portance are other fundamental studies demonstrat- ing that treatment of myocardial infarction in ani- mals with insulin and anabolic steroids resulted in marked increases in protein synthesis. Treatment with the mixture first proposed by Sodi-Pallares (glucose, insulin and potassium) increased protein synthesis in infarcted muscle seven hours after infarction by 173% Treatment with insulin or anabolic steroids prevented development of cardiac aneurysms, while animals fed a protein-free diet had a significantly thinner heart muscle, and frequently developed aneurysms (55). Although not directly connected with smoking. some fundamental work has been done on metabolic changes in infarcted and non-infarcted myocardium , (56-67). Some of these deal with reparative processes after myocardial infarction. Correlation is made between the morphological and biochemical changes following myocardial infarction (67). The other papers deal with problems of myocardial infarction in general, and are concerned with met- ` 2 TIMN 0115806 6 T200297 k

Cardiovascular Sc•stetn closed-chest animals. Early papers described the use of coincidence counting techniques (79. 80). studies which led to the differentiation between normal individuals and patients with coronary arterv dis- ease (81-8-1). A critical analysis of this work has appeared in a review article (85). Although these studies were not directlv concerned with the action of nicotine. they have made possible the measure- ment of total blood flow: in addition. coincidence counting has become important in the development of new and more accurate scanning techniques as well as the determination of regional coronarv blood flow. In the dog. where coronary flow can be directly determined. studies have dealt with the interactions of chemoreflexes and reflexes induced by pulmo- nart• inflation and the coronarv circulation. Responses to chemoreflex stimulation (nicotine) were compared when ventilation was allowed to increase or was controlled. The major component of coronarv dilatation was observed following an in- crease in the depth of respiration in which the efferent component of this reflex appeared to involve withdrawal of alpha-adrenergic constrictor tone (86). These studies relating nicotine to the coronarv circulation have been also extended to the microcir- culation. Using the bat wing (87). the effect of inhalation of cigarette smoke on the capillary cir- culation was observed. Unfortunatelv. the rapid onset of the inhibitorv condition that was also reflected bv cardiac slowing and an abrupt fall in central blood pressure made it difficult to evaluate any contribution of the initial increase in vasomo- tion of the arcuate and terminal arterioles in the tobacco smoke response usually observed in human subjects. This problem. the study of the response of the peripheral circulatory bed independent of cen- tral effects. has been discussed in a previous para- graph. Additional studies on the microcirculation in the ventricle of the dog and turtle (88) have dem- onstrated that red cell velocity in the capillary circulation increases with systole and diminishes with diastole in contrast to the arterioles. where the opposite occurs. Diameters of arterioles. capillaries and venules in the turtle ventricle all declined about 34% during systole. Apparently a shift in flow in the 64 blood vessels of the heart occurred at the transition from arterioles to the capillaries. This 180` change is the result of increased total capillary vascular capac- itv during diastole and of the squeezing action of the Ventricular muscle during systole (88). The effect of nicotine on the microcirculation of the cat was also studied bv direct observation (89). In doses of from 50-250 µg/kg. infused over a period of two minutes, nicotine significantly increased capillary red cell velocity and mean aortic pressure. Adrenergic block- ing agents and ganglionic blockage with hexome- thonium inhibited this effect of nicotine. This work grew out of fundamental studies dealing with efforts to visualize the microcirculation of the mammalian heart directly (90, 91). Other methods to measure regional coronary blood flow. using isotopes have also been devel- oped. Thus. regional blood flow has been deter- mined with 86rubidium. demonstrating that n itro- glycerine results in a redistribution of capillary blood flow. These studies have demonstrated that capillary flow is considerably higher in the inner than the outer myocardial half. When partial occlu- sion of coronary arteries is followed by infusion or noradrenaline. subendocardial flow is less than cap- illary flow in the subepicardial layers. Under these conditions. the administration of nitroglycerin re- sults in a redistribution of capillary flow toward normal (92). The effect of nicotine and vasopressin on the capacity of the terminal vascular bed in heart and skeletal muscle was also studied (93). The highest values for terminal vascular capacity in the normal heart were obtained in the apex and nicotine had no significant effect unless the blood pressure increased to very high values. Capillary blood flow determined in heart. lungs and kidneys increased after nicotine. This was later confirmed by direct observation of the coronary microcirculation. Dif- ferences in capillary blood flow between right and left ventricles disappeared after the administration of nicotine (93). The technique of measuring terminal vascular capacity with I131-albumin was used in the study of factors influencing capillary growth (94). A more detailed report on the effect of nicotine on capillary flow and terminal vascular capacity of the heart in normal dogs and in animals with restrictive coro- r.ary circulation showed that in dogs with normal coronary circulation, nicotine infusion increased both capillary flow and terminal vascular capacity. However, repeated nicotine administration had no 'I'ININ 0115808 T200299 ~

3 4 4 4 4 7 a 9 S 7 7 i ) I. I?hdrnlac:olo5lcal Studies ................ . ........................................ 152 •> io, His'tamine Release in the Dog after Leukocy'te Lc'sate Injection ........................ 152 211. mediators of Histamine Release from Human Platelets. Lymphocytes. and Granuloc:rtes ..... 152 212. (n Vitro Effect of 6-HVdrotydopamine on Isolated Rat Atria .......................... t5:3 213. Effect of Prolonged Alcohol Administration on Calcium Transport in Heart Muscle of the Dog 15:3 '7i.}. Prolonged Cardiovascular Effects of Alcoholic Beverages ............................ 153 215. C.ardiorascular Effects of Intravenous Acetaldehyde and Propionaldehvde in the Anesthetized Rat ..................................................................... 154 Effects of Intravenous Acetaldehyde. Acrolein. Formaldehyde and Propionaldehvde on Arterial Blood Pressure Following Acute Guanethidine Treatment ........................... 154 217. Inhibition by Lanthanum of Some Calcium-Related Actions in Frog Rectus Abdominis Muscle .................................................................. 154 218. Modification of the "Alarm" Pattern bt• Nicotine .................................. 155 '19. The Effect of Potassium on Nicotine-Induced Contracture and Ca45 Movements in Frog Sartorius Muscle .................................................................. 155 320. On the Site of Action of Nicotine on Contracture in Frog Sartorius Muscle ............... 156 221. On the Site of Action of Nicotine in Frog Rectus Abdominis Muscle ................... 156 323. The Effect of pH on Nicotine-Induced Contracture and Ca;s Movements in Frog Sartorius \fuscle.................................................................. .. 156 223. Actions of Procaine on Nicotine-Induced Effects in Frog Sartorius Muscle ............... 157 224. Nicotine and Pyrrolidine-Induced Release of 5-Hvdroxvtrvptamine and Histamine from Neoplastic Mast Cells ....................................................... 157 225. Structural Requirements for Nicotine and Cyclic Imine-Induced Amine Release from Neoplastic Mast Cells ............................................................... 157 226. Further Evidence for Nicotinic and Muscarinic Receptors and Their Interaction in Dog Adrenal Medulla ................................................................. 158 227. A Correlation between the Inhibition of Monoamine Oxidase Activity and the Relief of Angina Pain bv Organic Nitrates .................................................... 158 228. Nitroglycerin (Glyceryl Trinitrate) as a Monoamine Oxidase Inhibitor ................... 159 I. Studies on Endocrine System, Including Catecholamines ................................ 159 229. Effects of Various Agents on the Mg2`-ATP Stimulated Incorporation and Release of Ca- techolamines by Isolated Bovine Adrenomedullary Storage Vesicles and on Secretion from the Adrenal Medulla .......................................................... 159 230. Mechanism of Secretion from the Adrenal Medulla. IV. The Fate of the Storage Vesicles Following Insulin and Reserpine Administration .................................. 159 231. Mechanism of Secretion from the Adrenal Medulla. III. Studies of Dopamine B-Hydroxylase as a Marker for Catecholamine Storage Vesicle Membrane in Rabbit Adrenal Glands ........... 160 232. Storage and Secretion of Adrenal Catecholamines 160 ................................. 233. Mechanism of Secretion from the Adrenal Medulla. V. Retention of Storage Vesicle Membranes Following Release of Adrenaline .............................................. 160 TIMN 0115801 T200292 57

_VP Cardiovascular System Studies have also dealt with the effect of gluca- gon on the roronarv circulation in man (108). Glucaoon was once thought of as a possible positive inotropic'drttQ useful in the treatment of cardiogenic shock. This study demonstrated that glucagon in- creased heart rate. mean arterial pressure. tension- time index/minute and left ventricular work. Cor- onarv blood flow increased significantly. while m\•ocardial oxt•gen extraction remained constant. suggesting that the augmentation in blood flow was sufficient to meet the increased mvocardial de- mands of oxygen. In another study it could be shown that in man cardioacceleration by right atrial pac- ing increased myocardial blood flow at approxi- matelv 130 heats/minute. but decreased again at a more rapid rate (150 beats/min) (109). D. Electropht•siological Studies and Studies of ContractilitY The publications on electrophysiology are di- vided into three parts: 1) purely fundamental con- tributions. 2) fundamental contribution with ref- erence to nicotine and smoking, and 3) clinical studies. Fundamental Studies These cover a wide range of topics. from ven- tricular action potential as related to cardiac hy- pertrophy and force development (110). to vasopres- sor outflow from the brain to the external carotid nerve (111). to action potentials. pharmacological effects of potassium (112. 113), correlation between contractile changes in mammalian and amphibian mvocardium (114). mechanism of ventricular fibril- lation (115). action potentials from specialized Pur- kinje fiber system (116). analysis of T-wave al- terations (117). exit block (118), paired stimulation (119). automaticity in its relation to acetylcholine (120) and digitalis and vagal stimulation _during atrial fibrillation (121). Manv studies have dealt with electrophysiologi- cal parameters underlying such diverse matters as the relationship between cycle-length and cardiac action potential (122). rate-dependent right precor- dial Q waves (123), cardiac automaticity (124), high fidelity recording of depolarization (125), atrial fi- brillation (126). exit block (127), Wenckebach struc- tures (128), the relationship between mechanisms of automaticitt• and electrocardiographic conse. quences and with exposure of the canine proximal AV conducting system (129). In these reports. a temporal relation between atrial action potential and contractile potentiation. comparable to that observed in ventricular tissue has been described and the mechanisms of transient precordial Q waves has been investigated (123): the relevance and propen- sih• of low concentration of nicotine (2-4 µ4/ml) in the initiation and enhancing of cardiac automaticity in in vitro preparations was studied (124). A tech- nique was described which permits superimposition of action potentials. either serially or at selected times. and which provides accurate comparison of data recordings (125). Of particular clinical interest have been reports on the relationship between ino- tropic variations and ventricular functions and atrial fibrillation (126). It has been suggested that the irregular ventricular performance observed during atrial fibrillation under experimental conditions mav be related to variations in the inotropic al- terations analogous to post-extrasystolic potentia- tion of contractility (126). Another subject of great interest to electrophysiologists is exit-block (127). defined as a failure of an impulse to excite the surrounding tissue when falling outside of the refractory period of the heart. Evidence was pre- sented that exit block most often is due to a failure of conduction rather than to an alteration of the im- pulse (127). An investigation of the mechanism wherebv conduction disturbances of the Wencke- bach type occur below the atrial ventricular junction has been also presented (128). The study was di- rected toward a demonstration of this type of con- duction delay within the canine specialized Purkinje fiber system and human ventricular tissue. using microelectrode techniques. The workers demon- strate electrophysiological evidence for Wencke- bach's structures at all levels of the canine Purkinje svstem and human ventricular muscle (128). A brief report on automaticity was presented (130) which describes that changes in the automatic process can result in a variety of arrhythmias which may be due to alteration of the normal sinus and pacemaker, induction and acceleration of latent pacemakers or a combination of several mechanisms. Of technical interest is a report (129) which describes techniques of exposing the canine proximal AV conducting system for electrophysiological studies. TIMN 0115810 T200301 66

fi1 t~1 fi1 62 62 62 r,3 ti 3 64 64 64 55 65 ii5 55 i6 -i6 i7 7 8 ,8 ;8 i9 ;9 ;9 '0 -0 '0 '0 lrttrndurtion-5tudies on the cardiovascular svstem ,ulnmarized in this section involve effects on (1) l,l,,,,d %•essels: (2) cardiac metabolism: (3) coronary. ,,,t,ill,an•. cerebral and peripheral circulations: (-1) ,Ircarophvsiological studies and studies of contrac- tilitv: (,-)1 atherosclerosis: (6) Hemodynamics: (7) t•;pidemiological studies: (8) Physiological action of ni<otine derivatives: (9) Pharmacological studies: t ttt) studies on the endocrine system: and (11) ( lt-trrttnlo-K•. ~. Rlnod Vessels .\ series of publications have dealt with fun- ,l,,mental aspects of cardiac and blood vessel me- t,,lmlitim. while others were concerned with more .l,:.t ific topics involving the effect of nicotine and its ,lo,ri%<<tixes on cardiac metabolism and blood vessel Fundamental studies dealing with blood vessel in(~taholism have been primarily concerned with uptake and localization of catecholamines in the ,u,rta. and in other isolated arteries. This question is Of cnnsiderable importance. since nicotine is known to ~ ha.•e a definite effect on blood pressure: these inxestigations therefore deal with the end organs of tht, effect of nicotine. the blood vessels. It has been .ho«-n. for example. that there exist considerable func tional differences between elastic and muscular .irteries (1). It is not surprising that most isolated .v.sf•ls exhibited spontaneous rhythmic activity. ,uxl responded to norepinephrine by an increase in tensirnn (2). Studying the distribution of noradrena- lint, in the wall of the rabbit thoracic aorta by means --f frozen sections. it has been found that nor- epinophrine is distributed in almost all compart- 1m nts of the blood vessels (3). Catecholamines are .p-c ificalh• bound to sympathetic nerve endings. As 1,i niost physiological processes dealing with ac- ti%atinn of membranes, calcium appears to play an imwnrtant role in the reaction of rabbit arteries to nnrt-ninephrine. In studying the biphasic contractile rt'sponse to 1-norepinephrine (4) it has been found that the second phase of contractile response is particularly affected by calcium depletion (4). Nor- t°pinephrine in the media of the artery is associated %rith mesenchymal tissue (5). As concerns the effect of nicetine on vascular «•all. it has been found in isolated pulmonary arteries of rabbits that the contraction elicited in this vessel by postganglionic nerve stimulation is blocked dimethrlphenylpiperazinium (D%IPP) (6). but not bv nicotine or the nicotine metabolitP cotinine. Apparently. Dw1PP and nicotine differ in their action on adrenergic nerve-smooth muscle transmission. It was also concluded that the effv<.t of nicotine can be considered as being due primaril}• to an effect on the pre-junctional adrenergic terminals in the vascular smooth muscle (7). Other fundamental studies concerning the effect of nicotine on the arterial wall dealt with the blockade of nicotine-induced norepinephrine re- lease by cocaine. phenoxvbenzamine and desipra- mine (8). Apparently. the sympathomimetic: action of nicotine on the pulmonary artery is mediated through the release of endogenous norepinephrine: this release is not dependent on the initiation and propagation of action potentials. It is likely that the action of nicotine requires an intact norepinephrine uptake mechanism (8). Since the effect of nicotine on the vascular wall is closely related to release of catecholamines, an investigation was also published dealing with the transmural and subcellular lo- calization of monoamine oxidase and catechol-O- methyl transferase in the rabbit aorta (9). Apparently most of the activitv of both enzvmes was located in the media. Monoamine oxidase was primarily lo- cated in mitochondrial and microsomal fractions. catechol-O-methvl transferase in microsomes and cell sap (9). Nicotine itself seems to be concentrated in the inner third of the adventitia. the region of the terminal sympathetic plexus (10). Additional work has dealt with the effect of chelating agents on norepinephrine uptake by rabbit aortas (11), on distribution of norepinephrine uptake within the aorta between adventitia and media (12). on distribution of norepinephrine released from adrenergic motor terminals in the arterial wall (13) and the release of H3 norepinephrine from the aorta (14). In addition, reports dealing with structural consideration of the reactivity of vascular smooth muscle (15). alpha receptor blockade in the rabbit (16), biphasic constrictor response of the rabbit aorta (17), distribution of inulin space in the rabbit tho- racic aorta (18), adrenergic transmitter release and distribution in blood vessels (19), frozen sections used for studying distribution of H' norepinephrine in the arterial wall f201, vascular response to adrenergic transmitter in experimental hypertension (21), and superperfused and transmurally stimulated artery preparation have been published (22). Finally. TININ 0115803 T200294 59

Cardiovascular Svstem hut the various catecholamines had different an- tagonists. Thus propranolol or lidocaine raised the fibrillatipn index (FI) following norepinephrine and epinephrine. but restored the isoproterenol FI only partially to baseline. This is an important study which may have considerable clinical implications (141). Frequent references have been made to the possible mechanisms of pressor responses elicited by small intravenous doses of nicotine (5-40 µg/kg). The role plaved in these responses by both pre- and postganglionic branches of the superior cervical ganglion was investigated (142). The pressor re- sponse was abolished by transection of the spinal cord or bv denervation of the aortic and carotid bodv chemoreceptor. Apparently the major component of the pressor response produced by small intravenous doses of nicotine is the consequence of facilitation of ganglionic transmission rather than a direct central effect of the drug on vasomotor areas. Contractiliti• Some general studies on contractility have been carried out. dealing either with special pharmaco- logic agents such as nitroglycerine and propranolol (143) or with the effect of oxygen (144). Other papers have dealt with general evaluations of myocardial force velocity and contractility (145. 146). One study (143) dealt with early comparisons between nitro- glycerine and propranolol. This study, which was one of the earliest to deal with the direct effect of propranolol on myocardial contractility. described the essential differences between the two drugs. While propranolol increased left ventricular end- diastolic pressure. nitroglycerine did not affect this parameter. The results indicate that propranolol acts on the heart muscle directly, while nitroglycerine does not affect heart muscle directly. Breathing of 100°t> oxvgen in patients with coronarv artery dis- ease. caused a rise in left ventricular systolic pres- sure and tension time index. and a fall in velocity of the shortening of the contractile elements of the left ventricular muscle at zero load (V,,,aX). Apparently oxygen breathing diminishes myocardial contractil- ity and consequently may reduce myocardial oxygen demand (144). More, broader investigations were carried out evaluating myocardial force velocity re)ation in closed chest dogs (145. 146). Analysis of the force - velocitv characteristics of the left ventricle was carried out bt• means of a newlv devised strain gauge catheter, which permitted instantaneous force-veloc- ity relation at a constant muscle length. The action of various drugs on these parameters was studied (1-l5). The same instrument was also used to define more closely the four variables that determine the mx•o. cardial contractile state (146). Several references were made in the preceding paragraphs to the effect of nicotine on cardiac contractility in the intact animal and in isolated preparation: for example. it has been found that nicotine may influence contractile force in depolar- ized cat ventricular muscle by releasing sarcolem- mal stores of calcium. This action may be effected br formation of nicotine-calcium complexes and the flow rate utilized during high potassium contracture (147). An isolated membrane effect of nicotine in isolated reserpine pretreated cat ventricular muscle was also discovered (148). Using a strain-gauge catheter, force-velocity relations in the heart in situ after the injection of nicotine were discussed (149). Nicotine augments myocardial contractile state as indicated bv an increase in both the force and the velocity of shortening. When (3-adrenergic blocking preceded nicotine administration, a rise in left ventricular systolic pressure was proportionally greater while velocity of shortening showed a re- ciprocal decline. Left ventricular end-diastolic pres- sure rose significantly and the increase in dp/dt was less marked. These findings suggest that propranolol impairs the norepinephrine-like effect of nicotine on the myocardium while its peripheral vasopressor action is enhanced (149). Action of nicotine on parasympathetic nerve endings within the auricles, which is responsible directly or indirectly for the release of norepinephrine from the sympathetic nerve terminals, was also discovered (150). How- ever, there exist marked species differences as to nicotine and trans-atrial stimulation. which suggests that there must be species variations in transmission of autonomic nerves or their activity (151). The left atrial appendage of the guinea pig heart was also used to study the effect of nicotine on the restoration of conducted action potentials and con- tractions to atrial depolarization by bathing it in 22 µm of potassium ion (152). Apparently nicotine was able to restore action potential, a property which was sensitive to blocking by propranolol and hexame- thonium but not by atropine (152). TIMN 0115812 T200303 68

k s n I i r f i 1 1 ;is:nificant rlffc'c:t on either. In contrast. in animals with impaired coronary circulation both a single intrax'enotcs infusion and repeated administration of nirotine resulted in a fall in capillary blood flow and ,,II increase in terminal vascular capacity. This was „ne of the early indications of the difference in reartit ih• of a restrictive coronary bed (95). During gro«~h of an animal. considerable in- , neist' in the vascular capacity in the first post-natal 1~vek occurred. Highest values were reached in ,inimals approximatelv 6 weeks old. From this time. vase•ular capacity gradually decreased in relation to thr increase in heart tceight. The growth rate of the trrminal vascular bed was very rapid during the first post-natal weeks. No growth was detected in adult ,and old animals. Reduced protein diet in animals resulted in diminished vascular capacity (96). Additional studies were of definite clinical rr1Nvance. For example. the effect of Vineberg pro- cedure was studied in the dog. determining both terminal vascular capacity and regional myocardial blood flow. Results indicate that the new source of r.tracoronarv blood supply brought about by im- plantation of a systemic artery into the ventricular wall. increases the rate and distribution of capillary hlood flow and improves oxidative metabolism in the myocardium. Hocrever. this improvement only affects the region directly adjacent to the implanted arten• (97). Cholinergic mechanism of the heart and coronarv circulation were also studied (98). In this work. the effects of rapid intracoronary injection of ac:etylcholine were investigated. Increasing doses of ac.etylcholine produced progressively larger in- c•reases in svstolic and diastolic coronary blood flow <ind progressive decreases in end-diastolic vascular resistance. These results suggest that the coronary dilator response. the negative inotropic response. and part of the positive inotropic response are mediated through "muscarinic" receptors. The re- maining component of the positive inotropic re- ,-ponse appears to involve catecholamine release (98). Dilantin has become an important antiarrhyth- mic drug. It also apparently has significant effects upon the coronary circulation (99). The drug in- creased coronary blood flow ascribed to direct va- sodilating effects on the coronary vessels. However. mvocardial oxygen consumption remained un- changed during a constant workload (99). An ana- tomical study has been carried out on the origin of blood supply to the sinoauricular and atrioventric:u- lar node (100). The arterv to the sinus node ori4- inated from the right in 60% and from the left in 38°•0 of the cases. with 1.5°'o from both. The artery to the atrioventricular node was from the right in 84.7% and from the left in 15.3%. The origin of the posterior descending artery was from the right in 81% and from the left in 15.8%. with a dual supply in about 3%. There were some differences between males and females as to the origin of the blood supply to these structures. Clinical Studies Much of this fundamental work has been ap- plied to clinical studies dealing with the effect of nicotine on the coronary circulation and with the recognition of coronary artery disease. Other studies have dealt with coronarv collateral circulation and myocardial blood flow reserve (101) and with stud- ies relating computer quantitated treadmill exercise electrocardiograms with arteriographic localization of coronary arterv disease (102). Other clinical studies were concerned with the effect of rehabilitation of the coronary patient (103) the effect of propranolol and long-acting nitrates. and with multistage electrocardiographic exercise test (104). It has been known for many vears that nicotine can cause changes in peripheral circulation. Ap- parently cigarette smoking can affect the pulmonary circulation in a more consistent but different manner than the systemic circulation (105). As to be ex- pected. cardiac output during cigarette smoking increases as does blood pressure and pulse rate. Skin temperatures of fingers and toes decrease (105). Nicotine caused onlv a mild and transient vasoconstriction of the cerebral circulation of cats. mediated primarily by stimulation of the superior cervical ganglion with a small direct cerebral va- soconstrictor component. The authors also observed tolerance to the cerebral vasoconstricting effect after repeated increments of nicotine (106). Where perfusion of nicotine was restricted (107) to the carotid bifurcation and the cerebral circula- tion, the reflex and direct central inhibitorv effects on the peripheral circulation depended to a large extent on the integrity of the afferent and efferent autonomic pathways. Actions of nicotine contribut- ing to the above effects included activation of carotid chemoreceptors, leading to both tachycardia and bradycardia, and to hypertension and hypotension. t TIMN 0115809 T200300 65

Isomers of nicotine such as metanicotine in the ,.i, or trans form have quite definable effects on ;,iux~th muscle. For example. the cis isomer has a ~.,1„Siderable lower order of acti~•it~• than the trans form. Trans metanicotine causes a contraction of r,,hhit aortic strips. effects which are blocked with t,rt°treatment with phentolamine or with hexame- thonium. The suggestion has been made that me- t,3holism of these compounds provides one route for terminating the pharmacological response to meta- nicotine (153). t:linical Studies Some reports have dealt with purely clinical tiuhjects. some of them with cardiot~ascular function and electrocardiographic changes as related to timoking. In one of these (154) a statistical im- pairment in duration of exercise and maximal heart rate and systolic blood pressure attained during r.ercise was observed in smoking subjects. At the same time. the incidence of exercise-induced ven- tricular arrythmias did not correlate with smoking habits (154). Other workers (155) have tried to cxirrelate smoking habits with vectorcardiographv. and concluded that the most sensitive early variable in smokers is the horizontal QRS vector (155). Some of the papers are of more generalized clinical interest. They deal with c•ectorcardiography and left ventricular hypertrophy (156). electrocar- i1io5ram and left ventricular hypertrophy (157). left anterior hemiblock (158). and sinus nodal echoes 1159). Of general interest is the description of a continuous electrocardiographic telemetry s~rstem t%hich permits simultaneous voice communication with patients with mvocardial infarction prior to .i~lmission. This paper. which was published in t1); 1. antedated a now universally accepted practice -)f monitoring electrocardiograms of patients with ,;uspected myocardial infarction, together with voice 1 ommunication between physicians in the emer- ,f1nct• room and paramedical groups (160). Of clinical interest are attempts to monitor the Plectrocardiogram by telemetric system and at the same time maintain voice communication. It is believed (160) that continuous ECG telemetry is a useful tool in the pre-hospitalization detection of arrhythmias in patients with coronary arterr disease. This technique is now probably superseded by newer studies using halter monitoring. The basis for the genesis. maintenance and suppression of arrhythmias has also been discussed (161). Here the description of mechanism of automaticitv and conduction disturbances in coinhi- nation with re-entry is of particular interest. In ad- dition, modification of nicotine-induced depolariza- tion in striated muscle by changes in hydrogen ions. calcium ions. and chloride ions. are of interest (162). In a more oblique way, of interest are the effects of hypothermia. hvpotension, and hypoxia on the heart (163) and also of interest are experiments dealing with a negative chronotropic effect in isolated guinea pig atria (164). From a clinical point of ~•iew. the production of cardiac arrhythmias observed dur- ing maximal treadmill exercise in clincial normal man are of interest (165). The authors conclude that the appearance of unifocal ventricular premature complexes during near maximal exercise testing should not be equated with the presence of clinically significant heart disease. E. Atherosclerosis Because of the possible epidemiological rela- tionship between nicotine and atherosclerosis, the effect of nicotine has been explored. Several ap- proaches are possible. Nicotine could possibly pre- dispose to the development of atherosclerosis. or cholesterol svnthesis in liver, heart and brain might be influenced by nicotine. Because of the important role of smooth muscle cells in production of athero- sclerosis. nicotine mav also affect smooth muscle cells. Smoking could also effect atherosclerosis through an increase in carbon monoxide. Finally. the effect of smoke, or one of its constituents. may contribute to the formation of intravascular clotting through an effect on platelet function or through action on some other clotting mechanism. Several investigators have dealt with the prob- lem of the combination of dietarv cholesterol, vita- min D and nicotine in the development of calcific atherosclerosis. For example. Haas'and his co-work- ers, in publications in the American Journal of Pathology (166-168) and Federation Proceedings (169). described that atherosclerosis resulting from dietary choiesterol, vitamin D and nicotine can bt TIMN 0115813 T200304 69

Cardiovascular Srstem F. Hemodrnamics Manr papers have dealt with fundamental as- pects of cardiovascular hemodc•namics. For exam- ple. the use of aggregated iodinated "'1 serum albumin. in the study of hemodvnamics was in- vestigated (191). By measuring the ratio of dis- tribution of the isotope in the lung, the authors came to the conclusion that valuable information can be obtained bv this method on pulmonary arterial and left atrial pressure. as well as on cardiac output. Thev also were of the opinion that idiopathic pul- monarv hypertension can be recognized by this method. The same authors, as well as others (192). studied the effect of pentobarbital anesthesia on certain hemodvnamic parameters: As to be expected. thev found that observations on trained. resting awake dogs were superior to those on anesthetized animals. and that the results are stable and provide a suitable baseline for determining the response of the intact animal to a varietv of stimuli. Pentobarbital administration to the unanesthetized animal abol- ished the normal sinus arrhvthmia. and resulted in tachvcardia even before complete lapse of con- sciousness. This was apparently due to inhibition of normal vagal activity. %Iore closelv related to smoking are other papers (193. 194) whose authors stress tested smokers as well as non-smokers. They found that response of blood pressure. heart rate and cardiac output were not different in smokers from non-smokers. One of these groups smoked beagles. and after a period of 22 months. determined left ventricular function and volumes (194). No difference was found in heart rate. stroke volume, left ventricular end-diastolic pressure and volume, and intraventric- ular contraction time. However, left ventricular ejec- tion fraction was diminished in the smoked dogss interstitial fibrosis was evident, and an alteration of elastic elements may have been operative. Mescaline. in low doses. produced a slight increase in heart rate (195). Higher doses induced bradvcardia and conduction disturbances. Tidal vol- ume and compliance decreased. As with nicotine, right ventricular pressure increased. Antihistamines were ineffective against mescaline. Apparently mes- caline does not owe its effect to stimulation of histaminic receptor. One raport (196) deals with determination of flow in a multi-compartment model. bv external detection of gamma radiation. An eight-compartment recirculation model was con- structed. which was connected in series to a com- partment «•hich served as a cardiac mixing chamber. Various methods were used to calculate compart- ment flow. G. Epidemiological Studies The question of whether or not ph%siopathologi- cal changes observed in smokers are due to the smoking habit. or are the result of genetic factors. is highly relevant. A long-range study dealing with this subject has been in progress by the Swedish group (197). This study was possible because of an accurate twin registry in Sweden. The authors con- firmed that an association between smoking and certain cardiovascular symptoms exist. However, the excess morbidity is not due to smoking itself, since it was not possible to reproduce the association be- tween smoking and cardiovascular symptoms when studying monozygotic smoking discordant twin- pairs. This agrees with other Scandinavian workers who, studying discordant twin pairs, could not find an increased prevalence of coronary heart disease in smokers compared to non-smokers. It must be con- cluded. therefore. that genetic factors are of greater importance in the development of coronary symp- toms. Similar conclusions were reached by the Scandinavian workers comparing mortality between smokers and non-smokers (198). They found that the mortality among the registered subjects was sig- nificantly higher than among non-registered. regard- less of smoking. They also concluded that the increased mortality among the registered subjects (twins) was significantly higher than among the non-registered. regardless of smoking. They also concluded that the increased mortality among smokers was not due to smoking per se, but to factors associated with smoking. Other papers by the same group have confirmed these conclusions (199). A study by the Swedish workers (200) has anal_vzed data from about 4.000 male twin pairs from the Registry of the National Research Council in Washington. This study is particularly relevant to smoking. The authors conclude that in both Sweden TIMN 0115816 i r

t~ is found that carbon monoxide. nitric oxide• and nitrogt'ct• all constituents of tobacco smoke, changed scstemuc arterial pressure. heart rate and electrocar- cliograms (376-179). The effect on heart rate de- pended to some extent on the concentration of these compounds. It was suggested that the pharmacologi- c al effects of carbon monoxide ivere not entirely due to the concentration of carboxyhemoglobin (HBCO) fc~nned. The effect of carbon monoxide independent of HBCO was investigated on the isolated isovolemic rahhit heart. Apparently hemoglobin-free systems physicall}• dissolve carbon monoxide and signifi- rantl%• affect mvocardial metabolism and functi.on. The authors found that carboxt•hemoglobin. rxen in low concentration, can compromise the c'voen delivery to oxygen-deficient brains and 11 0 arts with circulatory insuffiencv. The smooth muscle cell has become an im- luortant tool in the investigation of atherosclerosis. t'sing culture of these cells• the formation of mounds Ithat is. accumulations of altered smooth muscle cells). with the interior of the mound containing modified smooth muscle cells. much ground sub- stance. and fibrils. were described (180). The authors believe that this represents a"mini-lesion" resem- bling atherosclerosis. These mounds develop upon hYpoxia. This ecork is particularly interesting, since platelets are absent from these cultures and since others have maintained that a platelet factor is necessar~- for stimulation of smooth muscle cells. The work indicates that lesions can develop without this hypothetical platelet factor (180). The same atrthors (181)• working on smooth muscle cell cul- tures. found that the mounds stain positive with PAS. The mounds were surrounded by two to four layers of intact cells. The centers of the mounds were (omposed of extracellular material and cell debris. Studies on the effect of tobacco smoking and atherosclerotic vascular disease have been summa- rized (182. 183). A close relationship between blood lipids and atherosclerosis was found and the role ~vhich catecholamines and nicotine play on lipids • and vascular changes have been investigated (184- 186). It was shown that the greater lipid catechol- amine response to cigarette smoking as compared to cigar and pipe smoking. is due to the tendency to inhale cigarette smoke, with resultant greater nico- tine absorption (184). In addition, a highly sig- n ificant acceleration of cholesterol crt•stallixatioii was effected by the serum lipids of blood taken thirt}• minutes after heavy cigarette smoking. This did nc~t occur after cigar or pipe smoking. These observa- tions suggest a possible contributing mechanism to the higher incidence of severe coronarv atheroscle- rosis in cigarette smokers (185. 186). The liver is the main organ for cholesterol production, and several investigators have at- tempted to relate the occurrence of arteriosclerosis in animals with cirrhosis (187. 188). Production of cirrhosis with no supplement of vitamin D led to the development of aortic and peripheral arteriosclero- sis• resembling senile arteriosclerosis in man. Pro- ducing hepatocellular disease with carbon tetrachlo- ride. and maintaining their animals (rabbits) on diet productive of mild dietary hvpercholesterolemia, it was found that periodic administration of large doses of vitamin D results in medial calcific de- generation of several peripheral systems (188). This process in animals with normal hepatic function stimulated fibrocellular proliferation of the adjacent intima and• in the presence of sufficient hyperli- pemic hypercholesterolemia. led to an accumulation of lipids in the intima. In animals with reduced hepatic function. however, there was no decrease in calcific medial arterial degeneration or serum cho- lesterol levels, but there was inhibition of fibrocellu- lar intimal proliferation. It was concluded that there may be a common pathogenesis involving hepatic mediation of vitamin D action as it relates to the regulation of the role of calcium ions and release and activation of biogenic amines. The combination cholesterol• vitamin D and nicotine. which leads to peripheral atherocalcific thromboarteritis. was cor- related with the degree of nicotine-induced rise in serum-free fatty acids (189). However, though rise in serum FFA plays no singular role in pathogenesis of thromboarteritis, the magnitude of rise allows for prediction about how each animal is likely to be ' affected bv the vascular toxic action of the cho- lesterol-vitamin D-nicotine regimen. Inasmuch as nicotine, vitamin D or dietary cholesterol in the amounts used were innocuous when used alone, the interactions between the effects of at least these three factors need to be known in individual animals before the pathogenesis of the calcific atheroscle- rotic lesion with thrombosis can eventually be un- derstood (190). TIMN 0115815 t T200306 71

Cardiovascular Svstem prevented by total adrenalPctomy: they also dis- covered that mural lesions had to be present in order to result in atherosclerosis in the presence of thesee risk factors. This is in line with more recent work which stressed the role of injur}• to the endothelium as an initial step in the production of atherosclerotic lesions. It was discovered that vitamin D. or dietarv cholesterol in the amounts used ivere innocuous when used alone. The interaction betveen the effect of these factors needed to be known in individual animals before the pathogenesis of the calcific ath- erosclerotic 'iesion and thrombosis could be un- derstood. The role of nicotine is not entirelv clear. but it is possible that vitamin D action is potentiated bv nicotine. A significant diminution in the rate of choles- terol svnthesis in nicotine-treated animals was found with the greatest reduction occurring in the srnthesis of serum-cholesterol followed bv liver. brain and heart muscle (170). The authors are of the opinion that this is the result of a reduction in the rate of degradation with equal decline in the rate of cholesterol formation. In line with more recent studies bv Ross and his co-workers from the University of Washington is the pioneer work of Zemplenyi and co-workers from the L'nic•ersitv of Southern California (unpublished re- port to AMA-ERF) which dealt with the fundamental aspects of cell cultures of smooth muscle obtained from the arterial wall. Although no mention .ti•as made of the effect of nicotine on these cell cultures, the paper is of interest. since it deals with an i mportant innovation in the study of atherosclerosis. the culture of smooth muscle cells. Finally. the effect of nicotine on clotting was investigated by a series of authors. In a paper delivered at the Third Research Conference on Tobacco and Health, held by the AMA-ERF in 1972. it was reported that nicotine may alter the mu- copol}•sacharide content of platelets from indi- viduals who smoke. The same authors studied the effect of thrombopoietic stimulating factor in thrombocytopenic mice (171). They described a sensitive immunoassay procedure of the thrombo- poietic stimulating factor (172). The same group of workers also found (173) that clot retraction in platelet-plasma preparations was enhanced by nic- otine treatment of rats. Apparently, nicotine does not 70 affect platelets directly, but enhances clot retraction observed in the blood of nicotine-treated animals because of a plasma factor, and not because of changes in platelets themselves. Cigarette smoking appears to increase platelet response to a standard aggregating stimulus such as adenosine diphos- phate. This appeared to be specifically related to the inhaling of tobacco smoke. Apparently, smoking- induced potentiation of platelet aggregation mar help to explain the increased incidence of arterial thrombi in cigarette smokers (247). Carbon monoxide, an important constituent of tobacco smoke, has been implicated in the pro- duction of atherosclerosis by a series of workers. particularly the Danish group. They found that smoked rabbits have a greater tendency to develop atherosclerosis than control animals. Others. how- ever. could not confirm this (174). Working on Macaca fasicularis. exposure to carbon monoxide intermittently throughout the day for 14 months, thev found no change in plasma cholesterol or aortic and coronarv atherosclerosis attributable to carbon monoxide exposure. Recent references in the literature have indi- cated that carbon monoxide (CO) has a profound influence on the hemodynamics. and that CO mav increase capillary permeabilitv. thus predisposing to the development of atherosclerotic lesions. How- ever. this is still disputed. Landaw (175) stressed the great variability in body carbon monoxide in sub- jects with similar smoking habits. An accurate estimation of carbon monoxide can be obtained by measuring either the blood carbon monoxide he- moglobin saturation (HBCO) or breath CO concentra- tion. since the two are closely correlated. It has been claimed by a series of authors that the amount of carbon monoxide bound to hemoglobin is only a small part of the total CO. Other heme compounds are also known to bind CO; heme and similar compounds are distributed through the whole body. Such sites could be associated with alterations in cellular metabolism. These authors also found that smoking beagles showed rapid alterations in blood hematocrit follow- ing initiation of smoking. This could lead to higher blood viscosity, which might potentiate the adverse effect of carbon monoxide on cardiovascular func- tion. Several papers have dealt with the heaiudy- namic effects of carbon monoxide. For example, it TIMN 0115814 T200305

Cardiovascular System corresponding ratio of %1.aO. The specific activities o% MAO and COMT change in parallel through the greater part of the transmural profile obtained on 24 µ cryostat sections. The specific activity of COMT drops significantly in sections that include endothe- lium and subendothelial media, where no decrease in MAO is noted. Subcellular distribution studies indicate the presence of MAO in mitochondrial and microsomal fractions, compared to the predomi- nantly microsomal and cell sap localization of COMT. Other support: U. S. Public Health Service. 10. Uptake of nicotine by the sympathetic nerve terminals in the blood vessel John A. Bevan, and Che Su Department of Pharmacology, UCLA School of Medicine, Los Angeles. California Journal of Pharmacology and Experimental Therapeutics 182:419-426. 1972 Nicotine causes contraction of the rabbit aortic strip by releasing adrenergic transmitter from the terminals of the sympathetic nerves. After exposure of these strips on their adeentitial surface to tritiated nicotine for two minutes, the time to the peak of their contractile response, nicotine is concentrated in the inner third of the adventitia, the region of the term,inal sympathetic plexus. After exposure for 10 minutes, the levels of tritium activity in both ad- ventitia and media are greater than can be accounted for by saturation of the extracellular space. Although the nicotine-induced, contraction is transient, the nicotine content of the sympathetic plexus seen at two minutes was maintained during relaxation. Nicotine uptake into the sympathetic plexus was reduced by cocaine, desipramine, guanethidine, and phenoxybenzamine, but not by d-tubocurarine, pen- tolinium and hexamethonium. All of these drugs prevented the contractile response to nicotine. None of these agents influenced nicotine uptake into the media. The relationship of nicotine uptake into the sympathetic nerve terminal and the release of the adrenergic transmitter is discussed. 80 11. Effect of the chelating agents, EDTA, 2,2'- bipyridyl, 8-hydroxyquinoline and pyrophosphoric acid, on norepinephrine uptake by rabbit aorta Ove A. Nedergaard. Augustine Vagne, and John A. Bevan Department of Pharmacology. UCLA School of Medicine, Los Angeles, California Journal of Pharmacology and Experimental Therapeutics 163:136-146, 1968 The uptake of tritiated norepinephrine by rabbit isolated aortic rings after 1 hr was enhanced by the chelating agents. (ethylenedinitrilo)tetraacetic acid (EDTA), either as the disodium salt (Na2EDTA) or as the calcium disodium salt (CaNa:EDTA), and so- dium pyrophosphate. but not by 2.2'-dipyridine and 8-hydroxyquinoline. The enhanced norepinephrine uptake seen with CaNaZEDTA was inhibited by 8- hydroxyquinoline but not by 2,2'-bipyridine. In the absence of CaNa2EDTA, repeated replacement of the bath fluid with physiological salt solution to which tritiated norepinephrine had been added immedi- atelv before increased the uptake to a level close to that seen in the presence of CaNa,EDTA. The re- moval of Ca" and Mg'-. either singly or together. from the physiological salt solution had no effect on the EDTA-enhanced norepinephrine uptake. How- ever, in the absence of both cations this uptake was decreased when NaZEDTA was present in a con- centration much higher than that necessary for causing maximal enhancement of norepinephrine uptake. The neurogenic contractile response of the rabbit isolated nerve-pulmonary artery preparation was blocked by NaZEDTA, 2,2'-bipyridine and 8- hydroxyquinoline, but not by CaNa2EDTA. Ca'- reversed only the Na2EDTA-induced block. The contractile response of the artery to exogenous norepinephrine and serotonin was not altered by NaZEDTA, but was inhibited by 2,2'-bipyridine and 8-hydroxyquinoline. These results support the view that EDTA acts by preventing the oxidation of norepinephrine catalyzed by traces of heavy metals in the physiological salt solution. It is concluded that it is essential to use EDTA to stabilize nor- epinephrine when the uptake of this amine is studied. Furthermore. EDTA in concentrations suf- ficient to cause maximum enhancement of uptake does not interfere with adrenergic neuroeffector TIMN 0115824 T200315 t , I f

ytiIclieti deal with the effect of nicotine on 'Itiiti,•rtruphied and failing cat %-entricle (131). Ap- ~0 rrutl}- thp direct effect of the drug increases peak ,,,,nu•tric: force in hypertrophied ventricles of cats. t}„• duration of the action potentials lengthened durint,' f'\posure to nicotine. The effect of nicotine „ll ,,i, tic;n potential can be abolished by treatment ~rith (i,1(1rf'nergic receptor blocking agents such as Pr„l,rancilol and in muscles remored from reserpine tn.,,ted animals (132). The opinion was expressed i t a:;) that both the inotropic response and the effect „n the action potential effected by nicotine may be ,,~,,,t'iah~cl with an increase in K- efflux or mem- },ran„o permeability to potassium (133). In light of these changes on the action potential. it is not surprising that nicotine produces arrhvth- niiase some of them extremely complex (134). These ~irsrrhvthmias include premature ventricular beats. .\V dissociation with accelerated junctional and t entricular pacemakers. and ventricular tachycardia. These effects may be attributed to conducti.on. de- t>rrssion. and induction of automaticity. Since nic- (,tint, ciemonstrates the capabilit~~ of inducing dia- ,tolir depolarization. conduction may be depressed. in that propagation is proceeding down partially rF•tiolarized fibers (134). Other investigators (135) 1 (,nfirm that'small doses of nicotine reduce sponta- ",•nus c.•cle length of sinoatrial node pacemaker t,•ll, and increase plateau amplitude in duration of h•ft atrial action potential. As mentioned above, t,rupranolol had no effect on the ability of nicotine or norrpinephrine to augment action potential dura- tion in the left atrial cell. In the whole dog large dus,•s of nicotine (200 µg) produced initial tachycar- ,li.i. hypertension. and reflex bradycardia (136). In 0cpn larger doses (400 µg), the injection of nicotine i:i the dog resulted in a triphasic response. with •iiicis arrest sinus tachvcardia and reflex bradvcar- Iii.i 11a6). Some of these arrhythmias could be •iltrred or abolished by atropine, while propranolol li,ui little effect in prolonging the asystole occurring .,fter intravenous administration of nicotine (136). The effect of calcium on action potentials and (atecholamines has also been investigated (137). The (:onclusion was reached that catecholamines restore excitability by increasing membrane conductance to Ca-- (137). Studies within recent Years have also dealt with the effect of (3-ganalionic blocking agents on the heart. Relatively few studies have been published on the effect of ganglionic blocking agents on action potential repolarization. One of these reveals that when transmembrane action potentials are recorded from cells in the electrically driven left atrial ap- pendage. catecholamines modified certain phases of the action potential. Propranolol completely pre- vented changes in electrical and mechanical activity caused by isoproterenol. The conclusion was reached that atrial muscle cells in the guinea pig displayed two pharmacologically distinct adreno- ceptive sites (138). Fundamental Studies with Reference to Nicotine and SmokinQ Smoking or nicotine have been frequently im- plicated in the genesis and mechanism of ventricular fibrillation: one of these studies was concerned with the effect of inhalation of cigarette smoke on ven- tricular fibrillation threshold in normal dogs and in dogs with acute myocardial infarction (139). It was not surprising that myocardial infarction lowers ventricular fibrillation threshold. The effect of smoke inhalation on ventricular fibrillation was almost as pronounced. In normal dogs inhaling smoke. a decrease in ventricular fibrillation thresh- old. averaging 30-40%. was observed. This was explained by assuming a direct effect of nicotine on the myocardium. or of nicotine-induced adrenergic stimulation (139). These findings are of interest in view of the increased incidence of sudden death observed among coronary patients who are heavy cigarette smokers. A similar paper deals with the effect of hypoxia in the genesis of cardiac ar- rhythmias (140). In this report. changes in mem- brane function which occur with oxygen deprivation were studied and the conclusion was reached that pure hypoxemic hypoxia very rarely leads to ven- tricular fibrillation. Ventricular asystole occurs, due to either SA node failure or to cessation of atrioven- tricular conduction. It is probable that failure of hypoxemic hypoxia to lower fibrillation threshold is the result of compensatory mechanisms such as changes in coronary blood flow and alterations in arteriovenous oxygen extraction (140). Antifibril)atory agents and catecholamines were also investigated by low-amplitude high-frequency electric pulses to the heart of dogs (141). Catechol- amines reduced the fibrillation index by about 35%. TIMN 0115811 T200302 67

tobacco smoke. The findings suggest that oxide of nitro~en does not occur in cigarette smoke in suf- ficient conc'entrations to form methemoglobin in (252). Closely related to these studies are investiga- tions dealing with the chronic effect of smoking on c lutting mechanisms (253). The hypothesis for these :tudies has been that enhancement of the thrombotic }>roucess by smoke may underlie the pathogenic maThanism of increased cardiovascular disease. Bea- 1F swere smoked through a permanent tracheos- to,mv and subsequently were submitted to chronic sm0l.ina for a period of eighteen months. It was found that the turnover rate of the fibrinogen was significantly increased in the chronic smokers. In acldition. clotting time as well as PTT (partial thromhoblastin time) were accelerated. There oc- curred a gradual decrease in platelet count and there was a significant enhancement of platelet aggregation (253). The role of such changes in the pathogenesis or acceleration of cardiovascular dis- ease in the smoker. however. remains to be further dE>fi ned. A. Blood Vessels 1. Comparison of adrenergic mechanisms in an elastic and a muscular artery of the rabbit J. A. Bevan, R. D. Bevan, R. E. Purdy, C. P. Robinson, C. Su. and J. G. Waterson Department of Pharmacology and Pathology (Division of .\'europathology) University of California Los Angeles School of Medicine, Los Angeles, California Circulation Research 30:541-548, 1972 Elastic and muscular arteries are known to differ in function and in the magnitude of their response to vasoconstrictor influences. With the isolated tho- nm.ic aorta and ear artery of the rabbit as representa- tive arteries, the morphological, physiological, and pharmacological correlates of these differences have been sought among their presynaptic adrenergic mechanisms. The adrenergic nerve plexus in the ear artery is wider and the nodes are denser than they are in the aorta. There is some evidence consistent %.•ith the hypothesis that the thicker the plexus, the greater the reuptake of released transmitter, the smaller the transmitter overflow, and the lower the efficiency of uptake of exogenous norepinephrine. Measurements of fractional release of tritiated nor- epinephrine suggest that qualitative differences in the adrenergic transmitter storage and release mech- anisms may exist between the two vessels. Thus the considerable functional difference between the two vessels is in part, at any rate, a consequence of adrenergic mechanisms which differ both quantita- tively and qualitatively from each other. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 2. Direct method for recording tension changes in the wall of small blood vessels in vitro J. A. Bevan, and J. V. Osher Department of Pharmacology, UCLA School of Medicine. Los Angeles, California Agents and Actions 2:257-260, 1972 A method is described for the direct recording of tension changes in the wall of blood vessels of 100 µm internal diameter or less in vitro. Most vessels tested exhibited spontaneous rhythmic activity and responded to L-norepinephrine by an increase in tension. The response to transmural stimulation of the intramural nerves was variable. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 3. Distribution of components of 3H-noradrenaline uptake in the wall of the rabbit aorta J. A. Bevan, R. D. Bevan, J. V. Osher, and C. Su Departments of Pharmacology and Pathology, Division of Neuropathology, UCLA School of Medicine, Los Angeles, California European Journal of Pharmacology 19:239-245, 1972 An analysis of the distribution of tritiated nor- adrenaline ('H-NA) in the wall of the rabbit thoracic aorta has been studied by a frozen-section tech- nique. Some of the components of this uptake were defined by relating tritium distribution to histologi- cal structure and by determining the effect of tissue washing, electrical stimulation of the intramural nerves and pretreatment with phenoxybenzamine (PBZ) in a concentration that inhibits maximally 'H- NA binding. Five components of 3H-NA distribution T11V1N 4115821 T20o312 77

transmission. The inhibition of this process and of norepinephrine uptake seen with 2.2'-bipyridine and 8-h~~drox~'quinoline may be due to toxic metal che~lates formed by these agents with the trace metals. Other support: L'. S. Public Health Service. 12. Distribution of norepinephrine uptake within rabbit aorta between adventitia and media O. A. \edergaard. A. Vagne, and J. A. Bevan Department of Pharmacology, U.C.L.A. School of .tfedicine. Los Angeles, California E..perientia 25:150-151, 1969 The results of this study demonstrate that in vitro 'H-norepinephrine (3H-NE) is taken up by rabbit aortic rings into both adventitia and media. It has been often implied that rabbit aorta is innervated by sympathetic adrenergic motor nerves. the ad- ventitia probably containing most of the terminal nerve plexus. At low. but not at high concentrations of 'H-NF, most of this amine was localized in the adventitia. The increasingly larger proportion of the uptake localized in the media seen with concentra- tions higher than 10-eM. suggests that when the capacity of the adrenergic neurones to accumulate the labeled amine is exceeded, extraneuronal uptake becomes predominant. Other support: U. S. Public Health Service. 13. Distribution of norepinephrine released from adrenergic motor terminals in arterial wall J. A. Bevan, and J. V. Osher Department of Pharmacology, UCLA School of Nfedicine. Los Angeles, California European Journal of Pharmacology 13:55-58, 1970 The distribution of tritiated material remaining in the c,•all of the rabbit aorta previously soaked in 'H-norepinephrine and then washed, changes as a consequence of prolonged electrical stimulation of the motor nerve plexus. The increase in medial radioactivity is not uniform, but is highest near the adventitio-medi.al border and lowest at the intimal surface of the vessel. This increase could not be demonstrated after exposure to a dose of nicotine causing a contractile response of similar magni- tude. 14. The release of H'-norepinephrine in arterial strips studied by the technique of superfusion and transmural stimulation Che Su. and John A. Bevan Department of Pharmacology, U.C.L.A. School of Medicine, Los Angeles, California Journal of Pharmacology and Experimental Therapeutics 172:62-68, 1970 The release of tritiated norepinephrine (H'-\'E) was studied in relation to muscle contraction in the isolated rabbit main pulmonary artery. This arter}• was cut spirally into a strip. labelled with dl-H'-NE and superfused with Krebs-bicarbonate solution. Excitation of the nervous elements by electrical transmural stimulation caused contraction and a sharp rise in the overflow of H'-NE. Both these responses were blocked by tetrodotoxin and bre- tylium. Of the total tritium activitv in the superfu- sate. during rest approximately 30% and during stimulation 50% was accounted for by H'-NE. The remainder was attributable to metaboli.c products of which the non-catechols predominated over the deaminated catechols. In the presence of phenoxN- benzamine, the transmural stimulation-induced tri- tium overflow and the proportion of intact H'-NE were greatly elevated. The overflow declined rapidly upon continued stimulation. The combination of superfusion and transmural stimulation may be of value for concurrent measurement of the neurotrans- mitter release and effector cell response in a number of tissues. A preliminary report of this work appeared in Federation Proceedings 27:335, 1968. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 15. Some structural considerations of the reactivity of vascular smooth muscle John A. Bevan Department of Pharmacology, School of Medicine, University of California, Los Angeles, California. Microvascular Research 1:329-334, 1969 Some morphological characteristics of the neu- roeffector apparatus in the elastic blood vessels TIMN 0115825 T200316 81

Cardiovascular System \o stimulatorv activitx. was found with these com- pounds in either the aortic strip or ileal prepara- tions. , I. Pharmacolqgical Studies This particular portion of the report deals with specific aspects of the pharmacological action of nicotine. histamine. 6-hvdroxvdopamine (6-OHDA) and alcohol. The A%IA-ERF has sponsored research in purely fundamental aspects of pharmacology which. in addition to the action of the agents mentioned above. also deals with the mechanism of calcium uptake and transfer as influenced by pro- caine and lanthanum. Pharmacological properties of other smoke constituents such as nitric oxide will also be discussed. It was found that leukocvtes and platelets. as well as lymphocytes. have significant histamine- releasing activitv when administered intravenously to dogs (210. 211). The studv therefore provided a demonstration of the histamine-releasing activity of dog leukocvtes and platelets. It was shown that mescaline does not owe its activitv to histamine receptor stimulation. Fundamental studies were concerned with the effect of 6-hvdroxydopamine (6-OHDA). The com- pound failed to elicit the excitative response in the atria obtained from reserpinized rats or rats treated with 6-OHDA in vivo. Apparently in vitro treatment ~~ ith 6-OHDr1 may cause atrial stimulation by an indirect action involving the release of catechol- amines as a result of its displacement at the nerve endings (212). Some fundamental effects of alcohol were also investigated. Alcohol weakens the link between contraction and relaxation. involving myofibrillar calcium transport in dogs maintained on alcohol for prolonged periods of time and alcohol blocks the bradvcardia when patients were placed from the recumbent to the upright positions (213. 214). Acetaldehvde. an important breakdown product of alcohol. resulting from the activity of alcohol dehvdrogenase possesses sympathomimetic and cholinergic action (215). Higher doses of the two aldehvdes produce a sharp fall in blood pressure and severe bradvcardia. After vagotomv. high doses of acetaldeh-%•de and propionaldehvde resulted in an increase in blood pressure and a positive chrono- tropic response. Apparently the effect on the vagus is the result of stimulation of higher centers result- ing in bradvcardia and hypotension, especiall}• when higher doses of the aldehydes are emploved. After guanethidine treatment, acetaldehyde causes a significant pressure response (216). Lanthanum blocks movements of external i5ca1- cium through the cell. The calcium-dependent ac- tion bt• lanthanum may be important for the initial portion of the contractile responses as well as the effect of K'. nicotine or acetylcholine. It appears therefore that the Ca?' sources utilized bv n icotine to elicit contractile responses in frog muscle differ in part from those used by K' and acetyIcholine (217). Several investigations have dealt with the phar- macologic effects of nicotine (218). The effect of nicotine on the behavioral and autonomic response pattern was demonstrated with the help of im- planted recording devices. which permitted stimula- tion of the hypothalamus. The investigators found that n icotine, by altering the reactivity of both the central nervous system and peripheral effectors. could modifv the extent of behavioral and au- tonomic responses that ultimately may have some pathogenic role in the development of cardiovascu- lar disease. Nicotine modified the response to short hypothalamic stimulation, resulting in less tachvcar- dia. a marked stimulation of gastrointestinal motil- it_v and an increased number of somatic responses. Nicotine also influenced the ionic movements and tension in frog muscle (219). The same authors (220) also demonstrated that nicotine acts at sites in the transverse tubular membrane to alter'sCa move- ments and induce contracture. In a further report (221) it was demonstrated that n icotine acts at receptors at the neuromuscular junction (only a portion of these are acetylcholine receptors) that nicotine is active in both ionized and non-ionized molecular forms, and finally that n icotine does not exert these actions at sites beyond the neuromuscu- lar junction. Further studies involving the effect of nicotine on calcium movements were investigated at various pH (222). The observations indicate that the mag-, nitude of the nicotine-induced contracture and Ca" TIMN 0115818 T200309 74

r I- ie n ts is rj n c y distributed between the tunica adventitia and media. This «'as demonstrated in stripping experiments in kvhich the uptakes «•ere 0.56 -* 0.02 and 0.42 ± 0.01 mllg for adventitia and media respectively (M ± S.E.M.). Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 19. Adrenergic transmitter release and distribution in blood vessels Che Su. and J. A. Bevan Department of Pharmacology, U.CL.A. School of kledicine. Los .•ingeles. California Proceedings of the Symposium on the Physiology and Pharmacologr of Vascular Neuroeffector Systems. Interlaken. 1969. pp. 13-21 (Karger, Basel 1971) The authors present a review of current knowl- edge on the above subject, including studies from their laboratory. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 20. A double-labeled frozen section technique for studying distribution of H3-norepinephrine C. Su. J. A. Bevan, and J. V. Osher Department of Pharmacology, U.C.L.A. School of ltedicine. Los Angeles. California Experientia 28:991-992. 1972 The arterial cvall is stratified into tunicae intima, media and adventitia with differing diffusion char- acteristics and catecholamine uptake capacities. Cat- echolamines. either released from the sympathetic nerve terminals or applied exogenously, do not distribute uniformly throughout the arterial wall. Their distribution profiles are of physiological and pharmacological consequences. An improved method for differentiation of intra- from extracellular distribution of exogenous nor- epinephrine in the wall of the aorta is described. 21. The vascular response to adrenergic transmitter in experimental hypertension J. A. Bevan, R. D. Bevan. and C. Su Departments of Pharmacology and Neuropathology, UCLA School of Medicine. Los Angeles. California Federation Proceedings 32(3):749. 1973 Parameters of adrenergic transmission, smooth muscle proliferation and the arterial wall dimen- sions have been studied in rabbits in which a partly constricting ligature was placed around the upper abdominal aorta. Changes in vessels above the constriction where the arterial pressure was raised were compared with those below. A decrease in the fractional release of labelled adrenergic transmitter stores from the pulmonary artery was associated with an increase in the contractile response to nerve stimulation. Alteration in the contractilitv of the vessel wall and MAO and COMT content is,of the order expected from the increase in smooth muscle mass. Neuronal uptake of labelled norepinephrine was increased. The apparent disparity between transmitter release and response may be the con- sequence of an alteration in the extraneuronal dis- tribution of transmitter caused by reactive changes in the vessel wall consequent upon the increase in arterial pressure. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 22. A superfused and transmurally stimulated artery preparation C. Su (John A. Bevan) Department of Pharmacology, U.CL.A. School of Medicine, Los Angeles. California Proceedings of the Western Pharmacology Society 11:143-144, 1968 The aim of this work was to develop a simple, isolated artery preparation, suitable for studying simultaneously the release of norepinephrine from vasoconstrictor nerves and arterial muscle contrac- tion. The combination of superfusion with transmu- ral stimulation seemed to be suitable for studying nerve functions, especially the transmitter release, in a small preparation. Maximal and purely indirect TIMN 0115827 T200318 83

"M Cardiovascular System in the vessel wall were defined; that (i) in the extracellular space. (ii) loosely bound to all tissues, (iii) specifically bound to sympathetic nerves. (iv) bound to non-nervous tissues at sites sensitive to PBZ. (v) bound to all tissues at sites sensitive to PBZ. Other Support: U. S. Public Health Service, and the Los Angeles County Heart Association. 4. The bimodal basis of the contractile response of the rabbit ear artery to norepinephrine and other agonists J. A. Bevan, W. Garstka, C. Su, and M. O. Su Department of Pharmacology, UCLA School of Medicine, Los Angeles, California European Journal of Pharmacology 22:47-53. 1973 Strips of rabbit ear artery exhibit biphasic con- tractile responses to 1-norepinephrine (1 ,NE), his- tamine, and serotonin. Evidence is presented on the basis of an analysis of the response to 1-NE that the two phases of contraction are associated with differ- ent modes of excitation and can be influenced independently. The initial part of the response is phasic. The response after 4 sec agonist exposure is the same as that after 32 sec. It is probably associated with local radial propagation of excitation in that the initial excitatory period is short, the contraction is preceded by electrical change and is abolished upon tissue exposure to a solution in which all NaCl and KCl is replaced by potassium methylsulfate. In contrast the second contractile phase is related to the time of tissue exposure to the drug, is an equilib- rium-like response and is not dependent upon cell membrane polarization. Since this phase is more affected by calcium depletion than the first, the sources of activator calcium for the two phases may be different: that for the first phase may originate predominantly from intracellular and that for the second from extracellular sources. These findings support the hypothesis that the biphasic contractile response of the vessel reflects two different modes of excitation of the muscle wall by the agonists: the first, a triggered, propagated event; the second, a slon•, equilibrium-type, non-propagated response. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 5. Distribution of bound norepinephrine in the arterial wall J. A. Bevan, J. V. Osher, and R. D. Bevan Departments of Pharmacology and Pathology (Division of Neuropathology), U.C.L.A. School of Medicine. Los Angeles, California European Journal of Pharmacology 5:299-301, 1969 The distribution of bound tritiated norepineph- rine in the wall of the rabbit aorta was determined by estimating the radioactivity in 24 µ frozen sections cut parallel to the intima. Peak radioactivity oc- curred in the region of the adventitio-medial junc- tion; there was also significant binding in the adventitia and media. The shape of the distribution profile on the adventitial side of the peak may be explained by the presence of the primary adventitial plexus found throughout the adventitia, and the terminal effector plexus in the region of the ad- ventitio-medial junction. Since there is no evidence for motor neurones within the media, except for those nerves associated with the vasa vasora, medial norepinephrine must be associated with mesenchy- mal tissue. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 6. Effects of nicotine, dimethylphenylpiperazinium and cholinergic blocking agents at adrenergic nerve endings of the rabbit pulmonary artery Ove A. Nedergaard, and John A. Bevan Department of Pharmacology, UCLA School of Medicine, Los Angeles, California Journal of Pharmacology and Experimental Therapeutics 168:127-136, 1969 The contraction of the rabbit isolated pulmonary artery to postganglionic nerve stimulation was blocked by dimethylphenylpiperazinium (DMPP) but not by nicotine or the nicotine metabolite, cotinine. The DMPP-induced block was reversed by repeated washing of the preparation. The extent of the DIv1PP-induced block was the sa~ne whether the TIMN 0115822 78 T200313

J t ~) ~- he to to tlv he :es in he to 1)v 'n- -4). im un lid im ne lp- tic br ;IC ~rs )o- ;ic be rta h- 'le P n e n Nd 011 re ce Lit •d ,, a t,p,ither.tonn produced the presvnaptic type su- E„-rst•nsitiVitr for isolated atria and seemed to induce ()tl),• presynaptic. but also postsynaptic su- E,rrs'ensiti%•it>' for aortic strips (31). A dual svm- t,,1thonlimetic action of nicotine on isolated blood nanlely release of adrenergic transmitters ,,1i(l f,x•ilitation of adrenergic transmission was also ,h,,,,•n (;;'). Nicotine monomethiodide did not re- I,•,itit• tht• transmitter. but blocked nicotine action in t)l,, isolated rabbit pulmonary artery. :\ conlprehensive review of the effect of n icotine ,,,, I)lc,ncl vessels has been published in 1973 in the lnrtual Rerien• of Pharmacolog~, (33). lircause of the difference in chemical structure. it i: nut Surprising to find differences in the effect of m<otinf• and its derivatives on the vascular wall. '1'his aphlies to aldehydes. such as formaldehyde, ac- vtaltlehyde. acrolein. etc.. which are present in low ,on(entrations in the vapor phase of cigarette smoke 1.3-]I. Each of those compounds possesses indirect .\nlp<jthomimetic activity. Intravenous doses of thr,r aldehvdes produced a dose-related rise in bloxul pressure in the rat with acrolein being the nio»t potent. Adrenalectomy. reserpine or phentol- .mnino reduced this response. Apparently the pres- ,ur efff°c:ts of these compounds are primarily due to k,,.' )constriction mediated by norepinephrine re- :;isvc1 from s%•mpathetic nerve endings (34). These ,tiuiirs have led to the suggestion that these al- 'l1,hXIlrs. especially acrolein. are involved in the trdicnascular response to cigarette smoke (35). In :,its inhaling various concentrations of these al- doh\des- a significant increase in blood pressure at :,,%\ (nncentrations is observed. At higher concentra- ; i-,ns. heart rate also increases. Concentrations of aldehydes were higher than those which ...nilcl he encountered in cigarette smoking (36). Thesr• fundamental studies further confirm the ., "Il-1.nuwn pharmacological and clinical observa- 'j-1 that the effect of nicotine as well as some of its .l-ik,itixes. is due to the release of sympathomi- :n1'tit . amines. In patients. submaximal upright exer- iso before and after smoking a single cigarette influ- .•:ic 1•d the circulatory response to exercise beyond thhit resulting from upright exercise alone (37). In line with previous published data. it~was not surprising that smoking increased mean cardiac index and mean heart rate. while arteriovenous oxv- calen differences. stroke index and pulmonary arterv pressure remained unchanged (38). By decreasing the response of the stroke volume to exercise, smoking a single cigarette significantly alters the hemodynamic response in a direction opposite to that accomplished by physical training (38). Using the perfused hind quarter of the dog. a multiphasic change in perfusion pressure of the innervated extremitv was found. Several compo- nents in perfusion pressure resulted from the in- travenous administration of nicotine (10-40 µg/kg). It was concluded that reflex dilatation evoked by nicotine must be attributed in part to inhibition of sympathetic vasoconstrictor tone, and to activation of the sympathetic dilator system. Denervation of the carotid and aortic body chemoreceptors reduced the initial rise in perfusion pressure produced by nico- tine (39). B. Cardiac Metabolism iV1anv of the studies have dealt with fundamen- tal aspects of biochemistry, particularly with energy metabolism and the regulation of metabolic pro- cesses in mitochondria and sarcoplasmic reticulum. Thus, fluxes through the citric acid cycle in the heart muscle, such as the dual role of the succinvl-CoA as a negative feedback regulator of flux through alpha- keto-glutarate dehydrogenase and citrate synthetase were described in the literature (40-44). Other fun- damental papers have dealt with the functional significance of the malate-aspartate and alpha-glyc- eride phosphodehydrogenase shuttles in isolated perfused rat hearts• with the interaction of glutamate metabolism with transport steps across the mito- chondrial membrane and with enzyme steps of the citric acid cycle, with particular reference to the transfer of reducing equivalents into mitochondria via the malate aspartate cvcle (42). The discussion of the effect of hypoxia on intracellular myocardial acid hydrolases has a more direct bearing on nicotine. It is known that hypoxia causes a release of lysosomal enzymes from the heart muscle. Using the isolated perfused rat heart prepa- ration (45), a significant leakage of lysosomal en- zvmes was found after hypoxia. However• it was surprising that nicotine itself was associated with leakage of enzymes into the perfusate during aerobic perfusion without evidence of enhanced glycolysis TIMN 0115805 ~- T200296 61

Cardiovascular System ponents of the I~,i rureceptnr reflf,\es (2:3i)). The e\i,tencf' of a supr,thulhar systeni which functions to inhibit vabal hraciyc:arc(ia induced by baroreceptor activation is descrihed. It is concluded that tachvcar- dia associated cvith the pressor response evoked bx• h}•pothalamic stimulation is the result not only of increased cardiac sympathetic nerve activit'.- but also of inhibition of baroreceptor-induced vagal activation. In another report a relationship was discovered between certain catecholamines (epi- nephrine and isoproterenol) and the effect of po- tassium. Apparently catecholamines possess a hy- pokalemic action. Further experiments have revealed that beta-2 receptors subserve a hvpoka= lemic action which is responsible for the protective effect of sympathomimetic amines against potas- sium intoxication (240). While nicotine does not exhibit specific interactions with a solubilized pro- tein from elastic fibrils. a-elastin. it does specificall% - hind this protein which has previously bound cal- cium ion at neutral sites (241). Damage of heart muscle by catecholamines has been postulated for a considerable length of time. In a studr of the effect of s~~mpathomimetic amines on heart lactic deh~•drogenase isoenzvmes (242) it was found that isoproterenol reduces total heart lactic dehvdrogenase (LDH) during the first and third dav following the injection of this amine. Heart LDH isoenzvmes 3. 4. and 5 increased to a maximum on the second day and gradually returned to the control level bv the tenth day. LDH 1 and 2 isoenzyrne activities were reduced over this same period. These effects of isoproterenol were used as a measure of potential blocking effects of phentoiamine. This drug blocked the effects of epinephrine. norepineph- rine and phenvlephrine (242). Certain beta-adrener- Oic blocking agents also had an effect on the nor- adrenaline content of rat heart before and after noradrenaline hifusion (243). This conclusion was reached by injection of several beta-adrenergic blocking agents. Adrenalectomized animals failed to survive liver damage due to carbon tetrachloride reg- imen for longer than 4 weeks. and a severe cardio- myopathy was observed. The authors believe that these observations mav bear on the pathogenesis of common puzzling skeletal and cardiac myopathies occurring in people with a history of alcoholism (244). K. Hematologt' Smoking could affect formation of red cells and platelets as well as the oxy0en carrying capacity of the blood and clotting mechanisms. The effect of tobacco smoking on hinclinQ of oxygen by hemoglobin was investigated (2-15) in man. and it was found that smoking significkintl\• affects the allosteric properties of hemoglobin. B*V regression analysis. it could be shown that most of the effect obsen•ed in smokers can be explained b~~ increased concentration of carbox~~hemoolobi~i. There was no significant difference in the heat stabilitt• of hemoglobin obtained from blood donors who smoke and those v,•ho do not (246). The effect of cigarette smoke on platelet function was also investigated in a controlled double-blind study. Smoking of a single cigarette increases the platelets' response to a standard aggregating stimulus (adeno- sine diphosphate) (247). This phenomenon appeared to be specifically related to the inhalation of tobacco smoke. The effect on platelets was in- dependent of the rise in plasma free fatty acids which follows cigarette smoking. Platelets from nicotine-treated animals are more functional as far as aggregation and clot retraction is concerned than platelets from saline-treated controls (248). Quantifi- cation of the level of mucopolysaccharides (NIPS) show greater uptake of Na,'550., by platelets of nicotine-treated animals than bv control animal platelets without major alterations in the kinds of MPS. Apparently nicotine injection increases the amounts of MPS in rat blood platelets. Smoke exposure and nicotine treatment caused no change in the average size of the circulating platelets (2-t9), but seemed to result in a significantly increased platelet adhesiveness (249). In rats exposed to cig- arettes, a significantly increased percent platelet aggregation after challenge with ADP was found. This. however, depended on the number of cig- arettes smoked. Studies have led to more funda- mental aspects of immunoassay and bioassay of thrombopoietin (250). Apparently thrombopoi.etin stimulating factor (TSF) can be purified by various methods. and the factor can be detected and quanti- fied by several assay procedures in whole serum or plasma fraction (250. 251). Mention was made of the possible effect of smoking on hemoglobin and the formation of methe- moglobin. This could be affected by nitrous oxide in TIMN 0115820 76 T200311

movements are Lippendent upon the concentration of nonionized nicotine in the bathing solution. Thus thE, notliotltzed form of nicotine appears to be the molecule of importance. not only for penetration of tht, cell membrane. but for alterations in Ca's move- ments and contracture in the muscle. Additional studies were concerned with the action of procaine cln nicotine-induced effects in muscle (223). Nico- tine contracture is sustained by procaine at pH 8.4 and inhibited at pH 7.40 Procaine decreases uptake and increases efflux of nicotine-C". The presence of nonionized procaine molecule can be correlated with contracture and increased residual Ca;' uptake in a manner similar to that of nonionized nicotine. Nicotine stimulates the secretion of biogenic amine-c.ontaining granules from the adrenal me- dulla. the gastrointestinal tract blood platelets. and mast cells (224). The structural subunit of the n icotine molecule which triggers and maintains the release process has been identified (224). Further studies on the mast cells and release of biogenic amines from these structures (225) have shown that alkaline conditions are required to convert the releasing agents to the form of their free base. The potency of the compounds as releasing agents was directh• related to their basicitv. Interesting information has been obtained on perfused isolated adrenal glands of dogs_ (226). Perfusion with n icotine or acetylcholine signifi- cantl}• increased the proportion of norepinephrine in the effluent whereas muscarine did not alter the relative proportions of epinephrine to norepineph- rine. Apparently there are nicotinic and muscarinic receptors for acetvlcholine in the adrenal medulla <Ind cholinergic transmission is possible via both mechanisms. When one type of receptor is blocked hy continuous contact with an agonist the sensitiv- ity of the other type is increased; inactivation of one is thus compensated for by increased response due to potentiation of the other (226). Studies on the mechanism of the general action of organic n itrates have been carried out (227. 228). The studies may be summarized by stating that n itroglycerin mav be a monoamine oxidase inhibi- tor. This was shown in rat heart mitochondria as xvell as rat liver and brain. 1. Studies on Endrocrine SYstenl. Including Catecholamines Several papers have been publitihed deaIinG, with the release of catecholamines from thc~ adrenal gland. Some of these publications deal with thr effect of nicotine on the adrenal gland. The activity of dopamine-beta-h}•clrnx' vIaso (DBO) activity on MA'- and other metals was stuc(ic'cl (229-235). Earlv studies confirmed that DRO k largely localized in the catecholamine storage ve,i- cles. and that the enzyme is more easily soluhilizPci than previously thought. Acet}•(choline as wcdl as nicotine. stimulates the release of DBO and the release of the enzyme parallels the release of c<Itc,- cholamines. Neurogenic secretion from the adrenal gland was characterized by the release of the soluble content of the storage vesicles directly into the exterior of the cell. and retention of the storage vessel membrane within the cell. DBO was also usf~d as a marker to trace the fate of the catecholaminc, storage vesicle membrane following secretion of adrenaline. During neurogenic stimulation of the adrenal gland. the entire soluble content of the stor- age vesicles. including DBO. is secreted by exocx•to- siss leax•ing the vesicle membrane within the med- ullary cell. An interesting report (236) describes evidence that neurogenic secretion from the adrenal medulla occurs b_v an all-or-none release from the storage vesicles. The effect of various doses of nicotine on the secretion of various catecholamines and their ex- cretion in the urine of rats were also studied (237). Doses of nicotine ranging from .1 to 1.0 mg/kg weight produced a significant increase in the ex- cretion of epinephrine and its derivatives. They conclude that the release of epinephrine from the adrenal medulla by nicotine is much more important in producing the various pharmacological responses than is the corresponding release of norepinephrine. It was also stated that the stimulation of adrenal cortical activity is due to enhanced corticotropin release resulting from a nicotine-induced increase in sympathetic and catecholamine activity (238). Closely related to the field of catecholamines is the neuro-control of catecholamine release and of reflexes regulating central sympathetic outflow. In this vein, the effect of hypothalamic stimulation was studied on the cardiac and vascular efferent com- TIMN 0115819 T200310 75 t

f i(u,li( ~,hanres in infarcted and non-infarcted myo- I ,t,ttliun1 during the post-infarction period (58). with ,letermination of glycolvtic metabolism. the assess- :nt,nt of hypoxia in human hearts (59). with high ,,11t,roy phosphate levels after coronary artery li- :,,tictn (60). and with storage and metabolism of ,itecholamines in experimental myocardial infarc- tion (61-63). Some work in this ffeld deals with i;,,i•tiicmes and infarcted heart muscle (65). and t%-ith <t study of the functional compartmentation of .\'1"P and creatine phosphate in heart muscle (64). Srveral papers have appeared dealing with the iit,.t hanism of chronic heart failure. Two of these are „f ~;rneral interest: one dealing with myrosin svnthe- .is in chronic heart failure (66) and the other with tht~ tuetabolism of the heart in failure (69). The latter i~ a review article in which it is stated that heart f,tilure can be enzvmatically characterized by spe- ( ific eniYme lesions. In the second report on chronic he.irt failure (68). it was found that there was no .i,nific:ant difference between the rate of synthesis 1)f Ivft-k~entricular mvosin and other protein fractions i6tained under conditions of heart failure. as com- pared to the same protein fraction in the normal .1nirnal. We know now in the light of more recent .tuctie•s. that the main deficiency in failure is in e~.c:itation-contraction coupling. and probably ATpase-related to mvosin receptors. In cardiac hvpertrophv. experiments were th-;c ribed (70) in which it could be demonstrated th.tt inhibition of cardiac hypertrophu by means of .it tinom}•cin D or puromycin. which inhibit protein -;vitthesis. led to development of myocardial failure i'ttf. A review article summarizes current concepts 1,f tm•ocardial failure (71). Several studies have been unclertaken dealing with the pharmacological as- i-t ts of nicotine and its relationship to catechol- .,mintrs. They have dealt with the effect of pronetha- no~L a bYta-adrenergic antagonist, on the myocardial ut>take of norepihephrfne (72) on the effects of beta- ddrt>nergic receptor blocking agents in antagonizing thW positive chronotropic response of the heart to it„repinephrine (73) and on the effect of nicotine and ~1'mpathomimetic amines on potassium intoxication 1%-t). It was found that intravenous infusions of n icotine protect anesthetized dogs against death produced by the infusion of potassium chloride. This appeared to be the result of the sympathetic discharge elicited by the drug. C. Coronart•. Capillan•. Cerebral and Peripheral Circulations The papers published in this field have dealt with both fundamental and applied aspects of these circulatorv systems. Fundamental Studies Perfusion of the heart with nicotine causes 8 transient release of norepinephrine presumably from postganglionic adrenergic nerve terminals. The question has often been asked whether this release is associated with morphological changes. ['sing flu- orescent histochemical and electron-microscopic studies on coronary arteries of dogs. it could be shown that the intracoronary infusion of nicotine (100 µgJmin) has no noticeable effect on the inten- sity of the specific fluorescence or on the staining properties of the granular and agranular vesicles. Apparently the coronary arteries have a dense adrenergic innervation. and exposure to nicotine has no detectable structural or histochemical effect on the nerves (75). Since it is known that n ic:otine causes an increase in coronary flow of normal individuals. the question was examined whether or not this effect was the result of direct action of nicotine on the coronarv arteries. or whether it %.•as mediated through central effects of the drug (rise in blood pressure, heart rate). Apparently nicotine has a direct effect on the coronarv arteries. since rapid. direct injection of nicotine into the coronarv arterv does not produce an immediate systemic effect. but results in increase in force of mvocardial contraction and in coronarv flow within four seconds of in- jection (76). This finding could be confirmed by others. using the adrenalectomized and decapitated cat. In these preparations nicotine also did not alter blood pressure and heart rate. but it significantlv increased coronary blood flow and coronary capil- lary red cell velocity (74). In related work (75). it could be shown that in the anesthetized dog intracoronary infusion of high doses of nicotine and acetvlcholine increased mv_ o- cardial contractile force: this could be prevented by pretreatment with desmethylimipramine or phen- oxybenzamine. The inotropic effect of nicotine was brief and subsided during the continuing infusion of the drug. It did not reduce the inotropic effects of cardiac sympathetic nerve stimulation. A considerable number of papers have been published dealing with the development of new methods of measurement of coronary flow in man or TIMN 0115807 T200298 63

frequency of nerve stimulation was 5 or 25 pulses/ sec. The neurogenic response of the artery was decreased 'slightlv by (+)-tubocurarine, potentiated ;lightly by mecamylamine and not altered by hexa- methonium. Prior addition of either of these cholin- ergic blocking agents or nicotine did not prevent the blocking effect of DMPP. The contractile response of the artery to exogenous norepinephrine was either increased slightly or unaltered by DMPP, depending on the concentration of the latter, whereas nicotine was without any effect. The data demonstrate that W,iPP and nicotine differ in their action on adrener- gic nerve-smooth muscle transmission. The results support the view that DMPP is an adrenergic neu- ronal blocking agent which does not act on classical nic:otinic receptors. The failure of nicotine to block adrenergic transmission in the pulmonary artery preparation contrasts with the blocking effect of this drug at other sites of sympathetic neuroeffector transmission. The absence of a blocking effect by either nicotine or mecamylamine cannot be ex- plained in terms of the "cholinergic link" theory of Burn and Rand. Other support: U. S. Public Health Service. 7. The effect of nicotine on the response of vascular smooth muscle to adrenergic nerve stimulation Ove A. Nedergaard, and John A. Bevan Department of Pharmacology, UCLA School of .\fedicine. Los Angeles, California Proceedings of the Western Pharmacology Society 10:, 4-7s. 1967 The isolated sympathetic nerve-pulmonary ar- trrti• preparation from rabbits was used. Nicotine 1 t0 " to 10-4%S) potentiated the contractile response Of the arterv to nerve stimulation. Addition of nicotine during the plateau of response to nor- epinephrine caused an additional rapid but transient increase in tension, the return of tension to control levels showing that nicotine does not alter the postsynaptic sensitivity to norepinephrine. It is concluded that the effect of nicotine can be con- sidered as being due primarily to an effect on the prejunctional adrenergic nerve terminals. Other support: U.S.P.H.S. 8. Blockade of the nicotine-induced norepinephrine release by cocaine, phenoxybenzamine and desipramine C. Su. and J. A. Bevan Department of Pharmacology, UCLA School of Medicine. Los Angeles. California Journal of Pharmacology and Experimental Therapeutics 175:533-540, 1970 The effect of some drugs on the svmpathomi- metic action of nicotine was studied with the su- perfused spiral strip of the rabbit pulmonary artery. Nicotine or transmural electrical stimulation evoked an increase in isometric tension and, in artery strips labeled with tritiated dl-norepinephrine (H'-NE). an overflow of H3-NE into the superfusate. Treatment of the rabbit with reserpine prior to isolation of the artery eliminated such contraction. Tetrodotoxin and bretylium, in concentrations which rapidly abolished the contraction and H3-NE overflow evoked by transmural stimulation, failed to affect the response to nicotine. Phenoxybenzamine, cocaine and desipramine selectively inhibited the nicotine- induced contraction and overflow. It was concluded that the sympathomimetic action of nicotine on the pulmonary artery was mediated by the release of endogenous NE; this release was not dependent on the initiation and propagation of action potentials in the axon. Since those agents which prevented the nicotine effect possess the common property of inhibiting the uptake of NE, the action of nicotine may require an intact NE uptake mechanism. 9. Transmural and subcellular localization of . monoamine oxidase and catechol-O-methyl transferase in rabbit aorta M. Anthony Verity. Che Su, and John A. Bevan Departments of Pathology and Pharmacology, and Brain Research Institute. U.C.L.A. Medical Center, Los Angeles, California Biochemical Pharmacology 21:193-201, 1972 A quantitative study of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) was made on homogenates of adult rabbit thoracic aorta. Most of the activity of both enzymes is located in the tunica media. The ratio of adventitia/media distribution of COMT is considerably less than the TIMN 0115823 = T200314 79

r Cardiovascular S%stem stimulation can be achieved with a conventional stimulator. and substances released from the tissue can be pbtained in relatively high concentrations. Using this technique, the effects of various drugs on the norepinephrine release and vasoconstriction are being investigated. Other support: U. S. Public Health Service. 23. Comparison of contracting agents on the isolated guinea pig aortic strip G. D. Maengwyn-Davies, T. M. Crisp, E. F. Daniel, and V . B. Thoa Departments of Pharmacology and Anatomy. Georgetown University Medical and Dental Schools, Washington. D.C. Archives Internationales de Pharmacodynamie et de Therapie 178:344-350. 1969 A comparison of contracting agents was made on isolated strips of descending thoracic aortae of the guinea pig cut spirally counter-clockwise start- ing just below the aortic arch. The guinea pig aortic strip, when compared to the rabbit aortic strip, was found to be about 10 times less sensitive to nor- epinephrine and 100 times less sensitive to 5- hydroxytryptamine while it was approximately equally sensitive to histamine and potassium chlo- ride. Nicotine failed to contract the guinea pig aortic strip. 24. Reactivity to biogenic amines of isolated aortic strips and electrically driven left atria from guinea-pigs after repeated treatment with nicotine G. D. Maengwyn-Davies, and N. B. Thoa Department of Pharmacology, Georgetown University Schools of Nfedicine and Dentistry, Washington. D.C. Archives Internationales de Pharmacodynamie et de Therapie 202:374-382. 1973 Nicotine (0.5 or 5.0 mg/kg) was administered to guinea-pigs subcutaneously once only (acute) or twice daily for up to 12 days (chronic). Dose- response curves were determined for potassium chloride, norepinephrine and histamine in isolated aortic strips and for norepinephrine and acetylcho- line in isolated electrically driven left atria. The responses of the aortic strips to potassium chloride remained unchanged whereas those to norepineph. rine and histamine were enhanced after one or two davs of nicotine administration (5.0 mg/kg). Atrial inotropic responses to norepinephrine were in- creased following acute nicotine (both doses) where- as 7 days of treatment (5.0 mg/kg) were necessarv to augment the negative inotropic effect of acetylcho- line. It is suggested that most of the acute and chronic nicotine effects were caused by release and by changes in the turnover of norepinephrine which occurred in the sympathetic nerve terminals of the aortae and of the atria. Nicotine's effects on the atrial response to , acetylcholine were attributed to an alteration by nicotine or by its metabolites in the allosteric configuration of the atrial muscarinic re- ceptors. Nicotine (5.0 mg/kg) produced tremors and convulsions to which the guinea-pigs became toler- ant after four days. This study was the subject of a preliminary report appearing in Federation Proceedings 28(2):286, 1969. 25. Bretylium potentiation of the contractor responses of isolated rabbit aortic strip to potassium and tyramine K. Kurahashi, and S. Shibata Department of Pharmacology. School of avfedicine. University of Hawaii, Honolulu, Hawaii British Journal of Pharmacology 43:210-221, 1971 Pretreatment of rabbit aortic strips with bre- tylium potentiated the contractor response to po- tassium and tyramine but not to noradrenaline. On the other hand, such pretreatment inhibited the response to nicotine. Pretreatment with the a-adre- noceptor blocking agent, phentolamine mesylate. also decreased the amplitude of the contractor re- sponse to nicotine, and strips obtained from re- serpine treated animals failed to show any contractor response to nicotine. Even in reserpinized or cold stored aortic strips, pretreatment with bretylium enhanced the contrac- tor response to potassium and tyramine. Pretreatment of fresh, reserpinized, or cold stored aortic strips with pheniprazine potentiated the contractor response to potassium and ty:a: :ine Pretreatment of aortic strips with bretylium or pheniprazine did not potentiate the response to 5- hydroxytryptamine. 84 TIlVIN 0115828 T200319

,ind the United States. in individuals disregarding t«•in relationships. alcohol drinking. lack of ex- ercise. frequent change of emplover, low occupa- tional adjustment and smoking are moderatPlv hut ,ignificantl}• related to angina. Howec•er. in mono- /,vgous hwins of the United States Registry. sig- nificantl}• different rates of angina appear onlv with alcohol drinking. Their findings suggest that alcohol clrinking. and to a lesser etitent. occupational ad- justment. are directly related to angina and not to their association with other factors such as age. oenetic background. smoking. physical exercise and tenvironment. In a study carried out in 1975 (201). life changes were further analyzed. The study in- dicates that fatal outcome in ischemic heart disease may to a large extent be the result of life changes. especially those connected with work situations. The same group (202) has further dwelled on the psychosocial status of smoking and genetic factors. in relation to angina pectoris. They feel that psv- rhosocial maladjustment was of great importance in the development of coronarv heart disease. The firmest associations were found with unspecific symptoms: that is. "nervous diseases." It is apparent. therefore. that there are many factors influencing the cause of coronarv heart disease. genetic disposition probably being one of the most important ones. The study also confirmed the opinion of many cardiolo- gists that environmental factors also may play an important role (203). As to be expected. blood pressure is important in the development of angina pectoris. It was found (204) that in studies on coal miners in the United Kingdom. as well as in the United States. of all the etiological factors examined. the most striking as- sociation was with blood pressure. Systolic blood pressure was roughly 20 mm and diastolic blood pressure 10 mm higher in the coronary group. as compared with the noncoronary group. Of some epidemiological interest is also the study by Goldbarg (205) showing that multi-stage treadmill exercise in healthy business executives showed a positive relationship with age. No cor- relation was found between abnormal exercise responses and coronary risk factors including cig- arette smoking. Finally. work is relevant here which determined the response of the coronary flow to nitroglycerine and used this as an epidemiolooical marker for the presence of coronary arterv disease (206). Apparetrtlv. in contrast to normal indiViduals. patients with coronarv artery disease failed to in- crease coronary flow following the ingestion ~~f nitroglycerine. H. PhYsiological Action of Nicotine Derivatives Manv studies of nicotine derivatives have been published in the Federation Proceedings (207. 208) and in the Journal of.tifedical Sciences (209). Studies have shown that the metabolism of nicotine in the dog results in urinary excretion of nicotine isome- thonium ion and cotinine methonium ion. The increase in vascular resistance produced by nicotine was primarily due to adrenal stimulation. Nicotine isomethonium iodide has a delayed effect on vascu- lar resistance. In contrast. cotinine methonium and cotinine itself are inactive. suggesting that metabo- lism of nicotine with breakdown to these two compounds assists in terminating vascular resis- tance produced by the parent alkaloid. Using the perfused partially isolated forelimb of the dog. 3-(2- methylaminoethvl) pyridine (3-2-MAEP) produced an increase in peripheral vascular resistance. This compound produced contractions of the isolated aortic strips. which were blocked by phentolamine. The effect is not mediated bv adrenergic receptors. since contraction of aortic strips occur also after depletion of norepinephrine by reserpine. Another breakdown product of nicotine. 3-prr- idylaceticacid (3-PAA) has marked pharmacological properties. including inhibition of lipolysis in iso- lated fat cells, inhibition of free fatty acid mo- bilization and hypocholesterolemic activit~•. This work is of particular importance, since it shows that some breakdown products of nicotine have marked pharmacological action, which may occur independent of the adrenergic activity of the parent alkaloid. The action of cis- and trans- isomers of metanicotine were observed on isolated rabbit aortic strips and ileal segments. Both of these preparations have nicotine-like action: however. the action of trans-metanicotine on the aortic strip is significantly less than that of nicotine (153). On the other hand, cis-metanicotine is less active than trans-metanicotine or nicotine itself. Additional studies have dealt with the effect of 4-pyridino compounds for both agonist and antagonist activity. TIMN 0115817 T200308 73

-W Cardiovascular S%•stem skeleton for FFA esterification in the hypoxic rat heart; endogenous glycogen served this_ function in ;the turtle mvocardium. Extraction of glucose by the turtle h1eart was limited and changed insignificantly during hypoxia. The rat heart increased glucose uptake five-fold during hypoxia. Glycogen stores in turtle heart were tenfold greater than they were in rat heart, and they provided greater metabolic flexibility and permitted the heart to function adequately under environmental conditions that are lethal to the mammal. Other support: U. S. Public Health Service, the New York Heart Association, and the Muscular Dystrophy Associations of America, Inc. 48. Differences in effects of cigar and cigarette smoking on free fatty acid mobilization and catecholamine excretion Alfred Kershbaum. Samuel Bellet, Jose Jimenez, and Leonard J. Feinberg Division of Cardiology, Philadelphia General Hospital. Philadelphia. Pa. fournal of the American kfedical Association 195:1095-1098. 1966 A study was made in normal male_subjects of the comparative effect of cigar and cigarette smoking on the mobilization of free fatty acids (FFA) and the excretion of catecholamines. There was a greater increase in FFA concentration in the serum with cigarette than with cigar smoking. Inhaling during smoking caused a greater FFA response than not inhaling, with both cigars and cigarettes. With tobacco containing glucose randomly labeled with radioactive carbon ('1C), there was a greater ab- sorption of "C with cigarette smoking. Urinary catecholamine excretion increased during smoking, significantly more with cigarettes than with cigars. The differences observed with the two forms of smoking probably result from the tendency to inhale with cigarettes and not with cigar smoking and from the effect of this on nicotine absorption. Other support: National Institutes of Health, and the Council for Tobacco Research-USA. 94 49. Effect of nicotine on the mobilization of free fatty acids from adipose tissue in vitro A. Kershbaum. H. Osada. D. J. Pappajohn. and S. Bellet Division of Cardiology. Philadelphia General Hospital. Philadelphia. Pa. Experientia 25:128. 1969 Release of free fatty acids (FFA) during in- cubation of epididymal fat pads of rats in Krebs- Ringer bicarbonate buffer containing 4% bovine albumin was studied. Addition of nicotine (1.0-8.0 µg/5 ml) did not significantly affect lipolysis com- pared to the control; with epinephrine (7.28 and 14.56 µg/5 ml), there was an increase in release of FFA of 66% and 89%. When nicotine (0.2 mg/kg, intraperitoneally) or epinephrine (0.2 mg/kg, in- traperitoneally) were administered 15 minutes be- fore sacrifice, subsequent incubation of the animals fat pads resulted in an increased FFA release of 45% and 54%, respectively, compared to saline control rats. It is concluded that nicotine has no direct lipolytic activity on rat adipose tissue and that the findings lend support to the view that mobilization of FFA by nicotine and tobacco smoke is a result of sympatho-adrenal stimulation. Other support: National Institutes of Health, and the Council for Tobacco Research-USA. 50. Some aspects of the effect of nicotine on plasma FFA and tissue triglycerides A. Bizzi, M. T. Tacconi, A. Media. and S. Garattini 'll4ario Negri' Institute of Pharmacological Research, 1?ilan, Italy Pharmacology 7:216-224, 1972 Nicotine given subcutaneously to rats raised the levels of plasma free fatty acids (FFA) and blood glucose, the content of FFA and glycerol in adipose tissue also being increased. The basal lipolysis in vitro of adipose tissue obtained from nicotine- treated rats was increased, while the lipolytic effects of norepinephrine or theophylline remained un- changed or were slightly diminisned. The rise in plasma FFA due to nicotine adminstration was inhibited by the administration of 5-carboxy-3-me- thyl-pyrazole, an antilipolytic agent, or of a R- adrenergic blocker, proPranolol. TIMN 0115838 T200329 -~

Cardiovascular System which have functional correlates are described. Since manv drugs act on blood vessels by modifying transmission between nerve and muscle cells, an undetstanding of this process should precede a study of their pharmacology. The slow response of the elastic vessel to drugs may be due to diffusion barriers between the surrounding medium and the receptor. The characteristics of the a-adrenergic receptor are not identical in different vessels; even greater variation is found when other receptors are studied. Other support: U. S. Public Health Service. 16. Distribution theory of resistance of neurogenic vasoconstriction to alpha-receptor blockade in the rabbit John A. Bevan, and Che Su Department of Pharmacology, U.C.L.A. School of !lledicine. Center for the Health Sciences. Los Angeles, California Circulation Research 28:179-187, 1971 The effects of a-receptor blocking agents on the contractile responses of isolated rabbit arteries to sympathetic nerve stimulation and exogenous I- norepinephrine (1-NE) were compared. In the pul- monary artery and aorta, yohimbine, phentolamine, and phenoxybenzamine blocked the response to nerve stimulation less than that to an equipotent dose of 1-NE. This resistance of neurogenic response was independent of the frequency and number of stimuli and persisted after inhibition of the nerve 1- NE uptake by cocaine. The neurogenically released transmitter 1-NE probably forms a high concentra- tion near the adventitia-media junction, whereas the exogenous NE is distributed evenly throughout the thickness of media. Thus higher concentrations of a- receptor-blocking agents would be needed to block the effect of neurogenic I-NE than to block that of exogenous 1-NE. This explanation of the resistance was thought to be more appropriate to the large vessels tested than that based on neuroeffector proximity. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 82 T 17. Biphasic constrictor response of the rabbit ear artery John A. Bevan, and John G. Waterson Department of Pharmacology. U.C.L.A. School of Rfedicine. Los Ange/es, California Circulation Research 28:655-661, 1971 The perfused rabbit ear artery shows a biphasic contractile response to intraluminal norepinephrine and sympathetic nerve stimulation. The peak of the first phase occurs after approximately 10 seconds of exposure to norepinephrine, and the second, after 1'/Z to 2 minutes. The magnitudes of the two responses are similar. The time course of the second phase of contraction is similar to the rate of satura• tion of the vessel's extracellular space as measured by tissue uptake of tritiated norepinephrine. On the other hand, the first phase of contraction occurs when norepinephrine has penetrated only partly into the media. It is proposed that the two phases of contraction are due to different mechanisms of excitation and that the first phase is associated with excitation of the surface layer of smooth muscle cells with subsequent myogenic propagation of excitation through the thickness of the vessel wall. The second may be related to the local concentration of nor- epinephrine in the extracellular space of the ves- sel. Other support: U. S. Public Health Service. 18. Distribution of inulin space in the rabbit thoracic aorta J. Torok, O. A. Nedergaard, and J. A. Bevan Department of Pharmacology, U.C.L.A. School of Medicine, Los Angeles. California Experientia 27:55-56, 1971 When a drug is added to the oxygenated physi- ological saline bathing an isolated organ in a tissue bath, it will mix rapidly with the saline and then more slowly penetrate the tissue or organ. If the rate of penetration of the drug is to be related to its pharmacological effect, the dimensions of the ex- tracellular space in the tissue must be known. In this paper, the dimensions of the inulin (extracellular) space in the rabbit thoracic aorta are described. The inulin uptake was found to be uniform along the whole length of the aorta, from its cardiac to abdominal end. The uptake was not evenly V TIMN 0115826 T200317 J.

d r- s, d d )r i- The results indicate that both bretc•lium and pheniprazine potentiate the action of h•ramine and potassium.t not by presynaptic mechanisms. but by posts}'naptic action. causing an increase in the sensitivitv of the effector cells to the stimulants. Other support: U. S. Public Health Service. 26. Adrenergic. innervation and cocaine-induced potentiation of adrenergic responses to aortic strips from young and old rabbits Shoii Shibata, K. Hattori. I. Sakurai. J. %fori. and \t. Fuliwara Depnrtment of Pharmacologv and Pathologjv. School of 11a°dicine. C'niversitt' of Hawaii. Honolulu. Hawaii fnurnal of Pharmacologl• and Experimental Therapeutics 177:631-632. 1971 \1'ith histochemical techniques, a catechol- amine-specific fluorescence was observed in the tunica media of the aortae of young rabbits (1.2-1.3 kg) but not of old rabbits (> 3.5 kg). The nor- epinephrine content of the tissues was investigated b}- a spectrophotofluorometric method, and the aor- tae of young rabbits were found to contain twice as much norepinephrine as those of old rabbits. The sensitivity of the young rabbit aortae to nicotine and tyramine was significantly greater than that of old rabbit aortae. Despite these differences, norepineph- rine-induced contractions were potentiated by co- raine (10-6-10-' M) in strips of both types. After five davs of cold storage at 2°C or reserpine treatment (4 mg/kg, 24 hours prior to sacrifice), these specific fluorescent substances disappeared from all layers of the tissue: such treatment was found to decrease the tissue norepinephrine content in both young and old rdbbit aortae. Cocaine-induced potentiation could still be demonstrated after five days of cold storage. After incubation for 20 minutes in a Ca"-free medium, and treatment with Mn-`, and Co-' (both i m.\i). no cocaine potentiation of norepinephrine- induced contraction could be observed. Cocaine potentiated the Ca"-induced contraction in a Ca'-- free medium with a high potassium content (30 mL1). Cocaine (10-6 M), but not pyrogallol (10-6 ~1j. could potentiate the norepinephrine-induced contraction of fresh strips but not that of cold storage strips. These results suggest that adrenergic nerve fibers penetrate into the smooth muscle layer of the rabbit aortae, but that the cocaine-induced potentia- tion may not be dependent on these adrenergic nerve terminals in the aortic media. External Ca-- ma}• possibly play a significant role in the development of cocaine-induced potentiation. Other support: U. S. Public Health Service: Hawaii Heart Association: Council for Tobacco Research- LT. S. A. 27. Pharmacologic nature of adrenergic innervation in rat aorta M. Kuchii. S. Shibata, and J. Ntori Department of Pharmacology. School of Medicine. University of Hawaii. Honolulu. Hawaii Comparative and General Pharmacologt• 4:131-138. 1973 No evidence for specific catecholamine fluores- cence was found in any tissue layer of rat thoracic aorta, even after combined treatment with iproniazid and L-DOPA. In contrast to the atria and abdominal aorta, reserpine (4 mg/kg) or 6-hydroxydopamine (50 mg/kg) did not affect the catecholamine content of the thoracic aorta. Cocaine (10-1M) or desipramine (10-5N1) exerted a similar differential inhibition on ['H] noradrena- line uptake by aortic tissues. After pretreatment with cocaine (10-51M) or 6- hvdroxydopamine (50 mg/kg) the dose-response curve for noradrenaline was not altered in the rat thoracic aorta. Moreover, this preparation was not highly reactive to nicotine (10-IM), tyramine (10-4M), or transmural stimulation. These results suggest poor adrenergic innerva- tion of rat thoracic aorta. 28. The neuromuscular mechanism of veins B. L. Pegram, J. A. Bevan, C. Su, and B. Ljung Department of Pharmacology, University of California. Los Angeles, California, and Department of Physiology. FACK S413 19 Goteborg, Sweden Proceedings of the Western Pharmacology Society 16:49-52, 1973 A survey of a variety of veins was carried out to gain some insight into the adrenergic mechanisms and the extent to which these vary. This survev TIMN 0115829 T20p320 85

Cardiovascular Svstem indicates that venous smooth muscle is not a ho- mogenous sc•stem. Hopefully, additional studies should disclose a pattern of variation in adrenergic mechanism throughout the venous system which is subservient to the different regional, anatomical and functional requirements of the circulation. Other support: U. S. Public Health Service, and the Los Angeles County Heart Association. 29. An unusual venoconstriction induced by acetylcholine A. J. Rice. and J. P. Long Department of Pharmacology. College of Medicine. University of Iowa. Iowa City. Iowa Journal of Pharmacology and Experimental Therapeutics 151:423-429. 1966 Experiments were designed to study the effect of acetvlcholine on the accessory cephalic vein of the dog. Injections of high doses of acetylcholine di- rectly into the vein perfused in situ produced a marked rise in perfusion pressure. Additional find- ings indicated that the response may be mediated through a release of catecholamines from extraneu- ronal stores. In this preparation, nicotine bitartrate had no effect on perfusion pressure in doses up to 1 mg. In other experiments using the vein perfused with Krebs' bicarbonate solution in an organ-bath, nico- tine bitartrate (500 µg) produced a transitory pres- sure rise of about 40 mm Hg. Other support: Council for Tobacco Research- U.S.A. 30. Localization of venoconstrictor responses A. J. Rice. C. R. Leeson. and J. P. Long Departments of Pharmacology and Anatomy, College of Medicine. L'nirersity of Iowa. Iowa City, Iowa Journal of Pharmacology and Experimental Therapeutics 154:539-545. 1966 Using a system for perfusion in situ, the responses of four veins in the dog, the accessory cephalic, lateral saphenous, external iliac and the splenic, to several vasoconstrictor stimuli were ob- served. The agents used were acetylcholine, nor- epinephrine and sympathetic nerve stimulation. 86 Only in two veins, the accessory cephalic and lateral saphenous, could c•asoconstriction be induced bt• these treatments. Nicotine did not produce venocon- striction in any of the preparations: however, it was effective in producing constrictions in isolated ve. nous segments. It was observed that the responses were of a localized nature. Histologic examination of these regions showed an area that contained valves and was richly invested with smooth muscle. Valves were present also in nonreactive veins; however, these veins lacked any appreciable amount of smooth muscle fibers in the tunica media. It would appear, then, that venoconstriction in these veins involves localized areas containing valves and rich in smooth muscle. Other support: Council for Tobacco Research- U.S.A. 31. The supersensitivity of isolated rabbit atria and aortic strips produced by 6-hydroxydopamine S. Shibata, M. Kuchii, and K. Kurahashi Department of Pharmacology. School of Medicine. University of Hawaii. Honolulu. Hawaii European Journal of Pharmacology 18:271-280, 1972 The effect of chemical sympathectomy with 6- hydroxydopamine (6-OHDA) on the sensitivity of isolated rabbit atria and aortic strips to several catecholamines and other nonspecific stimuli was examined. After 6-OHDA treatment, the inotropic and chronotropic responses of the isolated atria to nor- adrenaline and adrenaline were potentiated, the potentiation for noradrenaline being greater than that for adrenaline. 6-OHDA failed to potentiate the phenylephrine and isoprenaline responses of atria. On atria isolated from control rabbits, nicotine elicited a two-phase phenomenon consisting of a small negative inotropic effect followed by a large positive inotropic effect. In treated atria, nicotine failed to exert a positive inotropic effect, but the negative inotropic response was still demonstrable. The negative inotropic action was inhibited after treatment with atropine. The response to nicotine abolished by 6-OHDA treatment was not recovered even after a 90 min incubation in medium contain- ing noradrenaline. However, after similar incuba- tion, reserpine treated preparations exhibited an apparent recovery of the nicotine response. TIMN 0115830 T200321

Cardiovascular System of glvcine-2-C" only if protein synthesis was stimu- lated bv means of aortic stenosis. Actinomvcin D had no effect on the normal myocardium. Animals on protein-free diet also showed a decreased rate of glycine-2-C'a uptake into heart muscle protein after production of aortic stenosis, but an increased in- corporation into skeletal muscle protein. Inhibition of protein synthesis during the first state of cardiac hypertrophy was associated with the development of heart failure, regardless of whether the inhibition occurred as a result of actinomycin D, or puromycin or of protein-free diet. As compared to animals maintained on a regular diet, myocardial protein turnover rate in unoperated animals was not altered significantly by protein-free diet. Other support: U.S. Public Health Service, the Mich- igan Heart Association. Medical Research Fund, the Burroughs-Wellcome Fund, and the John A. Hart- ford Foundation. 71. Metabolism of the heart in failure John C. Fenton, Sigmundur Gudbjarnason, and Richard J. Bing Department of Medicine. Wayne State University School of Medicine, and the Harper Hospital, Detroit, Michigan American Heart Association Monograph No. 1. Second Edition, 1966, pp 32-48 A review of -current knowledge on the me- tabolism of the heart in failure, based on 120 literature references, is presented. Conclusions reached are as follows: It is apparent from this review that the metabolic mechanisms that underlie heart failure are not completely understood. In myocardial failure caused by prolonged mechanical overload or by intrinsic muscle disease, a defect in energy production may be present. Inefficient transduction of chemical en- ergy into tension and mechanical work is probably more important, and may be due to abnormality of the events that link excitation to contraction. After failure has developed, depletion of catecholamines and possibly relative hypoxia appear to follow, thus aggravating the primary problem. An inadequate supply of energy is believed to underlie the heart failure that occurs in anemia, hemorrhagic shock, thiamine deficiency, and pos- sibly hyperthyroidism Other support: U.S. Public Health Service, the %1ich- igan Heart Association, Life Insurance Medical Re- search Fund, the Council for Tobacco Research- U.S.A., the Burroughs-Wellcome Fund, and the John A. Hartford Foundation. 72. Accumulation of norepinephrine in rat tissue following treatment with three beta-adrenergic antagonists T. C. Westfall Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia Archives Internationales de Pharmacodynamie et de Therapie 167:69-79, 1967 The beta-adrenergic antagonist, pronethalol, was found to produce a significant inhibition of the accumulation or uptake of norepinephrine (NE) into the heart following NE infusion in the anesthetized rat. This same agent produced a signficant decrease in the endogenous levels of this amine following treatment once per day for 7 days. These two observations were seen only at doses larger than necessary to block the beta-adrenergic receptor and are probably unrelated to the blocking properties of the drug. Propranolol failed to alter the uptake of infused NE as well as failing to produce any alteration of the endogenous amine level. Iproveratril produced what appeared to be a slight but statistically insignificant inhibition of uptake of NE but did produce a significant decrease in the amine content of the rat heart after ad- ministration daily for 7 days. Other support: Council for Tobacco Research- U.S.A. 73. Action of a beta adrenergic receptor blocking agent on the positive chronotropic response and uptake of norepinephrine in the perfused guinea- pig heart Thomas C. Westfall Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia Journal of Pharmacology and Experimental Therapeutics 162:239-245. 1968 The influence of varying concentration of the beta adrenergic receptor blocking agent, MJ 1998 [4- TIMN 0115846 ' 102 T200337

, i t center of infarcted tissue 10 days after infarction with 8-13°'0 of control activities. Glycolytic enzymes showed a significant fall in activities with the sreatest diminution in activity of aldolase to 14% of control but a relatively smaller fall in glyceralde- hcde phosphate dehydrogenase activity to 32% of control. The increased activity of the HMP-shunt in infarcted tissue was also expressed in the relative oxidation of glucose-l-C" and glucose-6-C'{ to ''CO, Other support: U.S. Public Health Sen•ice, the Mich- igan Heart Association, and the Detroit General Hospital Research Corporation. 67. Reparative processes in heart muscle following myocardial infarction R. J. Bing Huntington Memorial Hospital, Pasadena, California, and the University of Southern California. Los Angeles, California. Cardiology 56:314-324, 1971/72 When for reasons of deficient blood supply or because of a discrepancy between supply and de- mand of oxygen, the heart muscle undergoes isch- emic changes, the injured area becomes a wound. The reparative processes taking place are those connected with wound healing. Wound healing may vary with the specific tissue in which it takes place. But in principle the reparative processes of wound healing are identical. The ensuing discussion at- tempts a correlation between the morphological and biochemical changes following myocardial infarc- t io n. Other support: Council for Tobacco Research- l".S.A.. and the Hoover Foundation, Los Angeles, Californa. 68. Myosin synthesis in chronic heart failure C. De Schn-ver. and S. Gudbjarnason Department of lfedicine, Wayne State Universit_v School u(.~ledicine and Harper Hospital, Detroit, Michigan Proceedings of the Society for Experimental Biology and Medicine 119:961-963, 1965 Left heart failure was produced in rabbits by constriction of the ascending aorta. Incorporation of DL-cystine-S15 into cardiac myosin. water soluble enzymes, plasma protein and skeletal muscle pro- tein was studied. There were no significant dif- ferences between the rate of synthesis of left ven- tricular myosin and the other protein fractions obtained under conditions of heart failure, as com- pared to the same protein fractions in the normal animal. Other support: U.S. Public Health Service, the Mich- igan Heart Association, the Council for Tobacco Research-U.S.A., the Burroughs-Wellcome Fund, the American Cancer Society, and the John A. Hartford Foundation. 69. The Metabolism of the heart in failure S. Gudbjarnason, and R. J. Bing Department of Medicine. Wayne State University School of Medicine and Harper Hospital, Detroit, Michigan Acta Cardiologica 20:371-380. 1965 Studies on the metabolism of the heart in failure are reviewed and implications are summarized as follows: Heart failure, which can be classified as de- generative, is enzymatically characterized by spe- cific enzyme lesions. A myocardial infarct, on the other hand, is a destructive lesion, a wound, which calls into play the cellular reparative mechanisms and results in increased synthesis of protein. The reparative process commences in the nuclear frac- tion of the cell. Other support: U.S. Public Health Service, the Mich- igan Heart Association, Life Insurance Medical Re- search Fund, the Council for Tobacco Research- U.S.A., and the John A. Hartford Foundation. 70. Inhibition of protein synthesis in cardiac hypertrophy and its relation to myocardial failure Volker Zuhlke, Wolfgang du Mesnil de Rochemont, Sigmundur Gudbjarnason, and Richard J. Bing Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan Circulation Research 18:558-572, 1966 The rate of myocardial protein synthesis was studied in hearts of rabbits with experimentally produced cardiac hypertrophy and treatment with actir.omycin D and gurorr.ycin as well as in animals maintained on a protein-free diet. Actinomycin D and puromycin inhibited myocardial incorporation TIMN 0115845 T200336 101

Cardiovascular Svstem infarcted heart at various levels of energy rich phosphates: and (b) in acutelv ischemic heart mus- cle. Mvocardial ischemia and infarction was pro- duced in dogs by ligation of coronary arteries. Biopsy samples were analyzed for ATP. ADP, CP, lactate and pyruvate at various intervals after coro- nary artery ligations. The relationship between the tissue content of ATP and CP in acutelv ischemic muscle differs markedly from that in non-infarcted muscle. In ischemic muscle there is a positive correlation between the rate of CP breakdown (-dCP/ dt) and the rate of lactate accumulation (+dLa/dt) during the first 30 sec of ischemia, whereas the rate of ATP depletion diminishes with the increase in the rate of lactate accumulation. The rates of glycolysis and high energy phosphate breakdown decreases markedly 30 sec after coronary artery occlusion. The ischemic muscle stops contracting at a relatively high ATP level of 4.5 µmole/g, when only about 20% of the ATP has been utilized, whereas the non- ischemic muscle survives and maintains contraction at ATP levels as low as 1.5 and 2.0 µmole/g. The rapid increase in anaerobic glycolysis coincides with an early inhibition of utilization of ATP stores without an interference with the utilization of CP stores. The kinetic heterogeneity of the ATP and CP depletion suggest an inhibition of transfer of in- tramitochondrial high energy phosphate to the ex- tra-mitochondrial compartment. The early cessation of contractile activity in ischemic myocardial cells appears to be the result of a reduction in the rate of regeneration of a small pool of extra-mitochondrial ATP available for muscle contraction and other ATP requiring processes such as ion transport. Other support: U.S. Public Health Service, and the Michigan Heart Association. 65. LDH-Isoenzymes in infarcted heart muscle S. Gudbjarnason, and D. M. Priver Department of Medicine, Wayne State University School of Medicine. Detroit, Michigan Life Sciences 7(Part II):623-627, 1968 The purpose of this study was to examine the effect of coronary artery occlusion and myocardial infarction on the lactic dehydrogenase (LDH)-iso- zyrne pattern of the ischemic heart muscle. N1%10. cardial infarction was produced in 20 dogs bc ligation of several branches of the anterior descend- ing and circumflex coronary arteries. Following myocardial infarction there was a significant change in the number of LDH-isoz}•mes in the ischemic tissue. In normal dog heart muscle LDH displays only three of its five isozyme molecu- lar forms. In the infarcted heart muscle all five isozymes can be observed. The quantitative changes in LDH of infarcted muscle heart muscle were accompanied by quantita- tive changes in LDH activity, in the ischemic area. The activity of LDH declined following coronary occlusion and diminished to 21% of normal levels ten days after infarction. The changes in the LDH- isozyme pattern were observed two days after in- farction and persisted during the period of obsen•a- tion (10 weeks), excluding leucocytes or other infiltrating cells as solely responsible for the altera- tions in the isozyme pattern. Possible interpretations of these findings are presented. Othersupport: U. S. PuF)1ic Health Service. the Mich- igan Heart Association. the Council for Tobacco Research-U.S.A., and the Detroit Hospital Research Corporation. 66. Changes in enzyme pattern of infarcted heart muscle during tissue repair S. Gudbjarnason, Ch. Cowan, W. Braasch, and R. J. Bing Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan Cardiologia 51:148-159, 1967 The activities of heart muscle enzymes were assayed during tissue repair following experimental myocardial infarction in the dog. The activities of 10 enzymes were determined in normal heart muscle and in the center and periphery of infarcted muscle. A significant increase in hexosemono phosphate (HMP)-shunt activity was observed in infarcted tis- sue 5 hours after infarction. The activities of glucose- 6-phosphate dehydrogenase and 6-phosphogluco- nate dehydrogenase increased 31-34 fold 10 days after infarction. In contrast to increased activity of the HMP-shunt, there was a rapid decline in oxi- dative and glycolytic enzyme activities in infarcted tissue. Oxidative enzymes reached the lowest in the TIMN 0115844 100 T200335

s , 1 i i Nicotine failed to raise plasma FFA in previ- ouslv adrenalectomized rats. Howe~•er. this effect ~ras restored by the administration of corticosterone, suggesting that other factors. in addition to epi- nephrine discharged from the adrenal medulla, are involved in the stimulation of the lipolytic activity of adipose tissue. No change of triglycerides, cholesterol and phospholipids concentrations were observed in liver. heart and plasma after single or repeated (9 times/day for 4 days) treatment with nicotine. 51. Effects of chronic nicotine, acute hypoxia, and their interactions on myocardial enzymes Duane G. Wenzel, and Mary H. Richards Department of Pharmacology and Toxicology. School of Pharmacy. University of Kansas. Lawrence, Kansas Toxicolo;r and Applied Pharmacology 16:656-667, 19: 0 %tale rats were treated with either 1.14 or 4.56 mg/kg/dav of nicotine alkaloid in the drinking water for 34 weeks. Half of each group and an equal number of untreated controls were then exposed to 6°o oxygen at 23°C for 12 hours. Immediately after the hvpoxia, and at intervals thereafter, hematocrits were measured, the hearts were examined for lesions and assayed for glucose-6-phosphate dehydrogenase (G-6-PDH), isocitric dehydrogenase linked to nico- tinamide adenine dinucleotide phosphate (ICDH- \ADP), and lactic dehydrogenase (LDH) isoenzyme activities in the soluble fraction of heart homoge- nate: and for acid phosphatase (AP) and (3-glucuroni- dase ((3-G) in the soluble and particulate fractions. No effects were observed on the hematocrits. Treat- ment with the larger dose of nicotine increased ICDH-NADP and particulate AP activities, and de- creased soluble (3-G activity. Hypoxia elevated ac- tivities of G-6-PDH, LDH,, particulate AP, and both soluble and particulate (3-G. Mortality during hyp- o\ia was increased by pretreatment with the larger dose of nicotine. The regression of hypoxic heart lesions became highly variable following nicotine pretreatment. Pretreatment with nicotine also re- duced the effect of hypoxia on R-G activity and altered some effects of hypoxia on LDH isoenzt•mes. The possible significance of these interactions is discussed, but whether a cause-effect relationship exists between the biochemical changes and those of mortality or myocardial lesions remains to be de- termined. This study was the subject of a preliminary report appearing in this Journal 14:651-652, 1969. 52. Increase in hexosemonophosphate shunt activity during tissue repair Sigmundur Gudbjarnason, Charles Cowan, and Richard J. Bing Department of Medicine, Wayne State Universih• School of Medicine. Detroit, Michigan Life Sciences 6:1093-1097, 1967 Myocardial infarction was produced in 45 mon- grel dogs by ligation of several branches of the anterior and posterior descending coronary arteries. The animals were sacrificed at various intervals after infarction, from 5 hours up to 10 weeks. Two samples were obtained from the left ventricle, a) center of infarcted tissue, representing 1/3 of the infarcted area, and b) periphery of infarcted tissue consisting of z/3 of the infarcted area. The control group consisted of 12 dogs without infarction. The activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were as- saved in control and infarcted tissue samples using standard test media. The findings showed a sig- nificant increase in hexosemono phosphate shunt activity during the reparative processes following myocardial infarction. Other support: U. S. Public Health Service, the Michigan Heart Association, and the Detroit General Hospital Research Corporation. 53. Changes in the activities of lysosomal enzymes in infarcted canine heart muscle Kurt G. Ravens, and S. Gudbjarnason Department of Medicine. Wayne State University School of Medicine. Detroit, Michigan Circulation Research 24:851-856, 1969 Experimental myocardial infarction was pro- duced in 32 mongrel dogs. The changes in activity of TIMN 0115839 T200330 95

t I I i i 1 after infarction by 173°o. Repeated infusion led to a further rise in incorporation 24 hours after infarc- tion. Treatment with ascorbic acid increased protein ;cnthesis in the center of infarcted area by 122",, 24 hours after infarctin and treatment with the anabolic steroid methandrostenolone augmented incorpora- tion into center of infarct by 249%. Long-term treatment with insulin or the ana- bolic steroid prevented development of aneurysm, whereas animals fed a protein-free diet had a sig- nificantly thinner scar and frequent development of aneurvsm. It should be reemphasized that the bio- chemical demonstration of protein synthesis in the infarcted myocardium does not imply synthesis of protein by necrotic heart muscle. Other support: U.S. Public Health Service, the John . A. Hartford Foundation, the Michigan Heart As- sociation. and the Council for Tobacco Research- [.'.S.A. 56. Acute alterations in energetics of ischemic heart muscle S. Gudb{arnason Science Institute. University of Iceland, Reykjavik, Iceland Cardiology 56:232-244. 1971/72 This is a somewhat amplified account of the studv described in the preceding abstract. 57. The use of glycolytic metabolism in the assessment of hypoxia in human hearts S. Gudbjarnason Science Institute. University of Iceland, Reykjavik, Iceland Cnrdiology 57:35-46. 1972 The recognition of myocardial hypoxia and ischemia in the intact heart has usually been related to changes in myocardial lactate metabolism and development of anaerobic metabolism. Three criteria have often been used to detect the presence of anaerobic metabolism: (1) diminution in myocardial lactate extraction. (2) release of lactate into the coro- nary sinus blood, and (3) increase in the lactate/ pyruvate ratio of blood perfusing the heart muscle. An attempt is made in this paper to illustrate that none of these criteria seem to be adequate to be used as a sole diagnostic tool to demonstrate the presence of myocardial hypoxia. 58. Metabolic changes in infarcted and non- infarcted myocardium during the postinfarction period S. Gudbjarnason, K. G. Ravens, and P. Mathes Departments of Medicine and Biochemistry. Wayne State University. School of Medicine. Detroit. Xlichiganh and Science Institute. University of Iceland. Reykjai'ik. Iceland Myocardiology 1:439-446. 1972. Volume 1 of the series entitled Recent Advances in Studies on Cardiac Structure and Nfetabolism. edited by E. Bajusz and G. Rona. University Park Press, Baltimore Following acute myocardial infarction in dogs, there is a significant, reversible diminution in tissue levels of ATP, creatine phosphate, and norepineph- rine in noninfarcted heart muscle. The diminution in ATP and CP levels of the noninfarcted muscle is accompanied by a significant impairment in mvo- cardial function as reflected in the decrease in rate of pressure rise (dp/dt) or stroke work. Treatment with anabolic steroids significantly increases the ATP level of noninfarcted muscle, whereas a protein-free diet results in a significant diminution in myo- cardial ATP level and increased mortalitv. Scar formation in cardiac muscle is markedly reduced in animals treated with the anabolic steroid, and col- lagen formation is reduced by 26%. These results suggest that diet and anabolic hormones may play an important role during tissue repair and muscle recovery, following acute myocardial infarction. Other support: U. S. Public Health Service, and the Michigan Heart Association. 59. Biochemical changes in non-infarcted heart muscle following myocardial infarction Sigmundur Gudbjarnason, Pritpal S. Puri, and Peter Mathes Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan, and Science Institute, University of Iceland, Reykjavik, Iceland Journal of Molecular and Cellular Cardiology 2:253-376, 1971 ' Changes in catecholamine and energy metabo- lism in the non-infarcted portion of the ventricle TIMN 0115841 T200332 97

I f i In aortic strips. 6-OHDA treatment potentiated the response not only to a specific vascular stimulant lnoradrenaline), but also to nonspecific stimuli tphenylephrine, potassium and barium). The con- tractile response to nicotine and tryamine was nearly abolished. The supersensitivity for noradrenaline was several times greater than for the nonspecific stirnuli. These results indicated that chemical svmpa- thectomt• by pretreatment with 6-OHDA produced the presynaptic type of supersensitivitv for isolated atria and seemed to induce not only presynaptic but also postsynaptic supersensitivitv for aortic strips. The inhibition of the positive atrial inotropic action of nicotine and tvTamine by 6-OHDA may be an indication of the depletion of tissue noradrenaline content and even the deterioration of the adrenergic nerve structure. Other support: U. S. Public Health Service, and the Hawaii Heart Association. 32. Nicotine monomethiodide and vascular adrenergic transmission C. Su. and J. A. Bevan Department of Pharmacologv. UCLA School of 1ledicine. Los Angeles. California Federation Proceedings 32(3):805, 1973 Our previous studies have demonstrated dual ,%-mpathomimetic actions of nicotine on isolated hlood vessels: release of adrenergic transmitter and f.icititation of adrenergic transmission (JPharmaco! Exp Ther 175:533. 1970). Nicotine monomethiodide (N%f%l) did not release the transmitter but blocked this action of nicotine in the isolated rabbit pulmo- nary artery. Like nicotine however, NMM poten- tiated the nerve stimulation-induced vasoconstric- tion reversibly and with little tachyphylaxis. NMM increased the contractile response to exogenous 1- norepinephrine and inhibited the neuronaJ 'H-1- norepinephrine uptake by an amount insufficient to account for the facilitation. The monoamine oxidase and catechol-O-methyltransferase activities in the vessel wall were not significantly affected. The facilitatory action of NMM is probably mediated primarily by an increase in the nerve impulse- mediated transmitter release. 33. Sympathetic mechanisms in blood vessels: Nerve and muscle relationships John A. Bevan. and Che Su Department of Pharmacology. UCLA School of Medicine. Los Angeles. California Annual Reriecr of Pharmacoloogr 13:259-285. 1973 The relationship between sympathetic nerve and muscle in blood vessels was reviewed. With additional information. a more complete description of blood vessels in terms of small number of com- mon but variant components ma}' be possible. Given a comparatively few data of primarily morphological nature, it may be possible to deduce the characteris- tics of the neurogenic response of even diverse vessels. Other support: U. S. Public Health Service. and the Los Angeles Heart Association. 34. Pressor effects of four aliphatic aldehydes and their interactions with 14C-norepinephrine in an isolated smooth muscle preparation J. L. Egle, Jr. Nfedical College of Virginia. Virginia Commonu•ealth University. Richmond. Virginia Federation Proceedings 32(3):795, 1973 The aldehydes studied (formaldehyde, acetalde- hyde, propionaldehyde and acrolein) are all present in low concentrations in the vapor phase of cigarette smoke. Each of these compounds has been shown previously to possess indirect sympathomimetic activity. Low intravenous doses of the four alde- hydes produced a dose-related rise in blood pressure in the rat. The order of potency was acrolein > formaldehyde > acetaldehyde > propionaldehyde. These responses were reduced by adrenalectomy and strongly antagonized by pretreatment with re- serpine or phentolamine. This indicates that the pressor effects of these compounds are primarily due to vasoconstriction mediated by norepinephrine (NE) released from sympathetic nerve endings. The effects of these aldehydes on t4C-NE accumulation and loss were compared in isolated rat vas deferens. 14C-NE efflux was increased by all four aldehydes at a concentration of 10-3M. The order of potency was acrolein > propionaldehyde > formaldehyde > acet- aldehyde. These findings confirm the observation TIMN 0115831 T200322 87

1W Cardiovascular System that the major action of these aldehvdes is via the release of NE from adrenergic nerve endings. How- ever, in addition to this action. these compounds appear to have the ability to inhibit NE accumulation by the nerve ending. This latter effect may play a significant role in their sympathomimetic activity. 35. Dose-dependent sympathomimetic and cardioinhibitory effects of acrolein and formaldehyde in the anesthetized rat John L. Egle. Jr., and Patricia M. Hudgins Department of Pharmacology. Medical College of Virginia. Hea1th Sciences Division. Virginia Commonwealth University, Richmond. Virginia Toxicology and Applied Pharmacology 28:358-366. 1974 Dose-effect relationships for i.v. formaldehyde and acrolein indicate that the cardiovascular actions of these aldehydes are qualitatively similar to acetal- dehyde and propionaldehyde. Pressor responses appear to result from the release of catecholamines from sympathetic nerve endings and from the adre- nal medulla. Concentrations of formaldehyde and acrolein required to produce significant pressor activity were found to be significantly lower than for acetaldehyde and propionaldehyde. The relative pressor potency for formaldehyde and acrolein was 10 and 100 times that of acetaldehyde, respectively. Higher doses of both formaldehyde and acrolein resulted in depressor responses which could be abolished by vagotomy. Thus, it appears that these aldehvdes also exert a cardioinhibitory effect which is mediated by the vagus nerve. Inhalation studies with acrolein revealed that this aldehyde has sig- nificant cardiovascular activity at concentrations below those which might be encountered in ciga- rette smoke. The predominant effect of inhaled acrolein at these doses was a significant increase in blood pressure and heart rate. It would appear that the aldehydes, especially acrolein, may be more significantlv involved in the cardiovascular re- sponse to cigarette smoke than was previously recognized. 36. Effects of inhaled acetaldehyde and propionaldehyde on blood pressure and heart rate John L. Egle, Jr. Department of Pharmacology, Medical College of Virginia. Health Sciences Division. Virginia Commomvealth University. Richmond. Virginia Toxicology and Applied Pharmacology 23:131-135. 1972 The objectives of this study .vere to determine whether blood pressure and heart rate are affected bv concentrations of acetaldehyde and propional- dehvde likely to occur in cigarette smoke. .-knes- thetized rats inhaled varying concentrations of ac- etaldehyde (0.5-30 µg/ml) and propionaldehvde (3.0-200 µg/ml) in air for 1 min intervals while changes in arterial blood pressure and heart rate were measured. With acetaldehvde. significant (P < 0.01) increases were seen in blood pressure at concentrations of 3.0 µg/ml and above. Concen- trations of 12 and 25 µg/mi significantly increased heart rate. Similar effects were seen at slightly higher concentrations of propionaldehyde. It appears that concentrations of acetaldehvde and pro- pionaldehyde producing significant changes in blood pressure and heart rate are somewhat higher than those that would be encountered in cigarette smoking. 37. Effects of cigarette smoking at rest and during exercise. II. Role of venous return Ronald J. Krone, Alberto N. Goldbarg, Maria Balkoura, Robert Schuessler, and Leon Resnekov Department of Medicine, Section of Cardiology, Pritzker School of Medicine, University of Chicago. Chicago, 711inois Journal of Applied Physiology 32:745-748. 1972 The cardiovascular response to submaximal upright exercise before' and after smoking a single cigarette was investigated in nine healthy male smokers, who had their legs wrapped in elastic bandages from the ankles to the thighs. Two exercise periods on a bicycle ergometer were separated by a 30-minute rest. Cardiac output was measured using the direct Fick method. Pulmonary and brachial artery pressures were measured directly. Previous studies from this laboratory [see preceding article, under identical conditions without bandages on the legs demonstrated an increase in heart rate and a TIMN 0115832 T200323 88

Cardiovascular Systein were studied in dogs during the post-infarction period. Metabolic chanjes in the non-infarcted myo- cardium were correlated with the functional status of the left ventricle. There was a marked fall in the ATP levels in the non-infarcted region of the left ventricle 24 h after infarction: a progressive rise till the tenth dat•. Parallel changes occurred in the creatine phosphate levels. The decrease in ATP and creatine phosphate levels were not accompanied by significant changes in lactate or pyruvate content of the non-infarcted myocardium during the period of observation. When changes in high energy phosphates in non-infarcted mvocardium were correlated with left ventricular function, a complex relationship was observed between the ATP content and the left ventricular stroke work; a similar relationship was found between ATP and the rate of left ventricular pressure rise (dp/dt). The results indicate that, following coronarv artery ligation, significant changes occur in the high energy phosphate me- tabolism in the non-infarcted portion of the left ventricle and that a relationship exists between these biochemical changes and left ventricular function. Determinations of the norepinephrine content in the dog heart were carried out following ex- perimenttrl myocardial infarction. Uptake. degrada- tion and subcellular distribution were determined after injection of tracer doses of D-r.(7-"C)norepi- nephrine. Normal left ventricular muscle contained 0.96 µg norepinephrine/g tissue; following coronary arterv occlusion. the infarcted tissue lost its norepi- nephrine content completely by the fourth day, and the non-infarcted tissue showed a marked decline during the first 10 days. reaching a level as low as 0.35 µg in the basilar and 0.14 µg norepinephrine/g in apical portion of the left ventricle. Norepineph- rine levels rose again 2 weeks after infarction and reached normal values 6 weeks after coronary artery occlusion. Despite these changes in endogenous norepinephrine levels, the myocardial uptake and degradation as well as the subcellular distribution of endogenous and exogenous norepinephrine re- mained unaltered. It is suggested that the increased sympathetic activity following infarction is respon- sible for the reduction in myocardial norepinephrine stores. Upon restoration of myocardial function, the cardiac norepinephrine content returns to normal. Other support: Michigan Heart Associaton. and the U. S. Public Health Service. 60. Changes in myocardial high energy phosphate levels and left ventricular function after coronary artery occlusion in anesthetized dogs Pritpal S. Puri, and Sigmundur Gudbjarnason Department of Medicine. Wayne State University School of Medicine, Detroit, Michigan Cardiovascular Research 5:451-457, 1971 Changes in metabolite and high-energy phos- phate levels in the non-infarcted portion of the left ventricle were studied in dogs from one to 10 days after coronary artery ligation. There was a marked fall in the adenosine triphosphate (ATP) levels in the non-infarcted region of the left ventricle 24 hr after infarction; a progressive rise occurred thereafter till the 10th day. Parallel changes were noted in the creatine-phosphate (CP) levels. The decrease in the ATP and CP levels was not accompanied by sig- nificant changes in lactate or pyruvate content of the non-infarcted myocardium during the period of observation. When changes in high energy phos- phates in non-infarcted myocardium were correlated with left ventricular function, a complex relation- ship was observed between the ATP content and the rate of left ventricular pressure rise (dp/dt) as well as stroke work. These results indicate that, after coro- nary artery ligation, significant changes occur in the high energy phosphate metabolism in the non- infarcted portion of the left ventricle and that a relationship may exist between these biochemical changes and left ventricular function. Other support: Michigan Heart Association. 61. Storage and metabolism of norepinephrine after experimental myocardial infarction Peter Mathes, Charles Cowan, and Sigmundur Gudbjarnason Department of Medicine. School of Medicine, Wayne State University, Detroit, Michigan American Journal of Physiology 220:27-32, 1971 Serial determinations of the norepinephrine (NE) content in all four chambers of the canine heart were carried out following experimental cardiac infarction. Myocardial NE retention and subcellular TIMN 0115842 T200333 98

(2-meth}'lamino-l-hvdroxvpropy 1) methanesulfon- anilide hydrochloride). in antagonizing the positive chronotropic response of the perfused guinea-pig heart to norepinephrine (NE) and the amount of NE taken up by the adrenergic nerves of this organ have been studied. All three concentrations of 1viJ 1998 (10-'. 3 x 10-' and 10-;:vf) significantly reduced the i ncrease in heart rate which resulted from a 20-min perfusion of NE (50 ng/ml, at a rate of 7 ml/min) in a dose-dependent fashion when the blocking agent cvas continuously perfused 30 min prior to and during the NE treatment. MJ 1998 blocked the increase in tissue NE and increased the content measured in the perfusate effluent. Therefore. it is concluded that MJ 1998 can antagonize NE uptake. The highest concentration of MJ 1998 (10-'Ivf) did not influence the positive chronotropic action of CaCl:. which suggest that blockade of NE uptake is not a result of myocardial depression but is probably a specific effect. The blockade of NE uptake cor- relates reasonab)y well with the receptor-blocking properties of this drug. Experiments using reserpine and the monoamine oxidase inhibitor, nialamide, seem to indicate that MJ 1998 is antagonizing the uptake of NE into the axonal membrane rather than the transport and/or binding in intraneuronal stor- age vesicles. Other support: Council for Tobacco Research- L'.S.A. 74. Effects of nicotine and sympathomimetic amines on potassium intoxication Bert K. Lum, and Raymond H. Lockwood Department of Phannacology, University of Hawaii School of Medtcine. Honolulu, Hawaii Journal of Pharmacology and Experimental Therapeutics 181:147-15-1. 1972 Intravenous infusion of nicotine was found to protect anesthetized dogs against death produced by the infusion of KC1. The protection induced by nicotine appeared to be the result of the sympathetic discharge elicited by the drug as indicated by the observations that (1) the protection by nicotine is abolished by chlorisondamine, (2) the protection is also produced by epinephrine and (3) the protection bv both nicotine and epinephrine is abolished bv propranolol. Atropine pretreatment did not affect the protective action of nicotine and phenoxyben- zamine did not affect that produced by epinephrine. Studies in anesthetized cats revealed that the pro- tection against KCI toxicity could be obtained with agents having beta adrenergic stimulant properties (isoproterenol and epinephrine); phenylephrine. an alpha adrenergic stimulant, failed to protect. Plasma potassium data indicate that the protection is related to the ability of the beta stimulant drugs to attenuate the hyperkalemia produced by the KCl solution. The latter as well as the protective action of the sym- pathomimetics against death produced by KCI in- fusion was abolished by propranolol. Acute ne- phrectomy did not affect the epinephrine induced protection or attenuation of the hyperkalemia pro- duced by KCl infusion. These effects of epinephrine are thus subserved by beta adrenergic receptors and may involve the entry of potassium into cells. This study was the subject of preliminary re- ports appearing in Federation Proceedings 29:476. 1970, and the University of Michigan Nfedical Cen- ter fournal 36:238, 1970. Other support: Hawaii Heart Association. C. Coronary, Capillary, Cerebral and Peripheral Circulation Fundamental Studies 75. Fluorescence histochemical and ultrastructural studies on the coronary artery of the dog Douglas G. Silva, and Gordon Ross Departments of Oral Medicine and Physiologv. Schools of Dentistry and Medicine, University of California. Los Angeles, California Anatomical Record 175:514, 1973 Perfusion of the heart with nicotine is known to cause a transient release of norepinephrine, pre- sumably from postganglionic adrenergic nerve ter- minals. To determine whether this release was associated with a morphological change, fluores- cence histochemical and electron microscopic stud- ies were carried out on the coronary arteries of normal adult dogs and dogs exposed to 10 minutes intracoronary infusions of nicotine 100 µg/min. In TIMN 0115847. T200338 103

78. Effects of intracoronary infusions of acetvlcholine and nicotine on the dog heart in v' 1V0 G. Ross Department of Phc•siology. C'CLA School of Medicine. Los Angeles. California British Journal of Pharmacology 48:612-619. 1973 The purpose of this investigation was to study the characteristics of the myocardial responses to infusions of catecholamine-releasing doses of ace- tvlcholine and nicotine in the dog heart in situ and to determine the effects of these agents on the response to cardiac sympathetic nerve stimulation and to intravenous noradrenaline. In anesthetized dogs intracoronarv infusions of high doses of nicotine and acetylchol.ine increased myocardial contractile force and this could be pre- ~-ented by pre-treatment with desmethylimipramine or phenoxybenzamine. The inotropic effect of nicotine was brief and subsided during the continuing infusion of the drug. The infusion of nicotine did not reduce the inotropic effects of cardiac sympathetic nerve stimulation. The inotropic effect of intracoronary acetylcho- line often fluctuated during prolonged infusions and was not altered by pre-treatment with atropine. Acett•lcholine infusions reduced the inotropic responses produced by cardiac sympathetic nerve stimulation and led to a substantial transient re- duction in the associated pressor responses. In- tracoronarv acetylcholine also reduced the pressor and inotropic effect of intravenous noradrenaline. The attenuation of these adrenergic cardiovascular responses by acetylcholine was prevented by atro- pine. Other support: American Heart Association. 79. The use of positron emitter in the determination of coronary blood flow in man Alberto Cohen. Edward J. Zaleski, Eide-Dittmar Luebs, and Richard J. Bing Department of Vedicine. Wayne State University School of Medicine. Detroit. Michigan Inurnal of Nuclear Medicine 6:651-666, 1965 The concepts underlying a coincidence count- ing method for the measurement of coronary blood flow using ";Rb, a positron emitter, is described. Coronarv blood flow is calculated by the logarithmic extrapolation of the myocardial clearance values of ";Rb to zero time. At this time, the myocardial extraction ratio is unitv. The accuracy of the method has been established in experiments on the isolated dog heart. All calculations are objectively performed by a digital computer. The resting coronary blood flows in individuals above and below the age of 40 and in males and females were not statisticallv different. In addition. there were no significant differences in the resting coronary blood flows in patients with and without coronary artery disease. The observation that sublingual administration of nitroglycerin increases the coronary blood flow in individuals without coronarv arterv disease while failing to increase it in patients with coronary artery disease has been used as a diagnostic test in the recognition of coronary artery disease. Other support: U. S. Public Health Service. the John A. Hartford Foundation, and the Council for Tobacco Research-U.S.A. 80. Measurement of coronary blood flow with radioisotopes Alberto Cohen. Edward J. Zaleski, and Richard J. Bing Department of Medicine. Wayne State University School of \fedicine. Detroit. Michigan Medical Times 95:573-579. 1967 Three main groups of methods are described: I. Methods Utilizing Radioactive Diffusable Inert Gases (Kr-85, Xe-133). II. Methods Utilizing Intravascular Non-Diffus- able Radioactive Substances (RISA, Dio- drast-131. Macroaggregated albumin. etc.). III. Methods Utilizing Diffusable Substances Which Actively Enter the Cell (K-42, Rb-86, and Rb-84). Other support: U. S. Public Health Service, the Michigan Heart Association, and the John A. Hart- ford Foundation. TIMN 0115849 T200340 105

r Cardiovascular System malate. citrate. ATP. and NAD oxidation-reduction state showed that increased flux associated with a State -1 to 3 transition could be accounted for largelv bv an increased availability of oxalacetate to citrate svnthase rather than bv ATP inhibition. On the other hand. comparisons between states in which phos- phate acceptor was not rate limiting for electron transport (State 3. uncoupled or uncoupled plus oligomvcin) showed that oxalacetate availability remained high because of the highly oxidized state of the prridine nucleotides. and indicated a reg- ulation of citrate svnthase under conditions of low acetvl-CoA availability by an energy-linked process dependent on substrate level phosphorvlation. Thus, a 55% inhibition of flux through citrate synthase was obtained after addition of oligomycin to the uncoupled state with acetylcarnitine plus malate,as substrate, but onlv a 25% inhibition was achieved with pyruvate plus malate as substrate. The in- tramitochondrial ATP:ADP ratio increased from 0.3 to 4:6 with either substrate under these conditions. A kinetic evaluation of the data indicated that the energy-dependent inhibition was not caused by a direct effect of ATP on citrate synthase, but was due to changes of the succinyl-CoA content relative to that of acetvl-CoA. This conclusion is based (a) on the finding of an accumulation of a short chain acetvl-Cor1 compound identifiable as succinyl-CoA bv direct enz«ne assay under conditions of di- minished citrate synthase flux, (b) a correlation be- tween decreased flux and lowered acetyl-CoA levels in the different experiments, and (c) the observation that succinvl-CoA is an inhibitor of citrate synthase competitive with acetyl-CoA. A comparison of succinyl-CoA levels in mi- tochondria incubated in the different metabolic states indicated that its content was regulated by the phosphorvlation state of the adenine nucleotides, presumably via the effect of altered GTP:GDP ratios on succinate thiokinase. Product inhibition of a- ketoglutarate dehydrogenase by succinyl-CoA was evidenced from an inverse relationship between a-keto?lutarate accumulation and succinyl-CoA lev- els. The conclusion is reached that feedback from the electron transport chain to the citric acid cycle is mediated by a combination of factors which include the phosphorylation state of the adenine and oua- nine nucleotides and the oxidation-reduction state of the pyridine nucleotides. Changes of these param- eters secondarily affect the intramitochondrial con- centrations of oxalacetate, acetvl-CoA. and succinvl- Cot1, which are the direct regulators of citrate synthase activity. Other support: U. S. Public Health Service. and the American Heart Association. 44. Inhibition of citrate synthesis' by succinyl-CoA and other metabolites Colleen M. Smith, and John R. Williamson Johnson Research Foundation. University of Pennsylvania. Philadelphia. PennsYlvania FEBS Letters 18:35-38, 1971 The kinetic experiments reported here with purified citrate synthase from beef heart and rat liver show that succinyl-CoA is a much stronger inhibitor of citrate production than ATP, strictly competitive with acetyl-CoA and noncompetitive with respect to oxalacetate. Furthermore, carefully controlled rate measurements show no kinetic indication of coop- erativity in the presence or absence of either ATP or succinyl-CoA. Citrate, propionyl-CoA and CoA are also specific inhibitors at concentrations of physio- logical significance. Other support: U. S. Public Health Service, and the American Heart Association. 45. Hypoxia and nicotine induced loss of intracellular myocardial acid hydrolases Erdogan Oran, and Norman Brachfeld Myocardial Metabolism Laboratory, Department of Medicine, Division of Cardiology, Cornell University Medical College, and the Institute for Muscle Diseases, Inc., ri'ew- York, Vew York Unpublished Report to the AMA-ERF The effect of hypoxia and nicotine on the leakage of intracellular enzymes, myocardial me- tabolism and hemodynamic performance was evalu- ated in the isolated perfused rat heart preparation. After a 30 min period of hypoxia, there was sig- nificant leakage of the lysosomal enzymes acid phosphatase and (3-glucuronidase and the intracyto- plasmic enzyme lactic dehydrogenase into the per- fusate. A simultaneous decrease in oxygen con- TIMN 0115836 92 T200327

. , lleUrease in stroke index during moderately severe ,Iyercise after smoking a cigarette. In contrast. in this study when the venous return was improved by leg bandages. there was no change in stroke index at nuoderately severe exercise (71 vs. 69 ml/m= before ,u1d after smoking at 600 kg-m/min, respectively. and 71 vs. 70 ml/m= at 750 and 900 kg-m/min). These data suggest. therefore. that cigarette smoking has effects both directly on the heart and on peripheral kenous return which alter significantly the circula- tnrr response to upright exercise. Other support: Chicago Heart Association. 38. Effects of cigarette smoking on hemodynamics at rest and during exercise. I. Normal subjects Alberto N. Goldbarg, Ronald J. Krone, and Leon Rrsnekov Department of 1~fedicine. Section of Cardiologl•. Pritzker School o,f ~fedicine. University of Chicago. Illinois ('hest 60:531-536. 1971 Cardiovascular responses to submaxi.mal graded upright exercise were investigated by pulmonary and subclavian arterial catheterization in nine healthy,voung men before and after smoking a single c: iaarette. At rest, after smoking, the mean cardiac index and mean heart rate increased, while arterio- venous oxygen difference, stroke index and mean pulmonarv artery pressure remained unchanged. During successively increasing levels of exercise, thN heart rate was greater and the stroke index lower than values for comparable work before smoking; the ,irteriovenous oxygen difference, oxygen consump- tion. pulmonary artery pressure, systemic vascular resistance. lactate levels, pulmonary ventilation and arterial Po, did not change significantly. By decreas- inp the stroke volume response to exercise, smoking a single cigarette significantly alters the hemody- namic response to exercise in a direction opposite to physical training. Other support: Chicago Heart Association. 39. Analysis of nicotine-induced vascular reflexes in the dog R. L. Tokar, and G. L. Gebber Department of Pharmacology. :blichigan State University. East Lansing, Nfichigan Archives Internationales de Pharmacodynamie et de Therapie 179:408--t18, 1969 The vascular consequences of the complex and widespread actions of nicotine on excitable tissue were separated and studied individually in the perfused hindquarters of the dog. Small intravenous doses of nicotine (10-40 µg/kg) produced a mul- tiphasic change in perfusion pressure of the in- nervated hindquarters. An initial fall in perfusion pressure (component 1) was followed by an increase in perfusion pressure (component 2) which in turn was succeeded by a second fall in perfusion pressure (component 3) and a more prolonged vasoconstric- tion (component 4). Components 1, 2 and 3 of the perfusion pressure response were abolished by acute sympathetic denervation of the hindquarters. Com- ponent 4 was markedly reduced by adrenalectomy. Vagotomy abolished the initial fall in perfusion pressure but did not markedly influence the, later occurring dilatation. Carotid sinus section produced a paradoxical increase in reflex dilatation (compo- nent 1 and 3) evoked by nicotine. The portion of nicotine-induced reflex dilatation which exceeded the fall in perfusion pressure produced by complete denervation of the hindquarters was blocked by the antihistamine, tripelenamine. Thus, reflex dilatation evoked by nicotine must be attributed in part to inhibition of sympathetic vasoconstrictor tone and, in part, to activation of the sympathetic dilator system. Denervation of the carotid and aortic body chemoreceptors reduced the initial rise in perfusion pressure produced by nicotine. B. Cardiac Metabolism 40. Feedback control of the citric acid cyde John R. Williamson, Colleen M. Smith, Kathryn F. LaNoue and Jadwiga Bryla Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania Energy Metabolism and the Regulation of Metabolic Process in Mitochondria, eds. Mvron A. Mehlman, and Richard W. Hanson, Academic Press, 1972, pp 185-210 The role of succinyl-CoA and other metabolites in the regulation of flux through the citric acid cycle TIlVIN 0115833 T200324 89

Cardiovascular Svstem has been investigated. Studies with purified pig ,heart a-ketoglutarate dehvdrogenase at saturating concentrations of NAD- and a-ketoglutarate showed that succinyl-CoA inhibited competitively with re- spect to CoA (Km for CoA of 3.6 µM; K, for succinyl- CoA of 10.5 µM). Studies of the kinetic properties of purified beef heart and rat liver citrate svnthase indicated a random order of addition of substrates, and demonstrated a number of new inhibitors. Inhibitions by succinyl-CoA (K; of 130 µM) and propionyl-CoA (K, of 50 µM) were competitive with respect to acetvl-CoA and noncompetitive with re- spect to oxalacetate. Inhibition by citrate (K, of 1.6 mM) was noncompetitive with respect to acetyl-CoA and competitive with respect to oxalacetate. Experiments with isolated rat heart mitochon- dria incubated with pyruvate or acetylcarnitine, in the presence of malate, under a variety of metabolic states which differed with respect to energy state, pyridine nucleotide redox state and rates of res- piration, are described which demonstrate the dual role of succinyl-CoA as a negative feedback regulator of flux through a-ketoglutarate dehydrogenase and citrate svnthase. It is concluded that flux through citrate synthase in the intact mitochondria is con- trolled by the intramitochondrial concentration of either oxalacetate or acetyl-CoA, depending on sub- strate and phosphate acceptor availability, and that regulation is achieved by feedback inhibition of metabolites which differentially increase the appar- ent Michaelis constant for the substrates. Other support: U. S. Public Health Service, and the American Heart Association. 41. Control of the transport of reducing equivalents across the mitochondrial membrane in perfused rat heart Brian Safer, Colleen M. Smith, and John R. Williamson Johnson Research Foundation, University of Pennsylvania. Philadelphia. Pennsylvania Journal of Liolecular and Cellular Cardiology 2:111-124, 1971 A comparison of the functional significance of the malate-aspartate and a-glycerophosphate hv- drogen shuttles was made in the isolated perfused rat heart. Amino-oxyacetate, a competitive inhibitor of aspartate aminotransferase, inhibited lactate and glucose oxidation in the normal rat heart. Although a-glycerophosphate oxidase activity was increased sixfold bv triiodothvronine, hydrogen flux through the a-glycerophosphate shuttle was limited by low tissue concentrations of a-glycerophosphate and low activity of oc-glycerophosphate oxidase. Significant flux was only achieved when the cvtosolic NADHI NAD ratio and the a-glycerophosphate concentra. tion rose to highly unphvsiological levels. It is concluded that the malate-aspartate shuttle is the predominant mechanism for reoxidation of cytosolic NADH in rat heart. Examination of changes in the tissue levels of citric acid cycle intermediates in rat heart under these experimental conditions also sug. gests that introduction or withdrawal of carbon moities from the pool can only occur by the con• certed action of both asparate and alanine ami- notransferases. Other support: U. S. Public Health Service, and the American Heart Association. 42. Regulation of glutamate metabolism and interactions with the citric acid cycle in rat heart mitochondria Kathryn F. LaNoue, Elzbieta I. Walajtys, and John R. Williamson Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania Journal of Biological Chemistry 248:7171-7183, 1973 The interactions of glutamate metabolism with transport steps across the mitochondrial membrane and enzyme steps of the citric acid cycle have been investigated in rat heart mitochondria in order to elucidate factors regulating transfer of reducing equivalents into mitochondria via the malate-as- parate cycle. With glutamate as substrate, net efflux of a-ketoglutarate from the mitochondria in Stage 3 was stimulated by addition of extramitochondrial malate, the concentration for a half-maximal effect being 0.36 ± 0.07 mM. Stimulation of a-ketogluta- rate-malate exchange by a decrease of the malate gradient resulted in a fall of the intramitochondrial a-ketoglutarate concentration and diminished flux through a-ketoglutarate dehydrogenase. A value of 0.67 mM for the apparent Km of a-ketoglutarate dehydrogenase for a-ketogiutarate was calculated from these data. Glutarate transaminase was in- hibited in the uncoupled state and the intramito- TIMN 0115834 T200325 90

Cardioc-ascular System vitro preparations of coronary arteries incubated for 15 minutes in a physiological salt solution contain- ing 100 µ'g/mi nicotine were also examined. In all animals studied large bundles of fluorescent fibers were seen to accompany the coronary arteries and a dense plexus of adrenergic fibers was also seen at the adventitio-medial junction. Ultrastructural studies revealed the presence of many bundles of nerve fibers at the adventitio-medial junction. Accurate counts of nerves undertaken on 25 randoml_v se- lected grids (5 grids from 5 different animals, each grid containing 140 µ of adventitio-medial junction) revealed an average of 8 fasciculi per 140 µ within 5 µ of the junctional zone. The closest distance be- tween a non-mvelinated nerve and a smooth muscle cell of the media was 4000 A. The non-myelinated nerves at the adventitio-medial junction contain a variety of granular and agranular vesicles. Small granular vesicles typical of adrenergic nerves were a prominent feature. Large granular vesicles were also present. In addition small and large agranular vesi- cles were observed. Exposure of the coronary arteries to nicotine had no noticeable effect on the intensity of the specific fluorescence or on the staining properties of the granular and agranular vesicles. This study indicates that the coronary arteries have a dense adrenergic innervation and exposure to nico- tine has no detectable structural or histochemical effect on the nerves. 76. The effects of nicotine on coronary circulation of dogs Gordon Ross, and Marta J. Blesa Department of Physiology. U. C. L. A. School of Medicine. Los .-ingeles, California : lmr•ric:nn Neart Journal 79:96-102. 1970 A study is reported of the action of nicotine on i nstantaneous coronary arterial flow in anesthetized dogs. such determinations being noted to provide much more information than average flow determi- nations used in most other studies. Drugs were injected directlv into the coronary artery. Nicotine in doses that produce no systemic effects and that do not elicit the Bezold-Jarisch reflex produced a si- multaneous increased force of myocardial contrac- t ion and an increase in coronary flow. These changes began within 4 seconds of injection, before the drug could have reached the systemic circulation, and were unaccompanied by changes in heart rate and 104 arterial pressure or by changes in myocardial force upstream from the point of injection. Examination of the instantaneous coronary flow records showed that the increased coronary flow was partly due to coronary vasodilatation as shown by the reduced end-diatolic vascular resistance and partly to an increase in the duration of diastole during which most of the coronary inflow takes place. Systolic flow was reduced and even reversed. Responses to norepinephrine were very similar to those of nico- tine and responses to both agents were blocked by intravenous propranolol, suggesting that the effects of nicotine on the myocardium and on the coronary flow are mediated by catecholamines. The nicotine responses were also blocked by intravenous pen- tolinium, but were not affected by atropine or vagotomy. 77. The effect of nicotine on the coronary microcirculation in the cat heart Harald Tillmanns, Shigeaki Ikeda, and Richard J. Bing Huntington Institute of Applied Medical Research and the California Institute of Technology, Pasadena. California. and the University of Southern California. Los Angeles. California Journal of Clinical Pharmacology 14:426-433. 1974 This report deals with a study of the direct effect of nicotine on the coronary microcirculation in- dependent of the systemic effects of the drug. The experiments were carried out on the adrenalecto- mized and decapitated cat. Capillary microcircula- tion was studied by means of trans il luminatio n of the left atrium, using an intravital microscope. Analyses of red cell velocity were carried out on a frame-to-frame basis using a camera speed of 400 frames per second. It could be shown that nicotine increases coro- nary capilJarv red cell velocity in the left atrium of the cat without significant changes in systemic blood pressure or heart rate. The findings suggest a direct effect of nicotine on the atrial capillaries through direct release of catecholamines, probably from their storage sites in heart muscle or atrial ganglia. Other support: The Margaret W. and Herbert Hoover, Jr. Foundation. TIMN 0115848 T200339 -- ,

and taking certain precautions. the mortalitv rate is low. and the complications are few. Changes in coronarc microcirculation may be implicated in the cause of atypical angina. Nonin~•asive techniques may be important in the presumptive diagnosis of coronary artery dis- ease. The fraction of LVET (left ventricular ejection time to EICT (externally determined isovolemic contraction) after exercise shows a good correlation to mvocardial contractility. The apex cardiogram and the kinetocardiogram, especially after exercise. offer additional help. The rubidium-84 coincidence counting technic is a simple method to determine coronary blood flow and coronary reserve in man. Using isoproterenol, recognition of severe atherosclerotic heart disease may be possible. Other support: Council for Tobacco Research- L'.S..'+.. the Los Angeles County Heart Association, and the Norris Foundation. 85. Coronary blood flow in relation to angina pectoris Richard J. Bing and Klaus Hellberg Huntington Memorial Hospital. Pasadena. California. and the University of Southern California. Los Angeles, California Circulation 46:1146-1154. 1972 The first portion of this paper represents a critical analysis of methods used in the determina- tion of coronary blood flow in man. Subsequent portions deal with changes in coronary flow in ischemic heart disease. Other support: The Margaret W. and Herbert Hoover, Jr. Foundation, and the Council for Tobacco Re- search-U.S.A. 86. Interaction of the chemoreflex and the pulmonary inflation reflex in the regulation of coronary circulation in conscious dogs Stephen F. Vatner, and Robert J. McRitchie Department of Medicine. Harvard Medical School and Peter Bent Brigham Hospital, the Department of Cardiology, Childrens Hospital Medical Center, Boston. .Wassachusetts, and the New England Regional Primate Research Center, Southborough, Massachusetts Circulation Research 37:664-673, 1975 The interaction of chemoreflex and pulmonary inflation reflex control of the coronary circulation was examined in conscious dogs by comparing the responses to chemoflex stimulation (intracarotid injection of nicotine) when ventilation was allowed to increase with those when ventilation was con- trolled. These responses were also compared with those elicited by both forced mechanical and sponta- neous hyperinflation. When the heart rate was constant. intracarotidly administered nicotine in- duced an increase in the depth of respiration fol- lowed closely by an increase in late diastolic coro- narv flow from 48 ± 2 to 106 ± 8 ml/min and a reduction in late diastolic coronarv resistance from 1.62 ± 0.08 to 0.78 ± 0.06 mm Hg/ml min-'. After beta-receptor and cholinergic blockade, a similar coronarv dilation in response to nicotine occurred onlv when ventilation was allowed to increase. Howec•er. when ventilation was controlled. intraca- • rotidlv administered nicotine increased coronar% resistance after combined beta-receptor and cholin- ergic blockade. The reflex coronary dilation was not observed after carotid sinus nerve section or after alpha-receptor blockade. Thus, nicotine stimulation of the carotid chemoreflex results in a striking coronary dilation that has two components. The minor component involves a chemoreflex with its efferent pathway in the vagi. The major component of coronary dilation follows an increase in the depth of respiration, and its efferent component appears to involve withdrawal of alpha-adrenergic constrictor tone. An almost identical period of reflex coronary dilation followed either forced mechanical or spon- taneous hyperinflation in the conscious dog. Other support: Council for Tobacco Research- U.S.A., Inc., and the U. S. Public Health Service. 87. The action of inhaled cigarette smoke on the microcirculation, heart rate and carotid pressure of the bat Paul A. Nicoll Department of Physiology, Indiana University .vfedical Center, Indianapolis. Indiana Angiology 17:851-858, 1966 The subcutaneous minute vascular bed in the bat wing is available for study at high magnification without the use of anesthesia or surgical prepara- tion. Unanesthetized small bats of the genus Myotis, TIMN 0115851 T200342 107

Cardiovascular Svstem 81. Myocardial blood flow in coronary artery disease; Correlation with severity of disease and treadmill exercise response Suzanne B. Knoebel. William C. Elliott. Paul L. McHenry, and Edward Ross Department of ~fedicine. Indiana University School of ~fedicine. and the Krannert Institute of Cardiology. Marion Count' v General Hospital. Indianapolis. Indiana American Journal of Cardiology 27:51-58, 1971 The severity of cinearteriographically demon- strated coronary artery disease was correlated with nutrient myocardial blood flow response to either right atrial pacing or isoproterenol infusion in 28 patients. Severity of coronary disease was estimated by percent of the 3 coronarv vessels remaining patent. and a numerical rating based on a possible 300 was assigned to each patient. Myocardial blood flow was measured using a coincidence co>snting technique and a single "bolus" of 114 rubidium. Re- sponse of the S-T segment during treadmill ex- ercise was also recorded. The results show good correlation between the severitv of coronary artery disease and the response of mvocardial blood flow to demand. In those patients with less severe disease (coronary artery indexes of greater than 200), myocardial blood flow increases to within the normal range with stress from either right atrial pacing or isoproterenol. Patients .vith more severe disease (coronary artery indexes of less than 200) were unable to increase mvocardial blood flow with either form of stress. The response to treadmill exercise paralleled the mvocardial blood flow results, being positive in the patients showing a decreased flow reserve. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociation. 82. Myocardial blood flow; Measurement by a coincidence counting system and single bolus of 8;Rb Suzanne B. Knoebel. and Paul L. McHenry Department of Medicine. Indiana University School of Medicine and the Krannert Institute of Cardiology. Marion County General Hospital. Indianapolis, Indiana Archives of Internal :Wedicine 127:767-772. 1971 Animal experiments demonstrated that myocar- dial blood flow (MBF) as measured by the coinci- dence system and a concomitant Fick determination were verv similar, varving from + 9.9°o to -7.9% with a mean absolute error of 5.3°'0. Human studies have demonstrated that (1) the average NIBF in normal subjects is 269 ± 61 ml/min/total heart, which represents 5.2 ± 1.6% of the simultaneous cardiac output, (2) MBF determined by the coinci- dence technique is a reproducible measurement and has appropriate directional change with stresses imposed to augment myocardial oxygen demand, and (3) patients with significant coronary artery disease have a decreased flow reserve. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 83. Measurement of coronary flow in ischemic heart disease Richard Bing and Klaus Hellberg Huntington Memorial Hospital, and the California Institute of Technology, Pasadena. California. and the University of Southern California School of Medicine, Los Angeles. California Atherosclerosis and Coronary Heart Disease, pp 146-149. William Likoff, Bernard L. Segal, William Insull, Jr., and John H. Moyer. Editors. Grune and Stratton, 1972 Current methods are reviewed and discussed. 84. Recognition of coronary artery disease Richard J. Bing, Klaus Hellberg, and Otmar Pachinger Huntington Memorial Hospital. Pasadena. California, and the University of Southern California School of Medicine, Los Angeles, California New York State Journal of lVedicine 72:1818-1822, 1972 This report deals with modern aspects of the recognition of coronary artery disease. History and the physical examination remain the keystone of the recognition of coronary artery disease. The serum cholesterol level and triglycerides are the most useful representatives of the lipid profile. Electrocar- diographic stress tests remain an important adjunct to the more quantitative tests. The well-standard- ized, graded exercise test is especially useful. Selec- tive coronary arteriography offers the best way to recognize coronary artery disease and to determine its severity. Using well••defined criteria for its use TIMN 0115850 T200341 106

p e e .e sumption. a fall in heart rate. and an increase in ;lucose consumption and lactate production pro- cided evidence for enhanced glycolytic metabolism. The presence of nicotine was also associated with leakage of enzyme into the perfusate, but these changes were noted during aerobic perfusion and %vere not associated with significant evidence of enhanced glycolysis. It is suggested that hypoxia and nicotine-induced myocardial cell injury may both be expressed as damage to the lysosomal and cell membrane. When both act simultaneously a sublethal. potentially reversible injury may be con- verted to a state of irreversible cell damage. 46. Free fatty acid metabolism of the human heart at rest Albert S. `tost. Norman Brachfeld, Richard Gorlin. and lohn %1'ahren Cardiovascular Division. Department of Xledicine. Peter Bent Brigham Hospital. Harvard :bledical School. Boston. Massachusetts: the Department of aledicine. Cornell t'niversity.kfedical College. 1Vew York: and the Institute for .tluscle Disease. Inc.. New York. Vew York /nurnal of Clinical Investigation 48:1177-1188, 1969 %fvocardial substrate metabolism was studied in 13 subjects at the time of diagnostic cardiac catheter- ization by means of palmitic acid-14C infusion with arterial and coronary sinus sampling. Two subjects trere considered free of cardiac pathology and all, %vith one exception, demonstrated lactate extraction across the portion of heart under study. Data for this single lactate-producing subject were treated sepa- ratelv. The fractional extraction of'}C-labeled free fatty acids (FFA) (44.4 ± 9.5%) was nearly twice that of unlabeled FFA (23.2 ± 7.8%) and raised the pos- ;ibility of release of FFA into the coronary sinus. FFA uptake. based on either the arterial minus :oronarv sinus concentration difference or the FFA-:4C fractional extraction, was directly propor- tional to the arterial FFA concentration. Gas-liquid ~hromatography failed to demonstrate selective han- dling of any individual FFA by the heart. Fractional oxidation of FFA was 53.5 ± 12.7% accounting for 53-2 = 14.4% of the heart's oxygen consumption while nonlipid substrates accounted for an addi- tional 30.0 = 17.3%. Determinations of both labeled and unlabeled triglycerides suggested utilization of this substrate bv the fasting human heart. Direct measurement of FFA fractional oxidation as ..vell as FFA uptake, exclusive of possible si- multaneous FFA release, would appear necessary in studies concerned with human mvocardial FFA metabolism. Othersupport: U. S. Public Health Service; Kriendler Memorial Foundation; Women's Aid for Heart Re- search, Boston, Massachusetts: Muscular Dystrophy Associations of America, Inc.; and the New York Heart Association. 47. Substrate preference and metabolic activity of the aerobic and the hypoxic turtle heart Norman Brachfeld, Yoshinaga Ohtaka. Irwin Klein. and %iasaka Kawade Mvocardial Metabolism Laboratory of the Department of Nfedicine. Division of Cardiology, Cornell University Medical College. and the Institute for Muscle Disease. Inc.. New York. Vew York Circulation Research 31:453-467, 1972 This investigation was undertaken to compare the relatively well-known pathways of mammalian substrate utilization and preferences for substrate extraction to those which serve the turtle mvo- cardium. Substrate utilization by the isolated aerobic (Po, 500 mm Hg) and hypoxic (Po2 50 mm Hg) turtle (Pseudemys scripta) heart was evaluated by retro- grade perfusion. During aerobic perfusion, the heart demonstrated active extraction and oxidation of free fatty acid (FFA) even in the presence of exogenous glucose. FFA metabolism was similar to that seen in the mammalian heart and was adequate to meet most of the myocardial energy requirements. When hyp- oxia supervened, FFA utilization did not compete with, but rather enhanced, concomitant carboxy- drate metabolism, and glycogenolysis provided suf- ficient substrate to meet myocardial energy require- ments with little change in observed hemodynamic function. During hypoxia. both rat and turtle dem- onstrated enhanced mvocardial esterification of ex- tracted [1-'}C]-pal.mitate into neutral lipid. Exoge- nous glucose provided the glycerophosphate TIMN 0115837 T200328 93

_r Cardiovascular Srstem occlusion of coronarv arteries was followed bv infusion of noradrenaline. subendocardial flow was less than capillary flow in subepicardial lavers. In these conditions, the administration of nitroglycerin resulted in a redistribution of capillary flow toward normal. Other support: Michigan Heart Association. Council for Tobacco Research-U.S.A., and Detroit General Hospital Research Corporation. 93. Effect of nicotine and vasopressin on the terminal vascular bed of the heart Karel Rakusan. Wolfgang du Mesnil de Rochemont. Jon Hanlon. Wolfgang Braasch. Hans Tschopp, and Richard J. Bing Department of Jledicine. Wayne State University School of Medicine. Detroit. Michigan Clinical Pharmacology and Therapeutics 9:24-30. 1968 The effect of nicotine and vasopressin on the capacity of the terminal vascular bed in heart and skeletal muscle of dogs was investigated. In normal heart muscle highest values were obtained in the apex. Nicotine had no significant effect unless the blood pressure increased to very high values. After vasopressin no significant changes were found in heart muscle, but vasoconstriction was present in skeletal muscle. Capillary blood flow determined in heart, lungs, and kidneys of rats increased in all organs after nicotine. In control rats, a slightly but significantly higher capillary flow was found in the right ventricle. This difference disappeared in nic- otine-treated animals and became more pronounced after injection of vasopressin. An earlier account of this study was published in Medicina et Pharmaco- logia Experirnentalis 17:119-128, 1967. Other support: U. S. Public Health Service; Council for Tobacco Research-U.S.A.; Detroit General Hos- pital Research Corporation. 94. Capillaries in heart and muscle of dog and rabbit K. Rakusan, and W. du Mesnil de Rochemont (R. J. Bing) Department of Medicine, Wayne State University School of Medicine, Detroit. Michigan Proceedings of the Society for Experimental Biology and .tifedicine 124:838-840. 1967 The capacity of the terminal vascular bed as an indicator of the capillary density in heart and skeletal muscles of the rabbit and the dog was investigated. Tissue samples were taken from two regions of the heart muscle (apex and septum) and from two different skeletal muscles (gastrocnemius and vastus lateralis). The capillary density in heart muscle was higher in the rabbit than in the dog. On the other hand, the capillary density of skeletal muscle was greater in the dog. Factors influencing capillary growth (physical activity and metabolic rate) are discussed. Other support: U. S. Public Health Service, the John A. Hartford Foundation, the Michigan Heart As- sociation, the Council for Tobacco Research- U.S.A., and the Detroit General Hospital Research Corporation. 95. Effect of nicotine on capillary flow and terminal vascular capacity of the heart in normal dogs and in animals with restricted coronary circulation Giancarlo Corsini, Pritpal S. Puri, Pedro V. M. Duran, and Richard J. Bing Department of Medicine, Wayne State University School of Medicine. Detroit, Michigan Journal of Pharmacology and Experimental Therapeutics 163:353-361, 1968 Effects of a single i.v. infusion and repeated i.m. injections of nicotine on capillary blood flow and terminal vascular capacity of the heart were com- pared in dogs with and without experimentally induced impairment of coronary circulation. Grad- ual coronary artery narrowing was produced by means of ameroid constrictors placed around the three main coronary arteries. Capillary blood flow and terminal vascular capacity were determined simultaneously from the activities of Rb86 and I13'- albumin, respectively, employing a differential counting technique. Results indicate that in dogs with normal coronary circulation, a single infusion of nicotine increases both capillary blood flow and terminal vascular capacity; repeated nicotine ad- ministration, however, has no significant effect on either. In contrast, in animals with impaired coro- nary circulation, both the single i.v. infusion and repeated administration of nicotine resulted in a fall in capillary blood flow but an increase in terminal vascular capacity. These findings suggest that im- paired coronary circulation resulting from coronary TIMN 0115854 T200345 110 ~

T Cardiovascular System when inhaling cigarette smoke, show a diphasic response of the minute vessels in their wing mem- brane. Initially there is an increase in vasomotion evidenced bv enhanced magnitude of contraction in the arcuate and terminal arterioles, frequently ac- companied by an increase in the duration of the contraction phase at the expense of the relaxation phase. This is soon replaced by an inhibitory action that blocks active contraction of the muscle cells in the minute vessels. resulting in greatly lengthened dilatation and even complete cessation of all va- somotion. The rapid onset of the inhibitory condition that is also reflected by cardiac slowing and an abrupt fall in central blood pressure makes it difficult to evaluate any contribution of the initial increase in vasomotion of the arcuate and terminal arteriole in the tobacco smoke response usually observed in human subjects. 88. Microcirculation in the ventride of the dog and turtle Harald Tillmanns, Shigeaki Ikeda, Herbert Hansen. Jonnalagedda S. M. Sarma. Jean-Marie Fauvel, and Richard J. Bing Huntington,Vemorial Hospital and the California Institute of Technology. Pasadena, California, and the University of Southern California, Los Angeles, California Circulation Research 34:561-569, 1974 Phasic red cell velocity and diameters of coro- nary arterioles, capillaries, and venules were mea- sured in the beating turtle and dog heart using high- speed cinematography'with transillumination of the left ventricle. In the turtle, arteriolar red cell velocity was diminished during systole, but during diastole arteriolar inflow increased, especially during the rapid and the slow filling period. Capillary and venule red cell velocity was increased during sys- tole, particularly at the time of ejection, however, during diastole red cell velocity declined and the lowest values occurred during isovolumic relaxa- tion. In dog arterioles, capillaries, and venules, the pattern of red cell velocity was similar. Thus, in the turtle and dog, the peak arteriolar red cell velocity occurred in unison with left coronary artery inflow, and the capillary and venule flow pattern followed that of the coronarv sinus. The diameters of ar- terioles. capillaries and venules in the turtle ventri- cle all declined about 34% during systole: similar results were obtained in the dog. Capillary arrange- ment appeared to be predominantly parallel and cocurrent; however, capillary loops with countercur- rent flow were occasionally observed. The data on microvascular phasic red cell velocity are consistent with the macroobservations of reduced coronary artery inflow and enhanced coronary sinus outflow during ventricular contraction. The results dem- onstrate that the shift in the flow pattern occurs at the transition from arterioles to capillaries. Other support: Robert E. and May R. Wright Founda- tion, the Margaret W. and Herbert Hoover. Jr., Foundation, the Council for Tobacco Research- U.S.A., and the George and Miriam Gould Dock Fund Foundation. 89. The effect of nicotine, propranolol, phentolamine, and hexamethonium on the microcirculation of the cat O. Pachinger, K. D. Hellberg, and R. J. Bing Huntington Memorial Hospital, Pasadena. California. and the University of Southern California. Los Angeles. California Journal of Clinical Pharmacology and 1,'ecr Drugs 12:432-439, 1972 The effects of intravenous infusion of nicotine on the coronary capillary red cell velocity were investigated by means of transillumination of the left atrium of the beating cat heart using high-speed cinematography. In doses of from 50 to 200 µg/kg infused over a period of two minutes, nicotine significantly increased capillary red cell velocity and mean aortic pressure. There appeared to be a relationship between capillary red cell velocity and amount of nicotine infused. The effects of nicotine on the coronary mi- crocirculation could be inhibited by adrenergic blocking agents, such as propranolol and phentol- amine, and by ganglionic blockade with hexame- thonium. These results suggest that nicotine exerts its effect on the microcirculation through coronary perfusion pressure as well as through the activity of the precapillary sphincters. TIMN 0115852 108 T200343 WARN

go. Studies on the coronary microcirculation by direct visualization Klaus Hellberg. Harold Wayland. Armin L. Rickart. and Richard J. Bing Huntington Memorial Hospital. Pasadena. California: California Institute of Technology. Pasadena. California: and the L'nirersitr of Southern California, Los Angeles, California .-lmerican Journal of Cardiology 29:593-597, 1972 With the use of high speed cinematography of the coronary microcirculation of cats. countercur- rents and asvmmetric capillary arrangement were found. This has far-reaching importance for the oxygenation of the heart muscle and makes doubtful previous calculations for the oxygen transport from the capillary to the surrounding tissue. Recruitment, denoting an increase in the number of capillaries. was observed with a rise in perfusion pressure in the arrested heart and after the administration of ni- troglycerin in the beating heart. It can occur in the presence of autoregulation. Definite patterns of red cell velocitv in the capillaries of the left atrium emerged. Nicotine and nitroglycerin had little effect on red cell velocity. However, after hemorrhage alone. and following the administration of nitroglyc- erin after hemorrhage, marked changes in red cell relocity were seen. Hemorrhage alone caused a marked diminution in red cell velocity; hemorrhage followed by nitroglycerin reversed red cell velocity to normal values despite a persistent diminution in blood pressure. Two major peaks in red cell velocity were obsen•ed, the first, large peak immediately preced- ing or at the onset of ventricular systole, the second small peak immediately after ventricular systole. Some alterations in this pattern were seen after hemorrhage and after the administration of nitro- Ox•cerin. Other support: Council for Tobacco Research- U.S.A.: Hoover Foundation; Los Angeles County Heart Association; Norris Foundation; U. S. Public Health Service. 91. The coronary microcirculation in the potassium chloride arrested heart K. Hellberg, A. Rickart. H. Wayland, and R. J. Bing Huntington Vemorial Hospital and the California Institute of Technology. Pasadena. California. and University of Southern California School of Medicine. Los Angeles. California Journal of Molecular and Cellular Cardiology 2:221-230. 1971 The experiments were concerned with the cor- onary capillary circulation of the left atrium in the potassium chloride arrested cat heart, using transil- lumination. Capillary red cell velocity was measured using cinematography and color films (16 mm 2-1 frames/s) and frame to frame analysis of red cell progression. Optical magnification on the film was from 20 to 30 times. Capillaries were visualized as lying on either side of the muscle fiber. Numerous intercapillary anastomoses which communicate through interconnecting loops of varying lengths were visualised. Capillary diameter varies from 4 to 8 µm. Constancy of capillary red cell velocity was present at from 75 to 210 mm Hg. With increasing perfusion pressure, the number of capillaries with discernible red cell movement increased as per- fusion pressure rose (recruitment). In some capillar- ies, abrupt and extreme changes in red cell velocity were observed at the same perfusion pressure. About 10 to 30% of all capillaries showed counter-current flow in adjacent capillaries. Other support: Council for Tobacco Research- U.S.A., the Los Angeles County Heart Association, and the U. S. Public Health Service. 92. Effect of nitroglycerin on total and regional coronary blood flow in the normal and ischaemic canine myocardium Peter Iviathes, and Jan Rival (Richard j. Bing) ih'ayne State University School of Jbfedicine. Department of bfedicine, Detroit, Michigan Cardiovascular Research 5:54-61, 1971 The effect of nitroglycerin on total coronary flow and its regional distribution was determined in normal conditions and in myocardial ischaemia. In normal hearts, capillary flow was 9.5% higher in the inner than in the outer myocardial half. When partial TIMN 0115853 T200344 109

:d ~a w tv nt i/ a- is ie ic ie at g- )n n- Ii- 1e 1 i ~ ; ~- iX 3 al ct 3- te 31 lx :)f te !d n- ~- , hoaidrial aspartate conten~rad enteacrosso thef a- mi- The increased aspartate o tochondrial membrane observed in the energy-de- pleted state was abolished, while aspartate efflux and glutamate transamination were restored either 1i%- direct addition of ATP or by addition of ace- t.•lcarnitine to increase substrate level of phosphory- l,,tion. These effects were prevented by oligomvcin. indicating that activation of asparate efflux required ,.nergy in a form closely associated with the energy transfer reactions of oxidative phosphorylation. Regulation of flux through citrate synthase, aspartate aminotransferase, and a-ketoglutarate de- h%drogenase as a function of the state of phosphory- lation of the mitochondrial adenine nucleotides was investigated at high NADH:NAD ratios produced by addition of uncoupling agents in the presence and absence of oligomycin. Acetylcarnitine and acetoacetate were used to provide sources of intramitochondrial acetyl coen- zyrne A. The following conclusions may be drawn from the data. In addition to the malate concentra- tion gradient, net a-ketoglutarate efflux is regulated b%, the rate of generation of intramitochondrial c:-ketoolutarate relative to the activity of ca-ketoglu- tarahe dehvdrogenase as determined by product in- hibition. Flux through a-ketoglutarate dehydrogen- ase was inhibited at high ATP:ADP ratios due to the high succinvl-CoA to CoA ratio, with the inhibition potentiated by an increase of the NADH:NAD ratio and relieved by an increased availability of in- tramitochondrial a-ketoglutarate. Production of a- 4.etoglutarate via citrate synthase was also regulated h%the phosphorvlation state of intramitochondrial ,Iclenine nucleotides as indicated by succinyl-CoA inhibition rather than by direct ATP inhibition. Si nce inhibition of citrate synthase by succinyl-CoA h,is been shown to be competitive with acetyl-CoA, it is proposed that regulation is exerted by changes .,t the succinvl-CoA to acetyl-CoA ratio. This reg- ulation is complementary to control of oxalacetate availabilitv by the intramitochondrial NADH:NAD ratio which also regulates a-ketoglutarate produc- tion via both citrate synthase and aspartate ami- notransferase. Coordination of flux between enzytnes which produce intramitochondrial a-keto- glutarate. namely citrate synthase and aspartate ami- notransferase, and a-ketoglutarate dehydrogenase which utilizes it. is determined by complex feedback relationships regulated indirectly both by the ATP:ADP ratio and the NADH:NAD ratio. These provide great flexibility to regulation of the over-all malate-aspartate cycle by controlling efflux of a- ketoglutarate from the mitochondria. Other support: U. S. Public Health Service, and the American Heart Association. 43. Feedback interactions in the control of citric acid cycle activity in rat heart mitochondria Kathryn F. La:v'oue, Jadwiga Bryla, and lohn R. Williamson Johnson Research Foundation. University of Pennsylvania, Philadelphia. Pennsylvania Journal of Biological Chemistrv 247:667-679, 1972 Factors regulating the citric acid cycle have been investigated in rat heart mitochondria oxidiz- ing pyruvate plus malate or acetvlcarnitine plus malate as substrates. Effects caused by changing the NAD oxidation-reduction state, the intramitochon- drial ATP:ADP ratio, and the respiratory rate were studied in five different metabolic states produced by additions of ADP, oligomycin, or uncouplers. The accumulation of cycle intermediates and the mi- tochondrial content of CoA derivatives and pyridine nucleotides were measured in extracts prepared from the whole incubation medium or after rapid separation of the mitochondria from the medium. These data, together with measurements of substrate utilization, were used to calculate flux through the individual steps of the citric acid cycle. Pyruvate dehydrogenase was found not to be rate limiting for citric acid cycle activity. Flux through citrate synthase in State 3 was approxi- mately the same (100 nmoles per min per mg) whether pyruvate or acetylcarnitine were used to generate acetvl-CoA, whereas acetyl-CoA levels were higher with pyruvate as substrate. Flux in State 4 with either substrate was diminished by 75 to 85% relative to State 3 and was associated during early incubation times with elevated levels of both NADH and acetyl-CoA, consonant with regulation of py- ruvate dehydrogenase by product inhibition. A comparison of flux through citrate synthase and changes in the levels of intramitochondrial TIMN 0115835 T200326 91

r Cardiovascular Svstem four lvsosomal enzymes (acid phosphatase. gluc- uronidase, deoxvribonuclease, and y-glutamyl- transpeptidase) were examined in the soluble and the particle-bound fraction. The pattern of changes in free and particle-bound enz«ne activity observed was similar for all four enzymes. During the first 48 hours after coronary occlusion, the particle-bound enzyme activity was decreased, while the free activ- ity was moderately increased, reflecting the auto- lytic phase of cell and tissue destruction. Between the second and the sixth day, the soluble hydrolytic enzyme activity was maximal and the particle- bound activity was slowly increasing. During this period. the main part of tissue degradation and removal of cell debris takes place. Ten days after myocardial infarction the free hydrolytic activity had returned to control values, but the particle- bound enzyme activity was four to ten times higher in the infarcted tissue than in control muscle. Other support: U. S. Public Health Service, the Michigan Heart Association, and the Detroit General Hospital Research Corporation. 54. Early changes in energy metabolism in the myocardium following acute coronary artery occlusion in anesthetized dogs Wolfgang Braasch, Sigmundur Gudbjarnason, Pritpal S. Puri. Kurt G. Ravens. and Richard J. Bing Department of Medicine, Wayne State University School of Nfedicine. Detroit, Michigan Circulation Research 23:429-438. 1968 The tissue content of energy-rich phosphates and gly colytic metabolites and the activity of myo- cardial enzymes were examined in the dog after producing myocardial infarction by ligating branches of the anterior descending and circumflex coronarv arteries. The pattern of systolic fiber move- ment. shown by a strain-gauge assembly, differen- tiated ischemic from nonischemic portions of myo- cardium. In ischemic muscle, 30 minutes after onset of ischemia, creatine phosphate content fell from 8.0 to 1.4 µmoles/g, ATP content fell from 5.8 to 1.5 µmolesJg, lactate content rose tenfold, and a-glyc- erophosphate content rose fivefold. The content of energy-rich phosphates and glycolytic metabolites did not change much in nonischemic muscle. The activities of myocardial enzymes were assayed in 96 extracts of tissue samples from ischemic and non- ischemic muscle 5 and 120 minutes after coronary artery occlusion. The activities of aldolase. lactic dehydrogenase (LDH), glyceraldehydephosphate de- hydrogenase, a-glycerophosphate dehydrogenase, malate dehydrogenase (MDH), and 6-phosphoglu- conate dehydrogenase did not change significantly in ischemic muscle during 2 hours of observation. In nonischemic muscle, phosphofructokinase activity increased 75% 5 minutes after coronary occlusion, followed by an increase in activity of isocitrate dehydrogenase, creatinephosphokinase. MDH, and LDH. The enzymatic changes in nonischemic mus- cle suggest metabolic changes in nonischemic mus- cle accompanying compensatory hyperfunction and increased energy requirements of surviving mus- cle. Othersupport: U. S. Public Health Service, the Mich- igan Heart Association, and the Detroit General Hospital Research Corporation. 55. Stimulation of reparative processes following experimental myocardial infarction Sigmundur Gudbjarnason, John C. Fenton, Paul L. Wolf, and Richard J. Bing Department of Medicine and Pathology, Wayne State University School of Medicine, and Harper Hospital. Detroit, Michigan Archives of Internal Medicine 118:33-40, 1966 The reparative processes in cardiac muscle fol- lowing experimental myocardial infarction were stimulated with anabolic hormones. Myocardial in- farction was produced in dogs by ligation of branches of the anterior and posterior descending coronary arteries. The rate of incorporation of gly- cine-214C into cardiac protein was used as a measure of the rate of protein synthesis. Samples were obtained from infarcted tissue, borderline tissue, and normal left and right ventricular muscles. Treatment with insulin increased the incorpora- tion of glycine-2'4C into protein of infarcted tissue by 94% 24 hours after infarction. Treatment with bovine growth hormone raised the incorporation by 168% and treatment with a combination of growth hormone and insulin raised the incorporation by 251%. Treatment with intravenous infusion of glucose, insulin, KCl, coenzymes and vitamins increased protein synthesis in infarcted muscle seven hours TIMN 0115840 T200331

Cardiovascular Svstem sician must treat not only the organic cardiac dis- order, but also the psychological and socioeconom- ical aspects of his illness. During the stage of acute myocardial infarction reassurance regarding the pa- tient's future is of prime importance in preventing cardiac neurosis. When possible, early activation and ambulation under supervised conditions will significantly shorten the convalescent period and allow him to return sooner to gainful employment. Management of lipid and carbohydrate disor- ders, abstinence from smoking, control of obesity and hypertension, and a program of enhancement of physical fitness are all indicated, not only to restore optimal function, but to prevent further complica- tions of coronary heart disease. A comprehensive approach to the patient and his problems in this way will be helpful in returning him to a useful, productive and enjoyable life. Other support: U. S. Public Health Service; National Heart and Lung Institute Training Grant: and the Chicago Heart Association. r 104. Multistage electrocardiographic exercise test Principles and clinical applications Alberto N. Goldbarg, John F. Moran, and Leon Resnekov Department of Medicine, University of Chicago. Chicago, Illinois American Journal of Cardiology 26:84-92, 1970 Multistage electrocardiographic exercise testing and the physiologic basis for this form of cardiovas- cular stress are discussed. When performed in the proper manner, the technique is safe and is a sensitive measure of cardiovascular performance. In addition, it is useful in the serial evaluation of patients and in the assessment of the effects of drugs, therapeutic regimens and the results of surgical intervention. Its use in diagnosing latent coronary arterial disease in "healthy" individuals is still unknown based on the results of 140 normal subjects studied, in 14 percent of whom abnormal S-T segment changes developed. The test is "noninva- sive" and therefore easily repeatable and is an invaluable aid in the management of patients with heart disease. Other support: U. S. Public Health Service, and the Chicago Heart Association. 105. Central and peripheral vascular effects during cigarette smoking Robert D. Allison, and Grace M. Roth Scott and White Clinic. Temple, Texas and Govelace Foundation. Albuquerque, New Mexico Archives of Environmental Health 19:189-198, 1969 Measurements of blood flow to finger, calf and toe segments, and changes in pulmonary blood pulse volume were obtained using a four-electrode impedance plethysmograph in healthy male smokers. Results of cardiac output changes during cigarette smoking using the impedance system were compared with the indocyanine green (Cardio- Green) dye dilution method of measurement. Smok- ing two cigarettes in succession resulted in a 10% increase in blood pressure, 31% increase in Pulse rate, 2% to 6% decrease in skin temperatures of fingers and toes, a 45% to 50% decrease in blood flow to fingers and toes, and a 28% decrease in pulmonary blood pulse volume. Duplicate measure- ments of cardiac output by dye dilution studies resulted in an increase followed by a decrease. This is compared with a consistent decrease measured by impedance techniques despite an increase in pulse rate. Results of comparative cardiac output studies suggest that cigarette smoking may affect the pul- monary circulation in a more consistent and differ- ent manner than the systemic circulation. 106. An analysis of the effects of nicotine on the cerebral circulation of an isolated, perfused, in situ cat brain preparation Alphonse J. Ingenito, James P. Barrett, and Leonard Procita Department of Pharmacology, Albany Medical College, Albany, New York Stroke 2:67-75, 1971 To determine the effect of nicotine HCl on the cerebral circulation of the cat, without the com- plicating action of the drug at other sites in the body, the drug was perfused at concentrations of 1, 10 and 100 µg/ml through a vascularly isolated, perfused in situ cat brain preparation. Cats having selective section of cranial nerves 9, 10, 11 and 12 and the cervical sympathetic trunks comprised various ex- perimental groups. Nicotine was also perfused TIMN 0115858 114 T200349 9~wn

artery constriction alters the response of coronary vasculature to nicotine. Othe~ support: U. S. Public Health Service; the .,tichigan Heart Association; the Detroit General Hospital Research Corporation. 96. Capacity of the terminal vascular bed during normal growth, in cardiomegaly, and in cardiac atrophy I:arel Rakusan. Wolfgang du Mesnil de Rochemont, Wolfgang Braasch. Hans Tschopp, and Richard J. Bing Department of Medicine. Wayne State University School r,f.kfedicine. Detroit. Michigan C,in:ulation Research 21:209-215. 1967 Vascular capacity representing the terminal vas- cular bed was determined by albumin t"I in the rabbit myocardium during normal and pathological nrowth of the heart. A considerable increase in the vascular capacity in the first postnatal weeks in- dicated growth of the terminal vascular bed during this period. Highest values were reached in animals approximately 6 weeks old. From this time, vascular capacity gradually decreased in relation to the increase in heart and body weight. The growth rate of the terminal vascular bed was very rapid during the first postnatal weeks and later became slower: no growth was detected in adult and old animals. Growth of the terminal vascular bed during patho- logical increase in heart weight followed the same trend. In young animals pathological growth of the heart was accompanied by an increase in the capac- i ty of the terminal vascular bed; in adult animals the total capacity remained unchanged. A decrease in heart weight in animals kept on a protein-free diet %vas characterized by a relatively small vascular c:apacitv. Other support: U. S. Public Health Service, and the Detroit General Hospital Research Corporation. 97. Physiological studies on revascularization of the dog heart Hans N1. Tschopp. Karel Rakusan, Sigmundur Gudbjarnason, and Richard J. Bing Department of Medicine, Wayne State University School of lfedicine. Detroit, Michigan Journal of Thoracic and Cardiovascular Surgery 55:466-478, 1968 Experiments were undertaken in order to evalu- ate the Vineberg procedure (internal mammary ar- tery implantation, epicardiectomv, seropericardiec- tomy, and omentoplexy) carried out at the time of triple coronary artery occlusion by means of Ame- roid constrictors. Capillary blood flow and vascular capacity in specific regions of the canine myo- cardium were investigated by means of rubidium-86 and iodine-131 used simultaneously. A significant reduction of capillary blood flow and a slight decrease of vascular capacity in the left ventricle were found following triple coronary oc- clusion. After revascularization, capillary blood flow and vascular capacity increased significantly in the implantation area. In other regions of the heart treated with epicardiectomy, seropericardiectomy, and omentoplexy alone, no significant changes of capillary flow or vascular capacity could be dem- onstrated. Myocardial enzymes representing the Embden- Meyerhof pathway, the citric acid cycle, and pentose phosphate shunt did not show significant altera- tions. AEh, used as an indicator of the state of cellular oxidative metabolism, was positive in control ex- periments and animals treated with the Vineberg procedure but was negative in non-treated animals with triple coronary artery occlusion. These results indicate that the new source of extracoronary blood supply, brought about by im- plantation of a systemic artery into the ventricular wall, increases the rate and distribution of capillary blood flow and improves oxidative metabolism in the myocardium. Other support: U. S. Public Health Service, the Michigan Heart Association, the Council for To- bacco Research-U.S.A., and the Detroit General Hospital Research Corporation. 98. Cholinergic mechanisms on the heart and coronary circulation Marta I. Blesa and G. Ross Department of Physiology, University of California at Los Angeles School of Medicine, Los Angeles, California British Journal of Pharmacology 38:93-105, 1970 The effects of rapid intracoronary injection of acetylcholine were studied in anesthetized open chest dogs. Changes in phasic coronary blood flow were followed with non-cannulating electromag- TIMN 0115855 T200346 ill

distribution were determined after injection of tracer doses of Dt.-norepinephrine-7-'}C. Normal left ven- tricular muscle contained 0.96 µg NE/g tissue; follotti'ing coronary artery occlusion, the infarcted tissue lost its norepinephrine content completely by the 4th day and the noninfarcted tissue showed a marked decline during the first 10 days, reaching a level as low as 0.14 µg NE/g in the apical portion of the left ventricle. The decrease in myocardial nor- epinephrine content extended to the right ventricle and both atria as well. Norepinephrine levels rose again 2 weeks after infarction and reached normal values 6 weeks after coronary artery occlusion. Despite these changes in endogenous norepineph- rine levels. the myocardial retention of labeled NE, as well as the subcellular distribution of endogenous and exogenous NE. remained unaltered. Upon res- toration of myocardial function, the cardiac nor- epinephrine content returns to normal. Other support: U. S. Public Health Service, and the Michigan Heart Association. 62. Changes in norepinephrine stores in the canine heart following experimental myocardial infarction Peter %tathes, and Sigmundur Gudbjarnason tt'a' i•ne State University. Department of Medicine. Detroi t. Michigan :lmerican Heart Journal 81:211-219, 1971 Serial determinations of the norepinephrine content in all four chambers of the canine heart were t.arried out following surgical myocardial infarction. The observed changes were related to myocardial function and metabolic alterations. Normal left ven- tricle muscle contained 0.98 µg NE per gram tissue, hut following coronary artery occlusion, the in- tarcted tissue lost its norepinephrine content com- pletely br the fourth day, and the noninfarcted tissue yhuwed a marked decline during the first ten days, reaching a level as low as 0.35 µg in the basilar and 0.14 µg NE per gram in the apical portion of the left t•entricle. The decrease in myocardial norepineph- rine content extended to the right ventricle and both atria as well. Norepinephrine levels rose again two weeks after infarction and reached normal values six weeks after coronarv artery occlusion. No correlation was found between norepinephrine content and tissue levels of lactate or ATP in the noninfarcted myocardium. Both the decline and recovery of left ventricular function after infarction were followed, rather than preceded or accompanied by similar alterations in the norepinephrine content. Other support: Michigan Heart Association. 63. Uptake and subcellular distribution of norepinephrine in the canine heart following experimental myocardial infarction Peter Mathes, and Sigmundur Gudbjarnason Department of Medicine, School of Medicine. iti'at•ne State University. Detroit, Michigan Proceedings of the Society for Experimental Biology and Medicine 135:504-508, 1970 Following surgical myocardial infarction in dogs, a marked decline in cardiac norepinephrine stores has been observed. In an attempt to elucidate the mechanism of the NE-depletion, the uptake, degradation, and subcellular distribution of nor- epinephrine in the canine myocardium were de- termined after myocardial infarction. The subcellu- lar distribution, uptake and accumulation of tracer doses of Dt.-norepinephrine-7-"C were unchanged and the rate of degradation was not significantly increased. It appears thus, that the decline in cardiac norepinephrine stores following infarction is neither due to a decreased ability of the sympathetic nerve endings to take up and accumulate NE nor to an increased rate of degradation. Other support: 'Michigan Heart Association 64. Functional compartmentation of ATP and creatine phosphate in heart muscle S. Gudbjarnason, P. Mathes, and K. G. Ravens Departments of llfedicine and Biochemistrt-, iVayne State University, School of Medicine. Detroit. Xfichigan, and Science Institute. University of Iceland, Reykjavik, Iceland Journal of Alolecular and Cellular Cardiology 1:325-339, 1970 The relationship between ATP and creatine phosphate (CP) in heart muscle was studied in two experirnental models. (a) in the intact area of the TIMN 0115843 T200334 99

I 1 ) a through an isolated. denervated hindlimb of the same cat for comparative purposes. Nicotine caused only a mild and transient vasoconstriction of the cerebral circulation, mediated primarily by stimula- tion of the superior cervical ganglia, and a small direct cerebral vasoconstrictor component. An acute tolerance to the cerebral vasoconstrictor effects of repeated increments of nicotine was also observed. The cerebral vasoconstrictor effect of nicotine was diminished in the presence of intact vagi, suggesting a cerebral vasodilator role for these nerves. In contrast. the effect of nicotine on the denervated hindlimb vasculature was a weak but sustained vasodilation. The mechanism of action of nicotine on the cerebral circulation is discussed along with the relevance of these findings to the potential health hazards of tobacco smoking in individuals with cerebrovascular insufficiency. It is concluded that in terms of the nicotine content of tobacco smoke. the hazards of tobacco smoking in the human with cerebrovascular insufficiency would appear to be minimal. 107. Direct central and reflexly mediated effects of nicotine on the peripheral circulation Alphonse I. Ingenito. James_P. Barrett. and Leonard Procita Department of Pharmacology, The Albany Medical College of Union University, Albany, N. Y. European Journal of Pharmacology 17:375-385, 1972 The direct central and some reflexly (chemore- ceptor) initiated cardiovascular actions of nicotine were studied using a vascularly isolated, perfused, in-situ cat brain preparation having neural control over the peripheral circulation. Four different groups were studied relative to the integrity of certain cranial nerves (CN): (a) all CN intact; (b) the carotid branch of CN IX only was sectioned; (c) CN X only was sectioned in the cervical region and (d) CN IX. X. XI and XII were sectioned at the level of the jugular foramina rostral to the nodose ganglia. The effects of perfusion of nicotine hydrochloride through the isolated brain at perfusion blood con- centrations of 1 and 10 µg/ml were as follows: In group (a) the effect appeared to depend on con- centration, a significant hypotension and bradycar- dia occuring only at 10 µg/ml. In group (b) the larger concentration caused a marked hypotension and bradycardia. In group (c) 10 µg/ml caused a sig- nificant hypertension but little effect on heart rate. At 1 µg/ml, the effect was qualitatively similar but not statistically significant. Finally, in group (d), 10 µg/ml caused a significant hypotension and bra- dycardia. These results indicate that when nicotine is restricted to blood perfusing only the carotid bifurcation region and the cerebral circulation, the reflex and direct centrally initiated effects on the peripheral circulation depend to a large extent on the integrity of the afferent and efferent autonomic pathways carried by CN IX and X and possibly to some extent XI and XII. Actions of nicotine con- tributing to the above effects included activation of carotid chemoreceptors leading to both tachycardia and bradycardia and to both hypertension and hypotension. What appeared to be a direct centrally induced hypotension and bradycardia was also dem- onstrated. There was no evidence of a direct cen- trally induced hypertension by nicotine at the above stated blood levels. 108. The effect of glucagon on the coronary circulation in man Nora Goldschlager, Erwin Robin, Charles M. Cowan, George Leb, and Richard J. Bing Department of Experimental Medicine. Wayne State University School of Medicine. Detroit, Michigan Circulation 40:829-837, 1969 Glucagon, 300 µg/minute, was infused intra- venously over 15 minutes in 27 subjects. The pa- tients were divided into three groups: group I, patients without heart disease; group II, patients with arteriosclerotic heart disease; and group III, patients with congestive heat failure. Hemodynamic measurements included observations on myocardial blood flow using bolus injections of 84rubidium and a coincidence counting technic. Myocardial oxygen consumption was determined after coronary sinus intubation in nine of the 27 patients. Significant increases were noted in heart rate, mean arterial pressure, tension-time index/minute and left ven- tricular work. Myocardial blood flow increased sig- nificantly while myocardial oxygen extraction re- mained constant suggesting that the augmentation in blood flow was sufficient to meet the increased TIMN 0115859 T200350 115

intervals (SZ S:). r1synchrony of conduction was frequently observed in response to narrow coupling intbrvals. These differential conduction times in- duced disoarities of activation times greater than 50 msec. In fibers exhibiting preferential depressed conduction, local block was observed with further decrease in the coupling interval. Disparities of activation times at very short S; S, coupling times could be markedly increased by minimal decrease in coupling intervals (in the order of 1 to 5 msec). Conduction depression was not clearly dependent upon the level of "take-off" potential or action potential duration. Asynchronous conduction may thus be induced by narrow coupling of premature beats and could account for reentry. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 117. T Wave alternans: An association with abrupt rate change Charles Fisch. Robert E. Edmands, and Kalman Greenspan Department of Medicine. Indiana University School of Medicine. and the Krannert Institute of Cardiology. Marion Count~• General Hospital. Indianapolis, Indiana American Hea, n: Journal 81:817-821. 1971 A case of alternation of T wave with reversal of polarity, independent of any changes of the QRS but apparently greatly influenced by abrupt cycle length change, is presented. Evidence suggests that this phenomenon is indicative of severe mvocardial disturbance. Other support: Herman C. Krannert Fund, U. S. Public Health Service, and the Indiana Heart As- sociation. 118. Electrophysiologic correlate of exit block Kalman Greenspan, Gary J. Anderson, and Charles Fisch l:rannert Institute of Cardiology (Marion County General Hospital), the Department of Medicine, Indiana t'niversity School of Medicine, Indianapolis, Indiana .-Imerican Journal of Cardiology 28:197-200, 1971 Electrophysiologic studies using canine and human tissue confirm, by direct recordings at the cellular level, the existence of exit block produced by artificial pacemakers and shed some light on the mechanism of the exit block. Both human ventricu- lar and canine Purkinje or ventricular tissue, or both, demonstrated Wenckebach structure from the stimu- lus electrode to the recorded action potentials. This conduction depression was induced by hypoxia. digitalis or infusion of 4 times the normal con- centration of potassium (10.8 mEq/liter). Both the exit block and the Wenckebach structure of the block recorded in these experiments are analogous to those seen in clinical situations. In addition, electrophysi- ologic evidence for unidirectional block is pre- sented. Other support: Herman C. Krannert Fund, U. S. Public Health Service, and the Indiana Heart As- sociation. 119. Role of the premature action potential in contractile potentiation: A Study of paired stimulation R. E. Edmands, K. Greenspan, and J. C. Bailey Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana L'niversih• School of Medicine, Indianapolis, Indiana Cardiovascular Research 6:368-374, 1972 Isometric contractions and transmembrane ac- tion potentials, recorded simultaneously from ca- nine ventricular tissue, revealed a consistent relation between action potential configuration and the in- tensity of the associated contractile performance during paired stimulation and the return to the control state. The nature of this association consisted of an inverse relation between the magnitude of the contractile response and the duration of phase two of the associated action potential. This relation persists despite a characteristically non-linear augmentation of contractile force effected by paired stimulation. A second correlation was also observed between the phase two duration of each premature action po- tential and the magnitude of the subsequent con- tractile increment. It is speculated, then, that the premature action potential may be indirectly im- plicated in this type of contractile augmentation. Variation in the phase 2 prolongation of premature action potentials may reflect a variable calcium influx which, in turn, yields the observed diversity of contractile increments in subsequent post-extra- systolic beats. Other support: U. S. Public Health Service, rhe Herman C. Krannert Fund, and the Indiana Heart Association. TIMN 0115863 T200354 119

I ,. septal fibers of the left bundle branch system and that this defect could underlie the transient right precordial Q waves seen in myocardial infarction or ischemia, as well as the fixed Q waves of many patients without septal infarction at autopsy. Other support: The Myocardial Infarction Research Unit. Government Contract. 124. Some effects of nicotine on cardiac automaticity, conduction, and inotropy [:alman Greenspan. Robert E. Edmands. Suzanne B. Knoebel. and Charles Fisch Department of Medicine. Indiana University School of .tifedicine. and the Krannert Institute of Cardiology. Marion Countv General Hospital. Indianapolis. Indiana Archives of Internal Medicine 123:707-712. 1969 Papillary and trabecular muscles were isolated from the right ventricle of dogs. Action potentials were recorded from either ventricular or Purkinje's cells. Nicotine administered in high concentrations produced a positive inotropic effect that appeared to be independent of adrenergic innervation. The ac- companying action potentials exhibited an abbrevia- tion of phase 2 without change in total action potential duration, and the extent of phase 2 short- ening bore a direct relationship to the relative magnitude of contractile augmentation. The pro- pensity of low concentrations of nicotine (2 to 4 µg/ ml) to initiate and enhance cardiac automaticity was also observed in these in vitro preparations. Possible consequences in terms of predisposing to the de- velopment of serious, possibly fatal arrhythmias are discussed. Other support: Public Health Service; Indiana Heart Association. 125. High-fidelity recording of cardiac depolarization Gary J. Anderson, Kalman Greenspan, Jack P. Bandura, and Charles Fisch Department of Medicine, Indiana University School of 1ledicine, and Krannert Institute of Cardiology, Marion County Geneial Hospital, Indianapolis, Indiana Journal of Applied Physiology 29:401-405, 1970 Transmembrane action potentials in dogs were recorded with rrlicroelecirodes and simultaneously displayed at rapid oscilloscope sweep speeds, thus enabling display of the more rapid components (phase 0) of the action potentials. With a memory oscilloscope, accurately synchronized to the tape system or pulse generator, oscilloscopic sweep speeds up to 5 x 107 mm/sec displaying phase zero could be attained, thus allowing careful examination of each component of the monophasic action po- tential. This technique also permits superimposition of action potentials either serially or at selected times, providing accurate comparison of data re- cordings. The determinants of accurate phase 0 recordings are those of adequate amplifier frequency response and accurate synchronization of data to oscilloscope. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociation. 126. The role of inotropic variation in ventricular function during atrial fibrillation Robert E. Edmands, Kalman Greenspan, and Charles Fisch Department of Medicine, Indiana University School of Medicine and the Krannert Institute of Cardiolo&•. Marion County General Hospital. Indianapolis. Indiana Journal of Clinical Investigation 49:738-746, 1970 A series of experiments were performed upon intact anesthetized dogs to determine the relevance of a variety of hemodynamic variables to the irregu- lar ventricular performance associated with atrial fibrillation. During experimentally induced atrial fibrillation central aortic pulse pressure was mea- sured in relation to the duration of the preceding diastolic interval, the relative degree of cycle-length change, the magnitude of the preceding aortic end- diastolic pressure, the rate of ventricular tension development (at a fixed diastolic tension), and to ventricular end-diastolic pressure. While all of the latter variables bore a significant relation to the chosen parameters of ventricular function, the most linear correlation lay with the rate of ventricular tension development. It has been suggested, as a consequence, that the irregular ventricular perfor- mance observed during atrial fibrillation under these experimental conditions, may be more directly re- lated to variation in the inotropic state of the ventricular myocardium than to an expression of the Frank-Starling concept, resulting from variable ven- TIMN 0115865 T200356 121

Cardiovascular Svstem netic flow probes and in contractile force with isometric strain gauges. Increasing doses of acetvlcholine from 0.01 to 100 µg produced progressively larger increases in systolic and diastolic coronary blood flow and progressive decreases in end-diastolic vascular re- sistance which were blocked by atropine but not by propranolol. Contractile force showed both negative and positive responses. The negative inotropic effect was small and was blocked by atropine but not by propranolol. The threshold for the negative inotro- pic response was higher than for the coronary vaso- dilator effect and the dose response curve was flatter. The positive inotropic response usually showed two components. One component reached its maximum 13 to 18 sec after injection, had a high threshold (over 1 µg), was potentiated by atropine and blocked by propranolol. The other reached its maximum 25 to 60 sec after the injection, had a threshold between 0.01 and 0.1 µg, and was blocked by atropine but not bv propranolol. These results suggest that the coronary dilator response, the negative inotropic response and part of the positive inotropic response are mediated through "muscarinic" receptors. The remaining component of the positive inotropic response appears to involve catecholamine release. 99. Effects of diphenylhydantoin (Dilantin) on peripheral and coronary circulation and myocardial contractility in the experimental animal D. N. Gupta. %i. O. Unal, F. A. Bashour, and W. R. Webb Departments of Surgery and Medicine. University of Texas Southwestern Medical School, Dallas, Texas Diseases of the Chest 51:248-255, 1967 The effects of diphenylhydantoin have been investigated in mongrel dogs with intact circulation and in situ heart-lung preparations in the open-chest dog. Diphenylhydantoin in increasing dosage re- sulted in a slight decrease in the cardiac output, systemic arterial pressure and heart rate. A transient negative inotropic effect lasting up to five minutes was observed. Coronary blood flow showed a marked increase which was ascribed to a direct vaso- dilating effect on the coronarv vessels. Mvocardial oxygen consumption remained unchanged during a constant workload. Other support: U. S. Public Health Service, and Parke-Davis. 100. Origin of blood supply to sinoauricular and atrioventricular node Donald W. Romhilt, Donald B. Hackel, and E. Harvey Estes, Jr. Department of lfedicine and Pathology. Duke C'nii•ersitt• :4fedical Center. and the Medical Service, Durham Veterans Administration Hospital. Durham. North Carolina t1 merican Heart Journal 75:279-280, 1968 A total of 192 hearts have been studied by postmortem coronary artery injection and the origins of blood supply to the sinus node, atrioventricular node, and posterior descending artery determined. The artery to the sinus node was found to originate from the right in 60.5 per cent and from the left in 38 per cent, with 1.5 per cent from both. The artery to the atrioventricular node was from the right in 84.7 per cent and from the left in 15.3 per cent. The origin of the posterior descending artery was from the right in 81 per cent and from the left in 15.8 per cent, with a dual supply in 3.2 per cent. Female subjects have an even greater frequency of origins from the right side, with the atrioventricular nodal artery occurring from the right in 93.75 per cent and the posterior descending artery as a continuation of the right coronary in 89.1 per cent. Other support: U. S. Public Health Service. Clinical Studies 101. Coronary collateral circulation and myocardial blood flow reserve Suzanne B. Knoebel, Paul L McHenry, John F. Phillips, and Ferrel J. Pauletto Krannert Institute of Cardiology, Marion County General Hospital, and the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana Circulation 46:84-94, 1972 This study was undertaken to assess the effect of collateral circulation on myocardial blood flow (MBF) reserve (ability to increase myocardial blood flow with a stress that increases myocardial oxygen requirements). One hundred patients had MBF mea- t c F d 112 TIMN 0115856 T200347

Cardiovascular System mvocardial demands for oxygen. The _ effects of glucagon on the coronary circulation resemble that 'of isoproterenol rather than norepinephrine without, however, leading to the production of arrhythmias seen with these catecholamines. Other support: U. S. Public Health Service, the Michigan Heart Association, and the Detroit General Hospital Research Corporation. 109. The effect of cardioacceleration by right atrial pacing on myocardial blood flow in normal human subjects Suzanne B. Knoebel, William C. Elliott. Edward Ross. and Paul L. McHenry Department of Medicine, Indiana University School of Medicine, and the Krannert Institute of Cardiology, Marion County General Hospital, Indianapolis, Indiana Cardiovascular Research 4:306-311, 1970 The myocardial blood flow response (MBF), measured by a coincidence counting technique and 84 RbC1, to cardioacceleration by right atrial pacing, was studied in 18 normal subjects. MBF was found to increase at approximately 130 beats/min but decreased at more rapid rates (150 beats/min). Fur- ther study will be required to resolve the problem of this narrow range of response in order to validate pacing studies in the evaluation of serial changes in coronary reserve. Other support: the Herman C. Krannert Fund, U. S. Public Health Service, the Indiana Heart Associa- tion, and the Marion County Heart Association. during the development of hypertrophy induced by pressure loading. Transmembrane potentials and isometric active force at optimal length (Po) were monitored in isolated right ventricular papillary muscles and trabeculae carneae after inducing chronic right ventricular outflow tract obstruction. Cats subjected to partial pulmonary artery occlusion or to sham operation were studied 1, 3, 7, 10, 21, and > 90 days after surgery. Right ventricular muscles were stimulated at 30/min at optimal length in Tvrode's solution (36°C). Muscles from sham-op- erated and 1-day pressure-loaded hearts had electri- cal and mechanical properties similar to normal muscles. Right ventricular peak systolic and end- diastolic pressures were increased for 1 and 3 day pressure-loaded hearts; the percentage of right ven- tricular water was increased for 3, 7, and 10 day pressure-loaded hearts. Muscles from 3 day pres- sure-loaded ventricles had significantly decreased Po, maximum rate of force development, time to peak force, and duration of contraction compared with normal muscles, and their action potential plateaus originated at more negative potentials (mean change was 24.7 mv for 11 muscles). The alteration in action potential configuration was less obvious in muscles studied 7 and 10 days after partial pulmonary artery occlusion and did not occur 21 and > 90 days after pressure loading; however Po and maximum rate of force development remained decreased, although time to peak force and duration of contraction returned to normal for 7, 10, 21, and > 90 day pressure-loaded ventricles. An account of this study has also been pub- D. Electrophysiological Studies and Studies on lished in Recent Advances in Studies on Cardiac Contractility Structure and Metabolism, Volume 4. Myocardial Fundamental Studies 110. Chronic partial ocdusion of the pulmonary artery in cats. Change in ventricular action potential configuration during early hypertrophy Arthur L. Bassett, and Henry Gelband Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York Circulation Research 32:15-26, 1973 Biology. N. S. Dhalla, Editor. University Park Press, 1974. pp 3-20. Other support: U. S. Public Health Service. 111. Electrophysiological studies on organization of central vasopressor pathways Gerard L Gebber, David G. Taylor, and Lynne C. Weaver Department of Pharmacology, Michigan State University, East Lansing, Michigan American Journal of Physiology 224:470-481, 1973 The relation of single cell electrical activity to A study was made of the responses evoked in generation of force was determined for myocardium the external carotid postganglionic sympathetic nerve of the cat by single shocks and trains of stimuli TIlliIN 0115860 T200351 116

cal-electrophy'siological correlation. During com- plete AV block the NH region showed the greatest degree of spontaneous phase 4 depolarization. Al- though the penetrating and branching parts can develop automaticity, their rate of activity was less than NH cells. These studies have demonstrated the feasibility of dissection and for intracellular studies of the conducting system to assess possible mecha- nism(s) of cardiac arrhythmias. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 130. Automaticity: Mechanisms and electrocardiographic consequences John C. Bailev. Gary J. Anderson. and Kalman Greenspan Krannert Institute of Cardiology. Marion Countr General Hospital. Department of Medicine. Indiana t'niversitr School of.kiedicine. Indianapolis, Indiana Journal of the Indiana State Medical Association 65:110-113, 1972 The property of automaticity, the consequence of complex ionic and electrical events at the cellular level. is influenced by a host of physiological, pharmacological and physical agencies. Changes in the automatic process result in a variety of arrhyth- mias which may be due to alteration of the normal sinus pace-maker, induction and acceleration of latent pacemakers, or a combination of both mech- anisms. The present paper describes and illustrates the above. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 131. Mechanical and electrical actions of nicotine on hypertrophied and failed cat ventricle Arthur L. Bassett. Jay R. Wiggins, and Henry Gelband Department of Pharmacology, College of Physicians and Surgeons of Columbia University. New York, New York, and Department of Surgery. Pharmacology and Pediatrics, University of Miami School of Medicine. Nfiami, Florida Proceedings of the Society for Experimental Biology and lfedicine 146:132-136, 1974 Nicotine increases peak active isometric force in sham operated and right ventricular hypertrophied (RVH) muscles by indirect (catecholamine-me- diated) and direct effects. In such muscles, action potential plateau and action potential duration even- tualh• lengthen during exposure to nicotine (6.2 x 10 }tii and 6.2 x 10-'M). vtuscles from cats in overt ventricular failure (RVF) showed significantly smaller transient positive inotropic responses dur- ing exposure to nicotine than sham and RVH mus- cles. RVF muscles demonstrated an increased main- tained peak active force during exposure to 6.2 x 10 ,VI nicotine. Other support: U. S. Public Health Service. 132. Nicotine and the action potential of cat ventricle Arthur L. Bassett, and Henry Gelband Department of Pharmacology. College of Physicians and Su beons. Columbia Universitr. New York. .1'eir York and Departments of Pharmacology. Sur;er}• and Pediatrics. University of Miami School of Medicine, Miami. Florida Journal of Pharmacology and Experimental Therapeutics 188:157-165, 1974 Nicotine has both indirect (catecholamine-me- diated) and direct effects on cardiac contractility. Microelectrode recordings showed that the action potential plateau often shortened during the tran- sient positive and negative inotropic effects induced by low concentrations of nicotine (3.1-6.2 x 10-'M) in normal cat ventricular muscles. Shortening of the action potential plateau was not evident in muscles treated with beta adrenergic receptor blocking agents, propranolol and alprenolol (10-6 to 10-5Ivi), and in muscles removed from reserpine-treated cats. However, higher concentrations of nicotine (6.2 X 10-'` to 6.2 x 10-'M) lengthened action potential plateau and action potential duration in beta adrenergic receptor-blocked muscles and in muscles from reserpine-pretreated cats. The direct effects of high concentrations of nicotine to increase isometric force may be related to increased inward calcium current flowing during longer action potential pla- teau durations and/or release of membrane bound calcium. A preliminary report of this study appeared in Circulation 42:suppl 3, 123, 1970. Other support: Program Project Grant from the National Heart and Lung Institute. TIMN 0115867 T200358 123

1 applied to pressor regions of the brain and spinal cord. The levels of the neuraxis explored included the hypothalamus, midbrain, medulla, and cervical spinal cord. The evoked potentials were analyzed with an average-response computer. Two distinct systems of vasopressor pathways were identified at each of the levels explored. Sympathetic nerve responses evoked from the more slowly conducting system of pathways were inhibited by baroreceptor reflex activation. Postganglionic potentials evoked from the more rapidly conducting system were not blocked by baroreceptor reflex activation. Potentials evoked from both systems also were distinguished on the basis of onset latency, duration, ability to follow high-frequency stimulation, and response patterns to single shocks and trains of stimuli. It is concluded that vasopressor outflow from the brain to the external carotid nerve is organized into two systems of parallel pathways, each of which is related differently to the baroreceptor reflex. Other support: U. S. Public Health Service. 112. The effect of change in cyde length on the ventricular action potential in man Charles Fisch. Robert E. Edmands, and Kalman Greenspan Department of Medicine, Indiana University School of Nledicine, and the Krannert Institute of Cardiology, Marion County General Hospital, Indianapolis. Indiana American Journal of Cardiology 21:525-529, 1968 Electrophysiologic studies of human papillary fibers disclosed a characteristic alteration in the configuration of action potential, associated with abrupt change in the cycle length. After a relative prolongation of the cycle, phase 2 was abbreviated and phase 3 lengthened. Conversely, a relative abbreviation of the cycle was terminated by an action potential with a more sustained phase 2 and a more precipitous phase 3. In either case the total duration of the action potential remained un- changed. The physiologic mechanism underlying these changes in action potential, although obscure, may represent a primary alteration in repolarization possibly caused by time-dependent changes in mem- brane permeability to K+. This pattern of change in action potential, associated with abrupt alteration in rate. is clearly at variance with the conventional concept of a direct relation between duration of phase 2, the total action potential and the length of the preceding diastole. The latter relation appears to hold true only if the preparation is driven at a constant rate. It is suggested that this interval-dependent al- teration in repolarization may account for this so- called change in post-extrasystole T wave that is occasionally observed in the surface electrocardio- gram. The absence of such T wave changes in normal subjects suggests that these changes in repolarization may be of insufficient magnitude in the normal subject to be recorded in the surface electrocardiogram. A temporal relation has been observed between the magnitude of contractility and morphology of the human action potential. Similar observations have been made in the dog. Whether the parallel between morphology of action potential and po- tentiation of contractility reflects a causal relation between inotropic phenomena and membrane per- meability remains to be seen. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociation. 113. The pharmacological effects of potassium: The occurrence of sino-ventricular conduction Kalman Greenspan, and Marcelo V. Elizari Krannert Institute of Cardiology, Marion County General Hospital, and the Department of Medicine, Indiana University School of Medicine. Indianapolis, Indiana Journal of Clinical Pharmacology 14:155-162, 1974 Sino-ventricular conduction persists at high levels of potassium while arterial activity and the P- wave on ECG are absent, and this has been attributed to the presence of potassium resistant fibers in the septum. The authors consider the relationship be- tween septal activation and P-waves and the effects of potassium infusion in dogs in terms of three stages of infusion:initial, intermediate, and late. In addition, a method of mechanically crushing the tissue around the SA node is described. Results and conclusions are, 1) the SA to AS interval remains constant with potassium infusion, whereas other intervals are increased. Also, right and left atrial activities disappear before SA and AS activities. Therefore, potassium infusion seems to inactivate TIMN 0115861 T20p352 117

Cardiovascular System 120. Effects of acetylcholine on automaticity and conduction in the proximal portion of the His- Purkinje Purkinje specialized conduction system of the dog John C. Bailey. Kalman Greenspan, Marcelo V. Elizari. Gary J. Anderson, and Charles Fisch Krannert Institute of Cardiology, Marion County General Hospital. Department of Medicine, Indiana University School of Medicine. Indianapolis, Indiana Circulation Research 30:210-216, 1972 Conventional intracellular recordings from the bundle of His and right bundle branch of the canine heart demonstrated that the slope of diastolic de- polarization is markedly depressed by superfusion with relatively small concentrations (4-8 µg/ml) of acetylcholine. As the cells become less automatic, take-off potential increases, rise time of phase O.is reduced, action potential amplitude increases, and conduction proceeds more rapidly. Other support: U. S. Public Health Service, Herman C. Krannert Fund, and the Indiana Heart Associa- tion. 121. Digitalis and vagal stimulation during atrial fibrillation: Effects on atrioventricular conduction and ventricular arrhythmias Kalman Greenspan, and Thomas J. Lord Krannert Institute of Cardiology, Marion County General Hospital. Department of Medicine, Indiana University School of Medicine. Indianapolis, Indiana Cardiovascular Research 7:241-246, 1973 The magnitude of vagal depression of AV con- duction in dogs with atrial fibrillation is enhanced by increasing doses of digitalis. For this reason, a decrease of ventricular rate with vagal stimulation cannot be used as evidence that additional digitalis can or should be administered. Vagal stimulation, by inhibiting atrial impulses from reaching the ven- tricules, may unmask digitalis toxicity in the form of enhanced His-Purkinje automaticity. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 120 122. Effects of cyde-length alteration on canine cardiac action potentials Kalman Greenspan, Robert E. Edmands. and Charles Fisch Krannert Institute of Cardiology, Nfarion County General Hospital, and Department of Medicine, Indiana University School of Medicine, Indianapolis. Indiana American Journal of Physiology 212:1416-1420, 1967 Electrophysiologic studies in canine hearts demonstrate a characteristic change in the atrial action potential following abrupt rate changes. The action potential terminating a relatively prolonged cycle length exhibits shortening of phase 2 and lengthening of phase 3. In contrast, the action potential which concludes a relatively short interval generally displays lengthening of phase 2, with acceleration of terminal repolarization. These latter changes appear comparable to those previously described in ventricular cells. Purkinje cells, how- ever, do not manifest this type of action potential change with abrupt rate alteration. Over a range of stimulus frequency, 30-200/min the duration of Purkinje fiber action potential varies directly with the length of the preceding cycle length. This relation obtains irrespective of abrupt rate changes and is in accord with the more orthodox concept of phase 2 dependency on the duration of the preced- ing interval. A temporal relation between atrial action potential change and contractile potentiation, comparable to that observed in ventricular tissue, is described. This relation, observed in atrial and ventricular tissue, is contrasted with the behavior of Purkinje fibers. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociation. 123. Rate-dependent right precordial Q waves: "Septal focal block" Miguel Gambetta, and Rory W. Childers Department of Medicine, University of Chicago, Chicago, Illinois American Journal of Cardiology 32:196-201, 1973 Rate-dependent pathologic Q waves in leads V, to V, were seen in two patients with myocardial infarction. Months later this conduction defect cculd be recalled with atrial premature beats, or as a continuing rate-dependent phenomenon. It is sug- gested that these cases represent a focal block in the - TIMN 0115864 T200355 ~

Cardiovascular System ordinarv atrial muscle but conduction between the sino-atrial node and atrial septum is preserved. This ,fixed temporal relationship could be related to the `presence of specialized fibers between the SA and AV nodes. that can maintain the resting transmem- brane potential necessary for conduction in an increased potassium environment; 2) whether or not a P-wave will be seen on ECG depends upon the degree of decremental conduction. With a minimal degree of same, a significant amount of septal tissue is activated and a P-wave recorded. Conversely, if the decrement is rapid, very little septal tissue will be activated and therefore no P-wave recorded, but enough septal tissue may be depolarized to maintain conduction and will be recorded as sino-ventricular conduction. However, as the potassium concentra- tion increases, conduction velocity decreases, and in some of the experiments AV dissociation occurred before sino-ventricular conduction was observed. Finally, mechanical crushing of the atrial tissue around the sino-atrial node differentiated sino-ven- tricular conduction from accrochage between SA and the ventricles. Other support: the Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart Association. 114. Electrophysiological correlates of contractile change in mammalian and amphibian myocardium Robert E. Edmands. Kalman Greenspan, and Charles Fisch Department of .tifedicine, Indiana University School of Medicine, and the Krannert Institute of Cardiology. .Llarion County General Hospital. Indianapolis, Indiana Cardiovascular Research 2:252-260, 1968 Studies of canine and frog myocardium disclose a consistent inverse relation between interval-de- pendent contractile alterations and the duration of phase 2 of the concomitant action potentials (AP). This is true despite the differing temporal patterns of contractile change in the two species. A relationship of both the inotropic state and AP configuration to K' flux is suggested. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociation. 115. Mechanisms of ventricular fibrillation Kalman Greenspan Krannert Institute of Cardiology. Marion County General Hospital, and the Department of ,'btedicine. Indiana University School of Medicine, Indianapolis. Indiana Cardiac Arrhythmias. Leonard S. Dreifus, and William Likoff. Editors. Grune and Stratton. Inc.. 1973. E)p 19S_ 202 Intracellular electrophysiologic techniques have provided little direct information regarding the gen- esis and perpetuation of fibrillation. A myriad of transmembrane recordings would be required to appreciate the simultaneous disparities of action potential duration, excitability, and refractoriness and the multiple sites of conduction delay and block that must exist if fibrillation is to be initiated and sustained. Nevertheless, numerous studies of fibril- lation utilizing two or more intracellular microelec- trodes have been reported, and there appear to be but a few underlying mechanisms that, alone or in combination, must operate to produce and maintain fibrillation. In this chapter, certain properties of the neces- sary premature stimulus are reviewed and how conduction depression favors this particular arr- hythmia is discussed. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 116. Asynchrony of conduction within the canine specialized Purkinje fiber system Gary J. Anderson. Kalman Greenspan, Jack P. Bandura, and Charles Fisch Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana Circulation Research 27:691-703, 1970 Asynchrony of conduction may prove to be an important mechanism for reentrant arrhythmias. The purpose of these experiments was to explore asyn- chronous conduction in the distal branches of the canine Purkinje system. Microelectrodes were placed in Purkinje bundle preparations resembling a T configuration, thereby permitting assessment of differential conduction times induced by premature beats (SZ). Equal conduction depression was observed in the post extrasystolic beat (S1) at wide coupling TITNIN 0115862 T200353 118

Cardiorascular Srstem 133. The effects of nicotine upon the cardiac action potential and contractile state K. Greenspan. R. E. Edmands. S. B. Knoebel. and C. Fisch Department of Medicine Indiana University School of Nledicine. and the Krannert Institute of Cardiologg}•. Marion CounhY General Hospital. Indianapolis. Indiana Physiolo6ist 10:187. 1967 __ r1 series of experiments was initiated to de- termine the effects of nicotine upon the inotropic state and action potential (AP) configuration of canine ventricular mvocardium. Initiallv obtained in the control state. recordings were made of AP and isometric contractile responses at a fixed stimulus rate. Nicotine was then introduced at concentrations varving from 100-1000 µg/cc. Contractile enhance- ment, without change in time-to-peak force develop- ment. was observed, and most strikingly with the higher concentrations of nicotine. In addition, a characteristic AP change was consistently observed in association with the inotropic enhancement; phase 2 abbreviation was noted, as was phase 3 prolongation with little or no change in total AP duration. A more prominent phase 2 abbreviation was observed with the more striking contractile enhancement produced by higher concentrations of nicotine. Comparable electrophysiological changes in canine Purkinje fibers were also observed, that is, -phase 2 attenuation, unattended by any significant change in resting potential. Less commonly ob- served changes included loss of resting potential and overshoot, decreased rate of rise, and develop- ment of a "step." The complex, thus altered, closely resembled that customarily recorded from AV nodal fibers. It is suggested, therefore, that both the ino- tropic response and the AP changes effected. by nicotine may be associated with an increase in K- efflux or membrane permeability to K-. Other support: Herman C. Krannert Fund; U. S. Public Health Service; and the Indiana Heart As- sociation. 134. Cardiac arrhythmias induced by nicotine: Electrophysiological mechanism(s) K. Greenspan Krannert Institute of Cardiology. Marion Countv General Hospital. and the Department of Medicine. Indiana t'niversih• School of Medicine. Indianapolis. Indiana Research in Phrsiology. F. F. Kao. K. Koizumi. and 14 Vassalle (Editors). Aulo Gaggi Publisher. Bologna. 1971. pp 189-196 This study reports some complex arrhythmias engendered by nicotine, in both the intact dog and in the isolated superfused, myocardial tissue. This dysrhvthmia included premature ventricu- lar beats, AV dissociation with accelerated junc- tional and ventricular pacemakers and ventricular tachvcardia with Wenckebach exit block. These adverse effects may be attributed to both conduction depression and induction of automatic- itv. And, the conduction depression was of sufficient degree to induce reentrance excitation. Furthermore, the effect of nicotine on propagation may not be related solely to the direct effect of nicotine on maximum rising velocity and resting potential. Since nicotine demonstrates the capability of inducing diastolic depolarization, conduction may be depressed in that propagation is proceeding down partially repolarized fibers, again a factor contributing to the development of decremental conduction. Variations in cellular susceptibility to the al- kaloid was noted and this mav have a clinical counterpart in that nicotine may demonstrate ad- verse effects readily in some patients, while other individuals appear to tolerate large doses. Other support: The Herman C. Krannert Fund; U. S. Public Health Service; and the Indiana Heart As- sociation. 135. Biphasic effect of nicotine on action potential repolarization in electrically driven guinea-pig atria Achilles J. Pappano Department of Pharmacology. University of Connecticut Health Center, McCook Hospital. Hartford. Connecticut Journal of Pharmacology and Experimental Therapeutics 172:255-265, 1970 Intracellular action potentials were recorded from cells in the spontaneously beating sinoatrial TIMN 0115868 124 T200359 ~. .

Cardiovascular System tive to large c:oncentrations of tetrodotoxin (3 x 10-`I) but antagonized by %In'- (0.2mM). These data support the proposal that the catecholamines restored excitabilitv bv increasing membrane con- ductance to Ca'-. In addition to the catecholamines, the divalent cations Ba'-. Srz-, and Ca2- restored action potentials to atrial muscle fibers depolarized by elevated K-. The effectiveness of the divalent ions in allowing action potentials was inversely related to the estimated hvdrated ionic radii. Like the action potentials observed in the presence of isoproterenol, those permitted by Ba'-. Sr--, and Ca'- were pre- vented bv Mn=- but insensitive to blockade b_v tetrodotoxin. 138. Propranolol-insensitive effects of epinephrine on action potential repolarization in electrically driven atria of the guinea pig Achilles J. Pappano Department o.f Pharmacology. Universitr of Connecticut Health Center. 1fcCook Hospital, Hartford. Connecticut /ournal of Pharmacology and Experimental Therapeutics 177:85-95. 1971 Transmembrane action potentials were recorded from cells in the electrically driven left atrial ap- pendage of the guinea-pig heart bathed in Tvrode's solution at 30°C. Epinephrine, isoproterenol and norepinephrine produced modifications in the pla- teau and the phase of rapid repolarization without changing the resting potential. All of the catechol- amines increased the duration of the plateau phase of the action potential. Modest concentrations (< 10-5%i) of epinephrine and norepinephrine in- creased action potential duration (measured when repolarization was 80% complete), whereas large concentrations (> 10-6M) tended to decrease the 80°o duration. At all concentrations examined, iso- proterenol did not appreciably change the 80% duration. Characteristically, the actions of isoproter- enol on the action potential were associated with an increase in developed tension and propranolol com- pletelv prevented the changes in electrical and mechanical activity caused by this agent. Large concentrations of epinephrine also increased de- veloped tension whereas modest concentrations of this agent were usually ineffective in this regard. 1,Soreoc er. the abilitv of modest concentratic;ns of 126 epinephrine to increase the 80% duration was in- sensitive to blockade by propranolol and was an- tagonized by phentolamine and dihvdroergotamine. It is concluded that atrial muscle cells in the guinea pig display two pharmacologically distinct adreno- ceptive sites. Fundamental Studies with Reference to .\'icotine and Smoking 139. The effect of inhalation of cigarette smoke on ventricular fibrillation threshold in normal dogs and dogs with acute myocardial infarction Samuel Bellet, Norberto T. DeGuzman. John B. Kostis, Laurian Roman. and Dietrich Fleischman Dic,ision of Cardiologl•, Philadelphia General Hospital. Philadelphia. Pa. American Heart Journal 83:67-76, 1972 The effect of inhalation of cigarette smoke on the electrical ventricular fibrillation threshold (VFT) was studied in normal dogs and dogs with acute myocardial infarction produced by ligation in two stages of the left coronary artery. The animals were subjected to inhalation of the smoke from 3 ciga- rettes, each containing approximately 2 mg nicotine, in 10 minutes. The electrical impulses were de- livered through the chest wall in one group of experiments, whereas in another group of experi- ments a gated series of impulses was delivered directly to the heart through previously implanted epicardial electrodes. A decrease in VFT that av- eraged 30 to 40% of the control value was observed in normal dogs as well as in those with acute myocardial infarction. This effect of inhalation of cigarette smoke was evident 30 minutes after smok- ing, became maximum at about 34 minutes, and lasted for about 90 to 120 minutes. In the dogs with myocardial infarction, the VFT was lower than in normal dogs and was decreased further after in- halation of cigarette smoke. Possible mechanisms by which the inhalation of cigarette smoke decreases the VFT might be the direct effect of nicotine on the myocardium and the nicotine-induced adrenergic stimulation. These findings are of interest in view of the increased incidence of sudden death observed among coronary patients who are heavy cigarette smokers. Other support: National Institutes of Health, and the Council for Tobacco Research-U.S.A. TIMN 0115870 T200361

i I 1 140. Hypoxia in the genesis of cardiac arrhythmias fohn C. Fen'ton. Sigmundur Gudbjarnason. and Richard 1. Bing Department of Nfedicine. Wayne State University School ()1'.lfedicine- and Harper Hospital. Detroit. Michigan .lfechanisms of Therapy of Cardiac Arrhythmias. pp 58- 63. L. S. Dreifus and W. Likoff, Editors. Grune and Stratton. 1966 Changes in membrane function which occur ~ti•ith oxygen deprivation can predispose or contrib- ute to almost any disturbance of rhythm. It is of interest and significance, however, that in the ab- sence of coronary occlusion, pure hypoxemic hyp- oxia cery rarelv leads to ventricular fibrillation. Ventricular asystole occurs, due either to SA node failure or to cessation of atrioventricular conduction. In the ischemic dog, heart excitability is the first of the vital cell functions to disappear. Othersupport: U. S. Public Health Service, the Mich- igan Heart Association, the Council for Tobacco Research-U.S.A.. the Burroughs-Wellcome Fund, the American Cancer Society, and the John A. Hartford Foundation. 141. Evaluation of antifibrillatory agents and catecholamines by a physiologic method Stennis D. Wax. Watts R. Webb, Roger R. Ecker, and tl'infred L. Sugg Department of Surgery. University of Texas .tiouthirestern 1ledical School. Dallas. Texas ,tiaraical Forum 19:149-151. 1968 Trains of low amplitude, high frequency electri- cal pulses were applied to the hearts of dogs to measure fibrillation threshold. This method simu- lates the pathophysiology of spontaneous ventricu- lar fibrillation. The various catecholamines all reduced the fibrillation index (F.I.) by about 35% but were differentiallv antagonized. Propranolol or lidocaine raised the F.I. following norepinephrine and epi- nephrine above control levels but restored the iso- proterenol F.I. only partially to base line. This suggests a dissociation of inotropic and chrono- tropic activity of these agents. Propranolol alone had no significant effect on F.L. indicating that the intrinsic cardiac sympathetic activity. in contrast to exogenous catecholamines. does not increase mvo- cardial irritabilitv. The antifibrillaton• action of lidocaine was independent of catecholamine activi- tv. The effect on F.I. of these two drugs was identical when exogenous catecholamines were given. Other support: U. S. Public Health Service. 142. Neurogenic basis for the rise in blood pressure evoked by nicotine in the cat Gerard L. Gebber Department of Pharmacology. Michigan State Universit_v, East Lansing, 1:fichigan Journal of Pharmacology and Experimental Therapeutics 166:255-263. 1969 The pressor response elicited by small intra- venous doses of nicotine (5-40 µg/kg) in the cat was accompanied by an increase in centrally emanating nerve activity recorded from both pre- and postgan- glionic branches of the superior cervical ganglion. The pressor response was abolished by transection of the spinal cord and was somewhat reduced after denervation of the aortic and carotid body che- moreceptors. Nicotine-induced enhancement of postganglionic activity recorded from the innervated ganglion also was somewhat reduced after chemore- ceptor denervation. However, chemoreceptor de- nervation abolished the enhancement of centrally emanating preganglionic activity produced by nico- tine. Although the doses of nicotine employed were too small to evoke a postganglionic discharge in the decentralized ganglion, they were sufficient to fa- cilitate the postganglionic action potential evoked by submaximal stimulation of the sectioned pre- ganglionic nerve. It is concluded that the major component of the pressor response produced by small intravenous doses of nicotine is the con- sequence of facilitation of ganglionic transmission rather than a direct central effect of the compound on vasomotor areas. This study also illustrates that abolition of an autonomic neuroeffector response by spinal cord section does not necessarily indicate that the response was of supraspinal origin. TIMN 0115871 T200362 127

Cardiovascular System The incidence of exercise induced ventricular premature complexes was determined for each sub- group according to age. No statistical differences were found in the incidence of exercise induced ventricular arrhvthmias in smokers versus non- smokers or former smokers. The duration of exercise and the maximally attained heart rate and systolic blood pressure were compared for each subgroup. The duration of maxi- mal exercise was significantly shorter in smokers (p = < 0.001) and former smokers (p = < 0.005). The maximal exercise svstolic blood pressures were greater in smokers compared to non-smokers (p = < 0.005) but there was no significant difference in this measurement between non-smokers and former smokers. The maximal exercise heart rates were significantly less in the smokers (p =< 0.001 and the former smokers (p = < 0.01) compared to the non-smoking group. In conclusion there is a definite statistical im- pairment in duration of exercise and maximal heart rate and systolic blood pressure attained during exercise in smoking subjects but the incidence of exercise induced ventricular arrhvthmias did not appear to be influenced by current smoking habits. 155. Cardiac vectors in women smokers and non- smokers Vernon H. Hoeppner, and Colin R. Woolf Department of Medicine, University of Toronto. and the Respiratory Research Laboratory, Toronto General Hospital. Toronto, Ontario Summary of a paper to be published Scalar electrocardiograms were recorded in 105 smoking and 165 non-smoking, apparently healthy ccomen, all of whom.had normal routine pulmonary function studies. Mean frontal QRS vectors did not demonstrate anv differences between the two groups. The initial mean horizontal QRS vector in smokers was -16.6 ± 11.5 which was significantly more posterior than non-smokers, -12.9 ± 12.5 (p < 0.01). A posterior shift was noted in both groups over 5 years, but the shift was significantly more posterior in smokers, 5.7 ± 3.3 than in non-smokers, 4.2 -* 2.9 (p < 0.01). These findings suggest that cardiac changes are associated with chronic ciga- rette smoking and that the most sensitive early variable is the horizontal QRS vector. 156. Vectorcardiographic diagnosis of left ventricular hypertrophy Donald W. Romhilt, Joseph C. Greenfield. Jr.. and E. Harvey Estes, Jr. Department of Nfedicine. Duke University .%fedical Center, and the Medical Service. Durham Veterans Administration Hospital. Durham. N'orth Carolina Circulation 37:15-19, 1968 The vectorcardiographic diagnosis of left ven- tricular hypertrophy, using the Frank lead system. was evaluated in 93 autopsied male patients (70 with hypertrophy of the left ventricle and 23 without such hypertrophy). The criterion of the maximal QRS magnitude in the transverse plane greater than 2.2 mv below the age of 50 years and 1.8 mv at the age of 50 or more was positive in 61% of hypertrophied hearts and negative for all the nonhypertrophied hearts. The criterion of the T loop vector greater than H°-70° in the transverse plane was positive in 82% of 49 hypertrophied hearts and positive in one (7°•0) of 15 normal hearts for which the T loop was available. The maximal QRS magnitudes in the frontal and sagittal planes had too low a sensitivity to be of value while the angles of the maximal QRS vector in the transverse and sagittal planes were too non- specific to be useful. By combining the QRS magnitude and the T loop vector in the transverse plane, the diagnosis of left ventricular hypertrophy was made in 90% of 49 hypertrophied hearts for which the T loop was available. Other support: U. S. Public Health Service. 157. A point-score system for the ECG diagnosis of left ventricular hypertrophy Donald W. Romhilt, and E. Harvey Estes, Jr. Department of Medicine, Duke University Medical Center, and the Medical Service, Durham Veterans Administration Hospital, Durham, North Carolina American Heart journal 75:752-758, 1968 A point-score system is presented for the di- agnosis of left ventricular hypertrophy from the ECG. It is evaluated in an autopsy seriPs of 150 hearts with hypertrophy designated on the basis of Zeek's criteria. Using this system, t1.ie ECC'r is positive in 60 per cent of the time when LVH is present at autopsy. LVH is diagnosed in 3.2 per cent of nonhypertro- phied hearts. The point-score system is significantly TI1YIhT 0115876 T200367 132

th nt ' in ic ze nt :). te g- al 0- ie ly in ;n Jr. 01 augmentation of the contractile state of the heart. Beta adrenergic blockade with propranolol led to a decline qf both force and velocity. Other support: U.S. Public Health Service. the Mi- chigan Heart Association, the Council for Tobacco Research-U.S.A., and the Detroit General Hospital Research Corporation. 146. Myocardial contractility of the intact heart p. S. Puri. and R. J. Bing Department of Xfedicine. Wayne State C`niversity School of.kfedicine. Detroit. Michigan Cardiologia 51:257-261. 1967 Current knowledge on the above subject is reviewed. From various studies on the isolated heart muscle, a profile of several of the factors which influence the nature and course of cardiac con- traction is now emerging. The activity of the con- tractile elements of the heart muscle is characterized by four factors: force, velocity, instantaneous muscle length and time. It is within the framework of these four variables that it has become possible to define precisely the term myocardial contractile state. 147. Nicotine and potassium chloride contracture in mammalian ventricle Jay R. Wiggins, Peter Danilo, Jr.. Henry Gelband. and Arthur L. Bassett Department of Pharmacology, College of Physicians and Surgeons. Columbia University, New York, New York Journal of Pharmacology and Experimental Therapeutics 185:457-467. 1973 For mammalian ventricle, the basis for the mechanical response to nicotine is complex. Results of experiments are presented which show that nico- tine can markedly influence contracture force in depolarized mammalian ventricle. Papillary and trabecular muscles dissected from the right ventricle of cats were maintained at 27-30°C and stimulated at 6 to 30 beats/minute under isomet- ric recording conditions. Contractures were induced by replacing NaCl with 100 to 140 mM KCl in the Tvrode's medium. Flow rate through the chamber during contracture was held constant at either 14 ml/ minute (low flow) or 140 ml/minute (high flow). Propranolol (1 x 10-6M) had little effect on maximal potassium-induced contracture force. Nicotine (0.03-12.4 m`i) did not induce contracture in normal or beta receptor blocked muscle maintained in control Tvrode's solution containing 4.0 m,%f po- tassium. Nicotine (6.2 and 12.4 m.M) increased contracture force in potassium-depolarized muscle in the presence and absence of calcium at low flow and in the absence of calcium at high flow: the same concentrations had little effect on high-potassium contractures in the presence of calcium at high flow. It is suggested that nicotine may influence con- tracture force in depolarized cat ventricular muscle by releasing sarcolemmal stores of calcium. This action mav be affected bv formation of nicotine- calcium complexes and the flow rate utilized during high-potassium contracture. Other support: U. S. Public Health Service. 148. Direct and indirect inotropic effects of nicotine on cat ventricular muscle Arthur L. Bassett, Jay R. Wiggins. Peter Danilo. Jr., Kristina Nilsson. and Henry Gelband Department of Pharmacology. College of Phcsicians and Surgeons, Columbia University. Vecr York. New York and Departments of Pharmacology. Sur,gerY and Pediatrics, University of Xqiami School of Medicine. Miami, Florida Journal of Pharmacology and Experimental Therapeutics 188:148-156. 1974 The inotropic effects of nicotine on papillary muscle from normal and reserpine-pretreated cats were evaluated by analysis of isometric recordings. Nicotine (3.1 x 10-' to 6.2 x 10-'M) at 22-36°C released endogenous cardiac catecholamine which then transiently increased isometric active force and rate of development of force in normal muscle. The transient positive inotropic effect induced by nico- tine (3.1 x 10-' to 6.2 x 10-'M) often was followed by a transient negative inotropic effect which was reversed despite continued exposure to nicotine. Nicotine (6.2 x 10-' to 6.2 x 10-'M) increased active force and rate of force development in reserpine pretreated muscles and in muscles treated with beta adrenergic receptor blocking agents, alprenolol (2 x 10-sM) or propranolol (10-6M). Additionally. for reserpine-pretreated muscle, 6.2 X 10-3 nicotine increased time to peak force and duration of con- TIMN 0115873 T200364 129

Cardiovascular Svstem tricular filling. The lability of the inotropic or contractile state has in turn been attributed to abrupt cycle-lerigth change effecting inotropic alteration analogous to postextrasystole potentiation of con- tractility and, at rapid rate, effecting an alteration of the contractile state. Other support: Herman C. Krannert Fund, U. S. Public Health Service, and the Indiana Heart As- sociation. 127. Exit block Charles Fisch. Kalman Greenspan, and Gary J. Anderson Department of Medicine, Indiana University School of Medicine, and the Krannert Institute of Cardiology, Marion County General Hospital. Indianapolis. Indiana American Journal of Cardiology 28:402-405. 1971 Exit block is defined as failure of an impulse to excite the surrounding tissue when falling outside of the refractory period of the heart. It may accompany either primary or latent automatic pacemaker tissue, may complicate reentrance arrhythmias and may be of either Wenchebach type I or type II variety. Evidence suggests that exit block is most often due to failure of conduction rather than to an alteration of the impulse. Other support: Herman C. Krannert Fund, U. S. Public Health Service, and the Indiana Heart As- sociation. 128. Electrophysiologic studies on Wenckebach structures below the atrioventricular junction Gary J. Anderson. Kalman Greenspan, and Charles Fisch Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana American Journal of Cardiology 30:232-236, 1972 Recent clinical reports have suggested that con- duction disturbances of the Wenckebach type occur below the atrioventricular junction. The purpose of this study was to demonstrate this type of con- duction delay within the canine specialized Purkinje fiber system and human ventricular tissue. Con- ventional microelectrode techniques were utilized in these experiments. Wenckebach periods with 4:3 condur.tion occurred within the His bundle in re- sponse to hyperkalemic (10.8 mEq/liter) Tyrode infusion. Impalement into the false tendons of the right and left bundle demonstrated typical 5:4 and 3:2 interelectrode Wenckebach periods after admin- istration of nicotine. Similar studies were performed in the distal specialized conducting system where microelectrodes were impaled in a terminal Purkinje cell and ventricular cell. With the administration of acetylstrophanthidin and subsequent development of automaticity, beats conducted in orthograde fash- ion demonstrated typical 3:2 Wenckebach conduc- tion while maintaining 1:1 stimulated retrograde conduction. When human ventricular tissue was exposed to Tyrode solution containing 4X normal potassium (10.8 mEq/liter), conduction times from the stimulating to recording electrodes demon- strated 4:3; 3:2 and, finally, 2:1 Wenckebach con- duction. These experiments demonstrated the elec- trophysiologic evidence for Wenckebach structures at all levels of the canine His-Purkinje system and human ventricular muscle. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 129. Exposure of the canine proximal AV conducting system for electrophysiologic studies Marcelo V. Elizari, Kalman Greenspan, and Charles Fisch Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana Journal of Applied Physiology 34:538-543, 1973 The mechanism(s) of AV and intraventricular conduction disturbances, and other cardia arrhyth- mias, has been related to the electrophysiologic properties of the specialized conduction system. However, the entire canine conducting system, en- dowed with such properties, has not been studied with intracellular microelectrodes, since the path- way has not been exposed by gross dissection at the proximal level of the intraventricular segments. The hearts were rapidly excised, placed in oxygenated Tyrode solution, and the conduction system dis- sected from the AV node to the beginning of the bundle branches. The exact position of the con- duction system was carefully mapped with empha- sis on the node and bundle of His. The latter, like the human heart, has two well-differentiated segments: a penetrating and a branching portion. These areas were impaled with microelectrodes for an ar.atomi• TIMN 0115866 T200357 - 122 ~w

Cardiovascular System 143. A comparative study of nitroglycerin and propranolol Erwin Robin. Charles Cowan. Pritpal Puri. Sunilendu Ganguly. Emile DeBorrie. Mila Ltartinez. Thomas Stock. and Richard J. Bing Department of Medicine. [t"arne State L'nirersity School of Medicine. Detroit. Michigan Circulation 36:175-186. 1967 The action of sublingual nitroglycerin (0.6 mg) and intravenous propranolol (0.1 mg/kg) on short- ening of right ventricular fibers was investigated in patients with and without coronary artery disease. This was accomplished with a newly devised strain gauge catheter. Hemodynamic parameters and changes in the oxidation-reduction potential of heart muscle were determined. Nitroglycerin results in decrease in shortening and velocity of shortening of ventricular fibers. heart rate, left ventricular end- diastolic and systemic pressure, dp/dt, tension-time index, peripheral resistance, and left ventricular minute work in normal and arteriosclerotic subjects. Intravenous propranolol leads to a fall in velocity of shortening of myocardial fibers, heart rate, dp/dt, stroke index, and left ventricular minute work. However, there is an increase in tension-time index and peripheral resistance with no change in sys- temic pressure. Left ventricular end-diastolic pres- sure rises significantly in arteriosclerotic patients. The mvocardial oxidation-reduction potential in- creases after nitroglycerin whereas it falls after propranolol in normal and arteriosclerotic patients. Other support: U. S. Public Health Service, the Michigan Heart Association, the Council for Tobac- co Research-U.S.A.. and the Detroit General Hospi- tal Research Corporation. Contractilitr 144. Reduction of myocardial contractility by 100% oxygen in patients with coronary disease Kinji Ishika%.•a. Radha Sarma, James H. Getzen, John D. McNair. Richard S. Cosby. Hilton Buggs, John L. Johnson, and Richard J. Bing Huntington Xfemorial Hospital. Pasadena. California, and the University of Southern California School of Nfedicine. Los Angeles, California Proceedings of the Society forEYperimer.tal Biolog3•and Nfedicine 145:99-102, 1974 Changes in the contractility indices for left ventricular muscle were calculated during air and oxy-gen breathing in 19 studies on 14 patients with coronarv arterv disease. The following significant changes were observed on oxygen breathing: rises in pO, of arterial blood, in left ventricular systolic pressure, and in tension-time index, and a fall in the velocitv of the shortening of the contractile element of the left ventricular muscle at zero load ("max). The fall in "max was similar even when heart rate was maintained constant. These observations sug- gest that oxygen breathing diminishes mvocardial contractility and consequently may reduce myo- cardial oxygen demand. It is recognized that the reduction in myocardial contractilitv may be only one of the factors responsible for the decrease in myocardial oxygen demand induced by oxygen administration. Other support: Council for Tobacco Research- U.S.A., and the Margaret W. and Herbert Hoover, Jr. Foundation. 145. Evaluation of myocardial force-velocity relation in closed-chest dogs Pritpal S. Puri, and Richard J. Bing Department of Medicine. Wayne State Universih• School of Medicine, Detroit. Michigan American Journal of Physiology 214:1273-1279. 1968 The force-velocity characteristics of the left ventricle in 35 closed-chest anesthetized dogs were studied by means of a strain-gauge catheter assem- bly. Instantaneous velocity of shortening was de- termined at an isolength point on the curve of fiber shortening, by drawing a tangent to the curve relating height of deflection to time. Ventricular pressure related in time to the isolength point was used as a measure of force. Instantaneous force- velocity relations at a constant muscle length could thus be studied. Alterations in afterload induced by inflating and deflating an intra-aorta balloon and by methoxamine led to reciprocal changes in force and velocity. Positive inotropic drugs, i.e., epinephrine, norepinephrine, and isoproterenol, on the other hand, shifted the force-velocity curve upwards, or upwards and to the right, indicating either an increase in velocity alone or an increase in force plus velocity, respectively. These results point to an TIMN 0115872 , T200363 128

Cardiovascular System be bv altered conduction. automaticitv, or a com- bination of the two and potential differences be- tween cells or groups of cells. Conduction is determined by the level of mem- brane potential at the point of excitation (take-off potential). This potential level from which the premature impulse takes off will then affect the rate of rise of phase 0 and the amplitude of the action potential. both of which will affect conductivity. In addition, the level of the threshold potential, degree of excitability of the cell membrane, and presence or absence of automaticity may influence propaga- t io n. Alteration, induction, and/or enhancement of automaticity in normal and latent pacemaker cells can occur spontaneously and may result from vagal influences or release from the control of a higher, faster firing cell. Automaticity can also be in- fluenced by many interventions, among which are catecholamines, hypoxia, hypokalemia, hypercal- cemia, isoproterenol, digitalis, and stretch. Automaticity and conduction disturbances in combination may lead to re-entry (i.e., repetitive stimulation consequent to a single, properly timed automatic or premature stimulus). The mecha- nism(s) responsible for re-entry may be either (1) conduction depression with asynchrony of conduc- tion; or (2) unidirectional block. Potential (voltage) differences between subadja- cent cells may result from "after-potentials" or any asynchronous repolarization which leads to a flow of current. Other support: Herman C. Krannert Fund, U. S. Public Health Service, Indiana Heart Association, and the Northeast Indiana Heart Association. 162. Modification of the nicotine-induced depolarization in striated muscle by altered [H+]o, [Ca*+]o and [Cl-]o Edward G. Henderson, and Linda Reynolds Department of Pharmacology, University of Connecticut Health Center. Farmington, Connecticut Naunvn-Schmiedeberg's Archiv fur Pharmacologie 278:81-90, 1973 In frog sartorius muscles, reduction of [Ca"]o enhanced the depolarization of the end-plate mem- brane caused bv nicotine while elevation of [Ca--lo reduced the nicotine-induced depolarization. A quantitative studv of the effects of [Ca'-jo on the nicotine-induced depolarization indicated that the interaction between calcium and nicotine was non- competitive. Reduction of the chloride concentra- tion or acidification of the Rin;er's solution en- hanced the depolarization caused bv nicotine (0.015-0.10 mM) and antagonized the influence of elevated (Ca"Jo. The enhanced sensitivity of mus- cles to nicotine at low pH and in Cl--free solutions was probably due to the increased effective re- sistance of the muscle membrane caused by these conditions. The effects of variation of [Ca-']o ap- peared to be independent of changes in the mem- brane resistance. 163. Direct and reflex effects of hypothermia, hypotension and hypoxia on the heart Erwin Robin, Sigmundur Gudbjarnason, and Richard J. Bing Wayne State University School of Medicine, Detroit, Michigan Pre- and Postoperative Management of the Cardiopulmonary Patient, pp 251-267. Wilbur W. Oaks and John H. Moyer. Editors. Grune and Stratton, 1970 In a review presentation on the above subject, selected experimental and clinical data are dis- cussed. Other support: U. S. Public Health Service, the Council for Tobacco Research-U.S.A., and the De- troit General Hospital Research Corporation. 164. Negative chronotropic effects of McN A-343 and nicotine in isolated guinea-pig atria: Insensitivity to blockade by tetrodotoxin Achilles J. Pappano, and Robert A. Rembish Department of Pharmacology, University of Connecticut Health Center, McCook Hospital, Hartford, Connecticut Journal of Pharmacology and Experimental Therapeutics 177:40-47, 1971 McN A-343 reduced the firing rate of pacemaker cells in the isolated spontaneously beating right atrium of the guinea-pig. Intracellularly recorded action potentials from these cells showed that the actions of McN A-343 were similaz to those of acetylcholine. The negative chronotropic effect of TIMN 0115878 T200369 134

more sensitive in the presence of combined hy- pertension and coronary artery disease than in either alone. Other support: U. S. Public Health Service. 158. Right precordial qrS pattern due to left anterior hemiblock Paul L. McHenry, John F. Phillips, Charles Fisch, and Betty R. Corya Krannert Institute of Cardiology. Marion Countr General Hospital. and the Department of Medicine. Indiana. University School of Medicine. Indianapolis. Indiana American Heart Journal 81:498-502, 1971 Five patients with left anterior hemiblock and a qrS pattern in V, were studied. In each instance, the q wave disappeared when V. was recorded in the fifth interspace and was accentuated when recorded in the third interspace. None of the patients had a history of myocardial infarction or were suspected of having arteriosclerotic heart disease. It is suggested that in the presence of marked left axis deviation, q waves in V: or V, and V, may not be due to myocardial infarction. Other support: Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart As- sociat ion. 159. Sinus nodal echoes: Clinical case report and canine studies Rory W. Childers, Morton F. Arnsdorf, Domingo J. de la Fuente, Miguel Gambetta, and Robert Svenson Department of Medicine. University of Chicago, Chicago, Illinois American Journal of Cardiology 31:220-231, 1973 Sinus nodal echoes are illustrated (1) in a case report. and (2) a study of the effects of atrial premature beats after atrial drive in dogs. When atrial premature beats confront the sinus node while it is still refractory, 3 types of response may be seen: (1) Complete interpolation-the subsequent sinus beat (or escape) comes precisely at the expected time; (2) incomplete interpolation-the subsequent sinus beat is delayed; and (3) sinus echoes-the sinus beat appears earlier than expected. In all 3 instances the node is entered, but the pacemaker fails to be reset. Although the echo has the form of a sinus beat, it is tollowed by a pause, presumably as a result of repenetration of the sinus node through pathways unused during exit. The curves character- izing the expansion by vagal stimulation of the nodal refractory period and total echo circuit time are defined, together with the latency of cholinergic effect on nodal refractoriness, sinus automaticitv and exit conduction of the echo. The secondarv concealment zone of a completely interpolated atrial premature beat is established. Atrial preexcitation (before the echo) sometimes evokes a second echo. The limiting factor on sustained sinoatrial reciproca- tion thus appears to be total echo circuit time rather than refractoriness of atrium or echo entrance path- ways. The repetitive echoes seen in this study maN • be the basis for some clinical cases of sinus or atrial tachycardia. Other support: U. S. Public Health Service. 160. Continuous prehospitalization monitoring of cardiac rhythm Gary J. Anderson, Suzanne B. Knoebel, and Charles Fisch Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana American Heart journal 82:642-646, 1971 A continuous electrocardiographic telemetry system is described which permits simultaneous voice communications. The data observed suggest that continuous ECG telemetry is a useful tool in the prehospitalization detection of arrhythmias in pa- tients with coronary heart disease. The instituting of earlier therapy for arrhythmias may reduce the incidence of early death. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 161. Electrophysiological basis of clinical arrhythmias Charles Fisch, Kalman Greenspan, and Suzanne B. Knoebel Krannert Institute of Cardiology, Marion County General Hospital, and the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana In: The Paul Dudley White Symposium on Cardiovascular Disease. New York, 1973, Williams and Wilkins Company, Baltimore, pp 309-319 The electrophysiological basis for the genesis, maintenance, and suppression of arrhythmias may TIMN 0115877 T200368 133

sured at rest and after isoproterenol. After classifica- tion by an~aiouIrfurthero compared by presence (C.~I). the b Ps ' tere or absence bf collateral vessels, and whether the collaterals were intercoronary or bridge collaterals. Forty patients (group A) had no coronary artery disease demonstrated by cineangiography. The in- crease in MBF with isoproterenol for this group was 870„ (P > 0.001). Fifteen patients (group B) had CAI of 175 or greater. These patients increased MBF 73% on infusion with isoproterenol, an insignificant difference from group A. Forty-five patients (group C) had Cr1I of 175 or less when an index of 300 represents no occlusive disease. Those with intercor- cmary collateral vessels (group C,.,) were unable to increase `iBF to the same extent as patients in groups A and B did. There was no difference between this group and those without collateral vessels and the same severity of disease. Fifteen patients with CAI of 175 or less (group C,,;) had bridge collaterals and were able to increase MBF to a greater extent than those with no collaterals or with intercoronary collateral vessels. This same group of patients. in a parallel observation, showed less S-T-segment depression on treadmill exercise than patients with intercoronary collateral vessels. The data suggest that intercoronary collateral vessels do not contribute significantly to myocardial blood flow reserve. Bridge collaterals, however, do seem to contribute in selected patients. Other support: the Herman C. Krannert Fund, the V. S. Public Health Service, the Indiana Heart As- sociation, and the Northeast Indiana Heart Associa- t inn. 102. Correlation of computer-quantitated treadmill exercise electrocardiogram with arteriographic localization of coronary artery disease Paul L. McHenry, John F. Phillips, and Suzanne B. f, noebel Dapartment of aledicine, Indiana University Schoof of .Vedicine. and the Krannert Institute of Cardiology, Marion Counh• General Hospital, Indianapolis, Indiana .American Journal of Cardiology 30:747-752, 1972 Graded treadmill exercise testing and coronary cinearteriographic studies were carried out on 86 patients with angina pectoris. At rest, all patients demonstrated a normal S-T segment on the modi- fied bipolar lead V; recording used. The computer- quantitated S-T segment response to exercise was correlated with the location and extent of obstructive coronary artery disease. The coronary cinearterio- grams were reviewed by 3 physicians and stenosis of 75 per cent or greater was considered significant. All patients showed at least this degree of stenosis in 1 or more major coronary arteries. and 83 of 86 exhibited 90 percent or greater stenosis in at least 1 artery. Thirty-one patients had stenosis in a single arterv, 43 had stenosis in 2 arteries, and 12 had significant lesions in all 3 major arteries. In 70 of the 86 (82 percent) patients, a positive S-T segment response developed during or immediately after exercise. In 12 of the 16 with a negative response. disease was limited to a single artery. In 11 of the 12 the disease was restricted to the right coronary or left circumflex arteries. Of the 12 patients with an isolated stenosis of the left anterior descending artery, 11 (92 percent) had a positive S-T segment response. Of 55 patients with 2- or 3-vessel disease. 51 (93 percent) demonstrated a positive S-T re- sponse. Graded treadmill exercise testing in 80 patients with chest pain normal coronary arterio- grams and normal left ventricular function revealed 4 (5 percent) with a false positive S-T segment response. The possible mechanisms underlying the high incidence of false negative exercise electrocardio- graphic tests in patients with disease isolated to the right coronary or left circumflex artery are dis- cussed. Other support: the Herman C. Krannert Fund, U. S. Public Health Service, the Indiana Heart Associa- tion, and the Northeast Indiana Heart Association. 103. Rehabilitation of the coronary patient Alberto N. Goldbarg Department of Medicine (Cardiology), University of Chicago Pritzker School of Medicine, Chicago, Illinois Medical Clinics of North America 57(1):231-241, 1973 The purpose of this paper is to review the information available in the area of coronary re- habilitation and to present the practicing physician with a simple, practical guide for the management of the coronary patient. This is summarized as fol- lows: In order to provide optimal care for the patient with coronary heart disease, the practicing phy- TIMN 0115857 T200348 113

Mc~,T A-343 was prevented by atropine but was not modified by hexamethonium or nicotine. Tetrodo- toxin. in concentrations sufficient to block impulse conduction in vagal neurons, did not oppose the cardiac slowing produced by McN-343, suggesting that the latter agent was acting at a site distal to vagal ganglion cells. Nicotine-induced depression of pace- maker activity was similarly unaffected by tetrodo- toxin although it was prevented by McN A-343. In addition, nicotine retained its sympathomimetic activity in the presence of tetrodotoxin and restored action potentials to hearts poisoned by this agent, probably by release of catecholamines from intracar- diac nerve terminals. 165. Cardiac arrhythmias observed during maximal treadmill exercise testing in dinic.ally normal men Paul L. McHenry, Charles Fisch, John W. Jordan, and Betty R. Corya Krannert Institute of Cardiology. Marion County General Hospital. the Department of Medicine. Indiana University School of Medicine, Indianapolis, Indiana American Journal of Cardiology 29:331-336, 1972 The incidence of cardiac arrhythmias observed during maximal treadmill exercise testing was studied in 650 men aged 25 to 54 years. In 561 there was no clinical evidence of cardiovascular disease; 89 were classified as having definite or suspected cardiovascular disease. The patients were divided into 3 age groups-25 to 34, 35 to 44 and 45 to 54 years-to define any age-related differences in the incidence of arrhythmias during exercise. Single or consecutive ventricular premature complexes were observed in 31 percent of the 25 to 34 year olds, 38 percent of the 35 to 44 year olds and 49 percent of the 45 to 54 year olds; the incidence of supraventric- ular premature complexes was 7, 10 and 14 percent, repectively. The incidence of both ventricular and supraventricular complexes increased with age. For any given age group the incidence of ventricular premature complexes was greater in patients with definite or suspected cardiovascular disease. These patients were more prone to demonstrate frequent ventricular premature complexes and had a higher incidence of multifocal ventricular premature beats and ventricular tachycardia. The ventricular pre- mature complexes were also more likely to appear at lower heart rates during exercise in patients with cardiovascular disease. However. the appearance of unifocal ventricular premature complexes during maximal or near maximal exercise testing should not be equated with the presence of clinically significant cardiac disease. Other support: The Herman C. Krannert Fund, the U. S. Public Health Service, and the Indiana Heart Association. E. Atherosclerosis 166. Assessment of catecholamine function in prevention of experimental arteriosclerosis by adrenalectomy R. E. Lee, Jr., R. A. Scott, M. Kedys, and G. M. Hass Rush-Presbyterian-St. Luke's Medical Center. Chicago. Illinois American journal of Pathology 78:35a, 1975 Young New Zealand albino rabbits on a "risk factor" regimen of dietary cholesterol, subcutaneous vitamin D in oil and subcutaneous nicotine in oil develop severe fibrocalcific atheroarteriosclerosis, often complicated by peripheral arterial thrombosis. This disease is prevented by total adrenalectomy. The question arises as to whether the prevention is due to loss of adrenocortical or adrenomedullary function or both. To assess deprivation of catechol- amine function as a factor, 98 rabbits were placed on the regimen. Reserpine, which depletes adrenome- dullary production and peripheral action of cate- cholamines, was given to 51 rabbits while 47 rabbits served as paired controls. Reserpine was given two to three times each week subcutaneously at dosages from 0.25 to 1.25 mg for 4 to 24 weeks. With increasing dosage, animals showed increased stu- por, increased convulsant sensitivity to nicotine in amounts well tolerated by controls, decreased rise in serum cholesterol and cardiac failure. Fibrocalcific arteriosclerosis was similar to that in paired con- trols, but controls had more arterial atheromatous deposition, mirroring the difference in serum cho- lesterol levels between reserpinized and control animals. Peripheral arterial thrombosis, though un- related to convulsant sensitivity to nicotine, oc- curred most often in animals given the largest doses of reserpine and much more frequently than in controls. These data indicate that loss of adrenocorti- TIMN 0115879 T200370 135

Cardiovascular Sc•stem traction. The action of nicotine to increase active force in isolated reserpine-pretreated cat ventricular muscle ma~• result from a relatively nonspecific membrane effect. Preliminary reports of this study appeared in Circulation 42:suppl. 3, 123. 1970, and The Phanma- cologist 13:299, 1971. Other support: Program Project Grant from the National Heart and Lung Institute. 149. Effect of nicotine on contractility of the intact heart Pritpal S. Puri. Danish Alamy, and Richard J. Bing Department of Medicine. Wayne State University School of Medicine. Detroit. Nlichigan Cardiology Digest 5:19-25, 1970 Effects of an intravenous infusion of nicotine on myocardial force-velocity relation, rate of left ven- tricular (LV) pressure rise (dp/dt), and LV end- diastolic pressure (LVEDP) were examined in closed-chest dogs with and without prior (3-adrener- gic blockade with propranolol. Velocity of short- ening was measured by means of an intracardiac strain-gauge catheter assembly. Results show that nicotine augments myocardial contractile state as indicated by an increase in both the force and velocity of shortening. When (3-adrenergic blockade preceded nicotine administration, rise in LV systolic pressure was proportionately greater while velocity of shortening showed a reciprocal decline. LVEDP rose significantly; the increase in dp/dt was less marked. These findings suggest that propranolol impaired the norepinephrine-like effects of nicotine on the myocardium while its peripheral vasopressor action became enhanced. An earlier account of this study was published in The lournal of Clinical Pharmacology 8:295-301, 1968. Other support: U. S. Public Health Service; the Michigan Heart Association; Council for Tobacco Research-U.S.A.; Detroit General Hospital Research Corporation. 150. Studies on the auricular stimulating action of nicotine J. P. Long, and E. G. Gross Department of Pharmacology. College of Medicine. University of lowa. Iowa City, Iowa Archives Internationales de Pharmacod_vnamie et de Therapie 161:30-37. 1966 When hemicholinium (0.5 mg/kg, i.v.) was ad- ministered to cats and the right vagal nerve stimu- lated for 10 sec every min for 50 min, there was complete abolition of vagal nerve induced bradycar- dia and hypotensive responses. When the auricles were isolated, 4 of 8 hearts so studied demonstrated no positive inotropic or chronotropic responses to nicotine, although the response returned if the preparations were washed repeatedly and allowed to stand for an hour. Nerve stimulation without pre- treatment with hemicholinium did not alter the response to nicotine. In 5 cats the right vagus nerve was sectioned near the heart, and, after two weeks, nicotine did not produce significant acceleration in the isolated au- ricles. In 5 other cats, the right vagus nerve in the neck was sectioned, and, after two weeks, nicotine induced significant acceleration. These findings suggested to the authors that nicotine acts on the parasympathetic nerves within the auricles and, directly or indirectly, produces release of norepinephrine from the sympathetic nerve terminals. Other support: Council for Tobacco Research- U.S.A. 151. Comparative studies of atrial responses following nicotine and transatrial stimulation E. G. Gross, T. S. Whitacre, and J. P. Long Department of Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa Archives Internationales de Phannacodynamie et de Therapie 166:273-280, 1967 Atrial reactivity to nicotine and transatrial elec- trical stimulation was evaluated in isolated prepara- tions from eight species (rat, guinea-pig, cat, turtle, chicken, dog, frog, rabbit). There was no consistent responses to nicotine or transatrial stimulation. Only the cat and rabbit demonstrated hoth positive and negative chronotropic and inotropic responses to nicotine (10 µg/ml). The cat also demonstrated TIMN 0115874 T200365 130

be unwise to predict such CO burdens in any one subject from mean values obtained from a group of subjects. On the other hand, blood COHgb saturation and breath CO concentration are closely correlated so an accurate estimation of one can be obtained from knowledge of the other. A number of observations suggest that the total body burden of CO may be significantly greater than that accounted for by CO attached to hemoglobin. Since other heme compounds are known to bind CO, it would be important to study further the size and turnover of such extravascular sites of CO binding in smoking subjects; such sites could be associated with alterations in muscle and cellular metabolism. Preliminarv evidence suggests that the turnover of CO attached to these sites is slower than that of CO attached to hemoglobin and more than three days of abstention from smoking may be necessary before such sites are cleared of CO. Studies in smoking beagle dogs showed rapid alterations in blood hematocrit following initiation of smoking. The present studies confirm these ob- servations, the rapidity of the changes suggests sudden alterations in plasma volume as soon as 30 minutes after smoking a single cigarette. The higher hemoglobin values in smokers resulting from this effect might be associated with higher blood viscos- itv which might potentiate other known adverse effects of CO on cardiovascular function. Other support: U. S. Atomic Energy Commission, and National Aeronautics and Space Administra- tion. 176. Comparison of the cardiovascular (CV) effects of some gaseous components of tobacco smoke L. Procita, and A. J. Ingenito Department of Pharmacology, Albany Medical College, .-l/banr. New York Federation Proceedings 32(3):805, 1973 To assess the CV effects of carbon monoxide (CO), nitric oxide (NO) and nitrogen (NZ), these tobacco smoke constituents were inhaled by a- chloralose-anesthetized cats at concentrations (in air) comparable to smoking levels. Systemic arterial pressure (SAP), heart rate (HR). electrocardiogram (EKG), perfusion pressure in a vascularly isolated neurally intact perfused hind-limb (HLPP) and the responses to common carotid occlusion (CC) were monitored. CO(.05-.2°fo), NO(.02-2%) and ti_(vari- ous conc.) caused only minimal CV effects at CO- induced carboxyhemoglobin (COHb) levels of < 20%, NO-induced methemoglobin levels of < 10%, and N_ induced oxyhemoglobin levels of not < 65°/0. However, these gases often changed the HR responses to CC from tachy- to bradycardia. At levels beyond these, CO and N, usually increased SAP and HLPP and decreased HR while NO decreased SAP and HR; all 3 gases caused T-wave changes in the EKG. The effects of CO on HLPP and the response to CC appeared to be unrelated to COHb formed. suggesting other pharmacologic effects of CO. Stud- ies with combinations of the gases are needed to assess their contribution to the effects of tobacco smoke in the smoker. 177. Negative inotropic action of carbon monoxide (CO) on the isolated isovolumic heart with a hemoglobin (Hb)-free perfusate A. J. Ingenito, P. C. Fiedler, and L Procita Albany Medical College, Albany, New York Federation Proceedings 33(3):503, 1974 To demonstrate effects of CO independent of HbCO, we used the isolated isovolumic rabbit heart preparation perfused with Krebs-Ringer bicarbonate solution equilibrated either with 48916 N_, 48 :•o 0, and 4% CO, (controls) or with 50% CO, 48% 0, and 2% COZ. Maximal systolic left ventricular pressure (LVP) and the positive rate of change of left ventricu- lar pressure with time (dP/dt) were used to evaluate myocardial contractility. The rate of decline in LVP and dP/dt during a 90 min experimental period was greater with the CO-containing perfusate than with that equilibrated with a similar concentration of N,. After 90 min, left ventricular ATP levels were significantly lower in CO hearts than in controls. CO partly prevented epinephrine-induced positive ino- tropy in these hearts, but it also partly prevented the negative inotropic effect which followed epineph- rine-induced positive inotropy. The results suggest that in this Hb-free system, physically dissolved CO can significantly affect myocardial metabolism and function. Possible sites of CO binding and action might include myocardial myoglobin and cyto- chromes. TIMN 0115883 T200374 139

/ s 1 r h e n 1 s j 1 node and electrically driven left atrial appendage of the ouinea-pig heart bathed in Tvrode's solution at 30aC. `ofepinephrine and small doses of nicotine reduced spontaneous cycle length of sinoatrial node pacemaker cells and increased plateau amplitude and duration of left atrial action potentials. Pro- pranolol antagonized the positive chronotropism of nicotine and norepinephrine but had no effect on their ability to augment action potential duration in left atrial cells. The increase in action potential duration was blocked by dihydroergotamine. Ace- tvlcholine and large doses of nicotine increased spontaneous cvcle length of sinoatrial node pace- maker cells and reduced action potential duration in left atrial cells. Atropine prevented these actions of nicotine and acetylcholine in the two atria. Since hexamethonium blocked the biphasic response of° both atria to nicotine, it appears that nicotine acts indirectly by stimulation of adrenergic and choliner- gic structures. Also. the sympathomimetic responses of left and right atria to nicotine are mediated by an action of norepinephrine on qualitativelv distinct receptor sites. - Other support: University of Connecticut Research Foundation. 136. Cardiovascular effect of nicotine in the conscious dog. Modification by changes in autonomic tone William J. Mandel, Michael Laks. Hirokazu Hayakawa, l:anji Obavashi, and Avile :vicCullen Drpartment of Cardiology. Cedars-Sinai Medical Center nnd the Department of Medicine. University of Calitornia. Los Angeles. California .lmerican Journal of Cardiology 32:947-955. 1973 Extensive studies of the cardiovascular pharma- i.ologic properties of nicotine conducted in isolated and partiallv intact animal preparations have pro- duced considerable information on the physiologic mechanisms responsible for the cardiovascular ef- fects. However, no studies have been made of these effects in the conscious animal with intact car- diovascular reflexes. Studies were performed in conscious dogs in which catheters had previously been positioned for long-term use in the right atrium, right ventricle, pulmonary artery and central aorta. Doses of less than 200 µg of nicotine injected into the right atrium produced no significant hemodvnamic effects. A dose of 200 µg of nicotine produced initial tachycar- dia. a subsequent hypertensive response and reflex bradvcardia. A 400 µg dose of nicotine produced a triphasic response: (1) a 2 to 4 second sinus arrest. (2) subsequent sinus tachycardia and marked h}•- pertensive response, and (3) reflex bradvcardia ~~•ith persistent increases in right ventricular end-dia- stolic pressure. Arterial administration of nicotine in similar doses produced only hypertension and reflex bradycardia. No asystole or tachvcardia was ob- served. Pretreatment with atropine eliminated the asys- tole produced by intravenous administration of nico- tine but did not alter the reflex tachvcardia occurring after intravenous or intraarterial administration of nicotine. Pretreatment with propranolol did not prolong the asystole occurring after intravenous administration of nicotine and did not diminish the peak increase in heart rate during phase 2. However, the reflex bradycardia was more pronounced after nicotine was given either intravenously or intraarte- rially. We conclude that, in the conscious dog, nico- tine (1) releases acetycholine in the right atrium, (2) may depress myocardial function, and (3) produces significant hypertension and reflex bradvcardia. Other support: U. S. Public Health Service. 137. Calcium-dependent action potentials produced by catecholamines in guinea pig atrial muscle fibers depolarized by potassium Achilles J. Pappano Department of Pharmacology, University of Connecticut Health Center, Hartford, Connecticut Circulation Research 27:379-390, 1970 Atrial muscle fibers of the guinea pig were depolarized and rendered inexcitable by elevation of [K+)o to 22mM. Isoproterenol, epinephrine, and norepinephrine restored propagated action poten- tials and contractions to atrial cells without chang- ing the resting potential. The peak value of the intracellularly recorded action potentials varied 28 mv for a 10-fold change in [Caz±]o. :soproterenol- induced restoration of action potentials was insensi- TIMN 0115869 _ T200360 125

Cardiovascular Svstem 178. Carbon monoxide (CO)-nicotine interactions on Xhe isolated perfused isovolumic rabbit heart A. J. Ingenito. J. J. Ryon. and L. Procita Department of Pharmacology. Albany Medical College, Albany, New York Federation Proceedings 34(3):793, 1975 To investigate possible CO-nicotine interactions at CO concentrations comparable to those in tobacco smoke, we studied the effects of repeated doses of nicotine (100 µg) every 30 min for 120 min on the isolated isovolumic rabbit heart perfused with Krebs Henseleit solution equilibrated with either 5% CO, 93% 0, 2% COz or with 5% N2, 93% OZ, 2% COz (controls). Maximal left ventricular pressure devel- opment (LVP) and the positive rate of change of left ventricular pressure with time (dP/dT) were used to evaluate myocardial contractility. CO did not affect the nicotine induced positive inotropic effect on LVP and dP/dT, nor did it affect the dose-response relationship for nicotine inotropy to doses between 20 and 100 µg. Curiously, however, CO prevented the decline in resting LVP and dP/dT (i.e. the negative inotropy) seen between successive nicotine doses over a 2 hr experimental period in the 5% NZ controls. The latter finding raises the interesting possibility that in the smoker this action of CO may actually protect the myocardium against some of the adverse effects of nicotine. 179. Effect of carbon monoxide (CO) on adenine nudeotides in brain and heart A. J. Ingenito, P. C. Fiedler, and L. Procita Albany Medical College, Albany, New York Pharmacologist 15:259, 1973 Low levels of blood carboxy-hemoglobin (COHb), e.g. 10%, due to CO-exposure, e.g. from heavy smoking may compromise OZ delivery to already OZ deficient brain and heart in those with circulatory insufficiency. If so, then synthesis of, e.g., ATP, may be reduced on CO-exposure, as reported for hypoxia. To assess this, CO(0.2 or 1.0% in air) was administered to anesthetized cats and rats, and at various times, certain brain areas (cat) or heart ventricular apex (cat and rat) were removed for analysis of ATP, ADP and AMP. At blood COHb levels > 25% (15 min), ATP was significantly decreased (18%) in the right parietal cortex com- pared to control levels in the left cortex. The ATP/ ADP ratio also decreased. COHb < 25% (15 min) did not significantly affect levels of cortical nucleotides. No significant changes in nucleotide levels in cere- bellum, medulla oblongata or myocardium were found at 25-75% COHb (30 min). The data suggest that biological effects of low levels of CO may be related to CO-actions other than its COHb-forming ability. 180. Effect of carbon monoxide and hypoxia on the morphology and metabolism of vascular smooth muscle cultures Tibor K. Zemplenyi, David H. Blankenhorn, Arnold F. Brodie, James F. May, Wendelin J. Paule. and Donald E. Rounds School of Medicine, University of Southern California, Los Angeles, California Unpublished report to the AMA-ERF Smooth muscle cells (SMC) were maintained under tissue culture conditions. Hypoxic cultures of SMC showed an altered morphology which con- sisted of a loss of myofilaments, and increase in rough endoplasmic reticulum, a great increase in free ribosomes, an appearance of smooth endoplas- mic reticulum accompanied by lipid deposits within the cytoplasm. Over an extended period of hypoxia, "mound" formation developed. In this case, isolated islands of cells formed large clumps that measured up to 150µ high. The mound was covered by a thin layer of altered smooth muscle cells while the interior of the mound contained modified smooth muscle cells, much ground substance and fibrils of an undetermined morphology. The mounds did not have the appearance of masses of necrotic material, but instead, seemed to be a response unique to smooth muscle cells under stressful conditions. Thus, in very broad terms, a mound resembled a "mini lesion." Time lapse cinematography indicated that the appearance of mounds is accompanied by contrac- tions of smooth muscle cells in the immediate area. The contractions were observed to occur at 10 to 15 minute intervals and to last 9 to 2 minutes. Con- tractions pulled smooth muscle cells free from their surroundings and into central cellular aggregates of TIMN 0115884 T200375 140

bon tetrachloride-induced hepatocellular disease with progressive cirrhosis. The results led to the following conclusions. 1. Chronic hepatocellular damage and subse- quent cirrhosis due to carbon tetrachloride did not produce sufficient change in levels of serum cho- lesterol to influence development of atheroarterio- sclerosis. Nfodifications which occurred were attrib- uted to inhibition of intercellular matrix formation by proliferating arterial intimal mesenchyme and to increased sensitivity to the vasculo-toxic action of vitamin D. 2. Mildy hvpercholesteremic animals without hepatocellular disease were unharmed by a periodic dosage of 50.000 IU of Vitamin D, while the same periodic administration of 100,000 IU promptly produced lethal generalized calcific atheroarterio- sclerosis. Animals with hepatocellular disease and cirrhosis proved to be exceptionally sensitive to Vitamin D so that dosages as low as 12,500 IU quickly caused lethal calcific atheroarteriosclerosis, complicated at times by thromboarteritis restricted to small vessels supplying muscle. The incidence of thromboarteritis and the severity of calcific arterial disease were directly proportional to the severitv of hepatocellular disease, the amount of Vitamin D given periodically, and, to a lesser extent, the degree of moderate hvpercholesteremia. 3, Periodic administration of 100,000 IU of Vitamin D always produced medial calcific degener- ation of several peripheral arterial systems. This degeneration in animals with normal hepatic func- tion stimulated fibrocellular proliferation of the adjacent intima and, in the presence of sufficient hyperlipemic hypercholesteremia, there was an ac- cumulation of lipids in the proliferating intimal mesenchyme. In animals with reduced hepatic func- tion due to administration of carbon tetrachloride, there was no decrease in calcific medial arterial degeneration or serum cholesterol levels but fi- brocellular intimal proliferation was inhibited. Hence, there was a conspicuous reduction in newly formed intimal matrices which favor intimal lipid accumulation. This seemed to be the principal factor preventing development of severe intimal atheroar- teriosclerosis in animals with hepatocellular dis- ease. 4. Two experimental conditions which greatly enhance the vascu)otoxic action of Vitamin D have now been defined. One is chronic hepatocellular damage as described in this report. The other, described in a previous report, is chronic adminis- tration of nicotine. The two conditions produce about the same result. This indicates that there ma% • be a common pathogenesis involving hepatic me- diation of Vitamin D action as it relates to the regulation of the role of calcium ions in release and activation of the biogenic amines. Other support: U.S. Public Health Service. and the Otho SA Sprague Memorial Institute. 189. Relations between nicotine induction of arteriosclerotic thromboarteritis and nicotine- induced rise in serum free fatty acids in rabbits Richard Scott. Donald Henson, and Anne Hemmens (George M. Hass) Presbyterian-St. Luke's Hospital, Chicago, Illinois American Journal of Pathology 59:72A. 1970 Rabbits given cholesterol, vitamin D and nico- tine often develop peripheral atherocalcific throm- boarteritis. Variations in the disease were correlated with the degree of nicotine-induced rise in serum free fatty acids (FFA). Among 21 rabbits, 13 devel- oped the disease, usually within 7-9 weeks. Six had calcified arteries but no thromboarteritis. Two had normal arteries. Though the nicotine-induced rise in serum FFA differed among animals, the magnitude of rise was repeatedly characteristic of each animal. Serum FFA levels rose about 100% in the most sensitive reactors, and severe atherocalcific throm- boarteritis occurred in this group. The least sensitive reactors had little or no rise in serum FFA and developed no arterial disease. Moderately sensitive reactors had a mild variable rise in serum FFA. Calcific arteriosclerosis developed in this group, but thromboarteritis did not occur or was mild and delayed. Stress of handling involved in using saline injections did not produce consistent increases in serum FFA. Although ACTH produced much greater rises in serum FFA than nicotine, no thromboarteri- tis occurred, nor could the use of epinephrine produce nicotine action. Therefore, though rise in serum FFA has no singular role in pathogenesis of thromboarteritis, the magnitude of rise allows for TIMN 0115887 T200378 143

biphasic responses to transatrial stimulation. The rabbit did not respond. The data demonstrate that care must be observed in interpreting results with isolated atria. The tremendous species variation observed with both nicotine and transatrial stimula- tion certainly suggests that there must be marked species variation in transmission of autonomic nerves or the reactivity to transmission in these c•arious species. Other support: Council for Tobacco Research- t`.S.A• 152. Nicotine-induced restoration of action potentials to cardiac tissue depolarized by potassium Achilles J. Pappano, and Robert A. Rembish Department o,f Pharmacology. Schools of Medicine and Dental Medicine, University of Connecticut. McCook Hospital. Hartford. Connecticut Life Sciences 9 part 1:1381-1388, 1970 In the isolated left atrial appendage of guinea- pig hearts. nicotine (1-2 X 10-' M) permitted the restoration of conducted action potentials and con- tractions to atria depolarized by bathing in 22 mM potassium ion. The ability of nicotine to restore action potentials was sensitive to blockade by pro- pranolol and by hexamethonium, but not bv atro- pine. The abilitv of nicotine to restore action po- tentials was dependent upon the external concentra- tion of calcium ions; nicotine did not restore action potentials when the concentration was reduced to less than one-half that normally used in Tyrode solution. It is tentatively concluded that nicotine can release catecholamines through a mechanism that need not be dependent upon changes in the resting potential of the cardiac adrenergic stores. 153. Nicotine-like actions of cis-metanicotine and trans-metanicotine Kendall L. Wilson. Jr.. Raymond S. L. Chang, Edward R. Bo%.•man, and Herbert McKennis, Jr. Department of Pharmacology. Medical College of 1'irginia. Richmond. Vi binia lournal of Pharmacology and Experimental Therapeutics 196:685-696, 1976 The actions of the cis- and trans-isomers of metanicoiine were observed on isolated rabbit aortic strips and ileal segments. The data are interpreted as showing a nicotine-like action on these preparations for both cis-metanicotine and trans-metanicotine. This hypothesis is supported in part by the dern- onstration that the action of the metanicotine isomers was affected bv hexamethonium, cocaine. phentolamine. reserpine and atropine in a manner similar to that previously seen in studies with nicotine. In dose-response studies on the aortic strip, trans-metanicotine was significantly less active than nicotine. cis-Metanicotine in turn was less active than trans-metanicotine and nicotine. Additionallv, four pyridino compounds, 3-pyridvlacetic acid. \- (3-pvridylacetyl) glycine, nicotinuric acid and trans- 4-(3-pyridvl)-3-butenoic acid, were tested for both agonist and antagonist activity. No stimulatorv ac- tivity was found with these compounds in either the aortic strip or ileal preparations. In aortic strip preparations, pretreatment with either 3-pvridvla- cetic acid or N-(3-pyridvlacetyl)glvcine provided a moderate to marked reduction in the contractile response to trans-metanicotine, whereas pretreat- ment with trans-4-(3-pyridyl)-3-butenoic acid caused a slight reduction. A preliminary account of this study appeared in Federation Proceedings 33:470. 1974. Other support: Council for Tobacco Research- U.S.A., the American Tobacco Company, and U. S. Public Health Service. Clinical Studies 154. Cardiovascular function and electrocardiographic changes in smokers and non- smokers in response to maximal exercise Paul L. McHenry Department of Medicine, Indiana University School of Medicine, and the Krannert Institute of Cardiology, Marion County General Hospital. Indianapolis, Indiana Unpublished report to the AMA-ERF Maximal treadmill exercise tests were carried out on 622 male members of the Indiana State Police Force who were free of clinical evidence of car- diovascular disease. The study group was catego- rized into three subgroups according to smoking history. Non-smokers constituted 28.3% of the group (176), current smokers 43.1% (268), and former smokers who had abstained for at least one year 28.6% (178). TIMN 0115875 T200366 131

~re tic ac- es- t~ rs !nt correlation coefficient for each method was greater than +0.98. There was no correlation between the calculated flows and cardiac output, compartment volumes or compartment distance from the cardiac chamber. Other support: U.S. Public Health Senice. G. Epidemiological Studies 197. Cardiovascular and respiratory symptoms in relation to tobacco smoking. A study on American twins Rune Cederlof. Lars Friberg, and Zdenek Hrubec Department of Environmental Health. University of Cincinnati: National Academy of Sciences, National Research Council. Washington, D.C.: National Center for Air Pollution Control. Public Health Service: and the Institute of Hygiene, Karolinska Institute, Stockholm .-Irchives of Environmental Health 18:934-940, 1969 A questionnaire study was carried out using a twin registry maintained by the National Academy of Sciences, established from birth certificates of white male multiple births in the years 1917 to 1927. The study procedure was similar to that employed in an earlier reported study of Swedish twins (Cederlof. et al: Archives of Environmental Health 13:726-737, 1966), and comparisons of findings are drawn. The following conclusions are reached: Both the Swedish and American twin studies show an association between smoking and certain cardiovascular symptoms [angina pectoris]. It is questionable, however, whether this excess morbid- ity is causal. It was not possible to reproduce the association when studying monozygotic smoking discordant twin-pairs. The number of such pairs admittedly is low. The data are in accord with the Swedish questionnaire studies, however, and fur- ther, Lundman (Acta Medica Scandinavica 180 (supplement 455):1-75, 1966) in his clinical study of smoking discordant twin pairs could not find an increased prevalence of coronary heart disease in smokers compared to nonsmokers. It seems that genetic factors are important in the development of coronary symptoms. We believe that along with social, dietary, and other environmental factors, the genetic factors may contribute to the higher prevalence among smokers than among non- s mokers. The above findings are, of course, not directly related to the increased mortality from coronary heart disease reported for smokers. We hope to be able to elucidate this question before long by ex- amining data on the mortality of the Swedish twins. Already a substantial number of these twins are deceased. The data on respiratory symptoms indicate a strong probability of a causal connection with smok- ing. Even these symptoms, however, seem to be influenced by genetic factors. It is probably at least as dangerous to have propensity for bronchitis as it is to smoke without propensity. Other support: U.S. Public Health Service. 198. Mortality in twins in relation to smoking habits and alcohol problems Lars Friberg, Rune Cederlof, Ulla Lorich, Torbj6rn Lundman. and Ulf deFaire Department of Environmental Hygiene. Karolinska Institute and the Medical Department, Seraphimer Hospital, Stockholm. Sweden Archives of Environmental Health 27:294-304, 1973 Results are presented from an 11-year study on 9,000 pairs. On a nonpair basis, a significant hy- permortality was related to smoking in men and women. Among 706 male dizygotic smoking-dis- cordant pairs born 1901 to 1925, 55 deaths or "first deaths" occurred in a high smoking group, against 31 in a low smoking group. Among 246 correspond- ing monozygotic male pairs, the numbers were 18 vs 18. For women, the numbers were 42 vs 31 among 781 dizygotic pairs, and 14 vs 13 among 326 monzygotic pairs. The hypermortality was mainly due to coronary heart disease, lung cancer, suicides, and accidents. Nonsmokers were registered at 10% in a nationwide "alcohol registry" as against 30% for high smokers. The mortality among the regis- tered subjects was significantly higher than among the nonregistered, regardless of smoking. Data imply that part of the hypermortality among smokers is not due to smoking per se, but to factors associated with smoking. Other support: Intramural funds from the Karolinska Institute, and the Swedish National Environmental Protection Board. TIMN 0115891 T200382 147

rosettes. Sudden flooding of the cultures with ni- trogen initiated marked increase in contraction. It appears that spontaneous contractions may play a role in mound formation. The hypoxic cells reveal in older cultures an increased activity of glycolytic and decreased activ- ity of TCA cycle enzymes. Furthermore, pulse la- beled studies with acetate show an increased in- corporation into lipids of hypoxic cells. Preliminary experiments with SMC cultures subjected to an atmosphere containing 0.02% CO showed a slight increase in the number of cells as compared to the controls. The experimental cells also were thinner and were not altered in such a dramatic fashion as the cells subjected to hypoxia. All enzyme activities had a tendency to decline under such conditions. Other support: U. S. Public Health Service. 181. The induction of atherosclerotic plaque-like mounds in cultures of aortic smooth muscle cells fames F. May, Wendelin J. Paule, Donald E. Rounds, David H. Blankenhorn, and Tibor Zemplenyi Departments of Anatomy and Medicine, University of Southern California, School of Medicine. Los Angeles, California Virchows Archiv B Cell Pathology 18:205-211, 1975 - Smooth muscle cells harvested from the tunica media of piglet aortae were maintained in con- tinuous culture for 10 months. When grown in the presence of 95% air and 5% CO2, they maintained a mature morphology as evaluated ultrastructurally. As these populations became confluent, the cells became oriented paralled to each other. When grown in the presence of 4% OZ, 91% N2, and 5% COZ, this polarized pattern was disrupted. Focal areas of lipid accumulation were observed, succeeded by mound formation at these sites. The mounds stained posi- ti-e with PAS, aldehyde fuchsin, and oil red 0. They were surrounded by 2-4 layers of intact cells. The centers of the mounds were composed of extracellu- lar material and cell debris. A preliminary report of this study appeared in Anatomical Record 178:413-414, 1974, and Clinical Research 22:110A, 1974. Some of the results are also in print in The Smooth Muscle of the Arterial Wall (S. Wolf, ed.), Plenum Press. New York, London. Other support: U. S. Public Health Service. 182. Smoking as a factor in atherosclerosis. A review of epidemiological, pathological, and experimental studies Alfred Kershbaum, and Samuel Bellet Division of Cardiology. Philadelphia General Hospital. Philadelphia. Pennsylvania Geriatrics 21:155-170, 1966 The role of tobacco smoking in the genesis and development of atherosclerosis and its clinical man- ifestations is examined by reviewing epidemiologi- cal, pathological, and experimental studies. Other support: NIH, Council for Tobacco Research- U.S.A. 183. Tobacco smoking and atherosclerotic vascular disease A. Kershbaum (S. Bellet) Division of Cardiology, Philadelphia General Hospital, Pennsylvania Malattie Cardiovascolari 8:1-19, 1967 The author presents a review of epidemiologic, pathologic and experimental studies. Other support: NIH: Council for Tobacco Research- U.S.A. 184. A comparative study of cigarette, cigar and pipe smoking effects on blood lipids, catecholamine excretion and nicotine content of the urine A. Kershbaum (S. Bellet) Division of Cardiology, Philadelphia General Hospital, Philadelphia, Pa. Acta Cardiologica 23:317-329, 1968 The author summarizes studies on the above subject by himself and his colleagues (see Kersh- baum et al., JAMA 195:1095-1098, 1966; Arch Intern Med 120:311-314, 1967). Additional data obtained in dogs are presented showing that with the inhalation factor constant, there was no difference in free fatty acids or tri- glyceride response to cigarette, cigar, or pipe smoke and the effects of nicotine were similar to tobacco smoke. Also, in dogs there was no difference in catecholamine excretion with cigarette, cigar, or pipe smoke. TIMN 0115885 T20p376 141

Cardiovascular System 199. Ischaemic heart disease in death discordant twins. A study on 205 male and female pairs C'lf de Faire (Lars Friberg) Department of Medicine. Karolinska Institutet at Serafimerlasare;tet. the Department of Environmental Hc•giene. Karolinska Institutet and the Swedish National Environmental and Protective Board. Stockholm. Swed en Acta lledica Scandinavica: supplement 568:1-109. 1974 From Januarv 1971 to March 1973 all twin pairs in the Swedish Twin Registry below the age of 70, who became death discordant were continuously recorded. 78% or 205 of the surviving co-twins were examined with respect to different manifestations of ischemic heart diseases (IHD) and several "environ- mental" and "biometric" risk factors. Among the death discordant pairs, the cause of death was IHD in 57 pairs and other than IHD in 148 pairs. Analyses revealed that the prevalence rate of myocardial infarction, angina pectoris, pathologic Q-wave and ST depressions in connection with exercise were significantly higher among the surviving co-twins whose partners had died from IHD than those whose partners had died from other causes. The same trends were seen for most of the risk factors mea- sured both singly and in combination although not very pronounced. The results indicate a substantial genetic influence in the development of IHD. The genetic influences are possibly transmitted not only through some of the risk factors measured but also through other factors. still unknown. Other support: Swedish National Association against Heart and Chest Diseases, and the Swedish Medical Research Council. 200. Background of angina pectoris: Social and environmental factors in relation to smoking Zdenek Hrubec. Rune Cederlof, and Lars Friberg Medical Follow-up Agency. National Academy of Sciences-National Research Council, Washington, D.C., and the Department of Environmental Health, Environmental Protection Board, Karolinska Institute, Stockholm. Sweden American Journal of Epidemiology 103:16-29, 1976 Questionnaire data on about 1200 male twin pairs from the Registry at the Karolinska Institute, Stockholm. and on about 4000 male twin pairs from the Registry of the National Research Council, Wash- ington, D.C.. have been used to study factors affecting angina pectoris. An operational definition of "an- gina pectoris" was developed from the question- naire. In the available data, alcohol drinking, lack of exercise, frequent change of employer, low occu- pational adjustment and smoking are moderately but significantly related to angina among individ- uals (disregarding twin relationships) in both Sweden and the U.S. In monozygous US twin pairs discordant for the above variables, significantly different rates of angina appear only with alcohol drinking. In discordant dizygous US twin pairs, significantly different rates of angina appear with alcohol drinking and low occupational adjustment. Of the independent variables only smoking and drinking are appreciably associated with each other. These findings suggest that alcohol drinking and to a lesser extent occupational adjustment are related to angina directly and not to their association with other factors such as age, genetic background, smok- ing, physical exercise and early environment. Other support: U.S. Public Health Service. 201. Life change patterns prior to death in ischaemic heart disease. A study of death- discordant twins Ulf de Faire (Lars Friberg) Medicinska kliniken, Serafimerlasarettet, Stockholm, Sweden Journal of Psychosomatic Research 19:273-278, 1975 Life change patterns have been analyzed in 27 male twin pairs [9 MZ and 18 DZ], death discordant with respect to IHD. The LCU [life change units] totals gradually increased throughout the 4-yr pe- riod prior to death discordance for both deceased and surviving twins. The deceased twins, however, consistently displayed higher scores than their sur- viving partners. The mean of the 4-yr LCU totals was significantly higher for the deceased MZ twins than for their surviving partners (p < 0.05), whereas the corresponding difference for the DZ pairs was not significant. It is concluded that these results indicate that life changes-especially those connected with work situations-may characterize the total psycho- 'I'IldIN 0115892 T200383 148

Cardiovascular System cal rather than adrenomedullary function is the means by which total adrenalectomy prevents de- rvelopment of fibrocalcific atheroarteriosc)erosis with thrombosis produced in rabbits by the "risk factor" regimen. Other support: Otho S. A. Sprague Memorial In- stitute, the Schweppe Foundation, and the U. S. Public Health Service. 167. The role of the hepatoadrenal axis in the pathogenesis of experimental arteriosclerosis in rabbits G. M. Hass, R. E. Lee. Jr., and R. A. Scott Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois American Journal of Pathology 74:51a. 1974 In a study of "risk factors" believed important in human arteriosclerosis, several hundred rabbits, 4 to 5 months old, were placed on chronic permutations of the following factors: dietary cholesterol, sub- cutaneous (sc) vitamin D, sc nicotine, sc reserpine, sc carbon tetrachloride, adrenal insufficiency and alloxan diabetes. Significant arteriosclerosis did not occur at levels of hyper)ipemic hypercho)esteremia, common in arteriosclerotic patients, unless arterial mesenchyrne was otherwise activated by mural le- sions. Mural lesions resembling those in human arteriosclerosis were produced by sufficient vitamin D. Insufficient levels of vitamin D became sufficient when combined with sc nicotine in oil and dietary cholesterol or, more dramatically, with chronic car- bon tetrachloride-induced cirrhosis, even without supplemental vitamin D. Converselv, sufficient vitamin D was not suf- ficient for production of arterial lesions in adrenalec- tomized animals despite corticosteroid replacement therapy. Nor was the effect of adrenalectomy re- produced by use of sc reserpine. Nor was the arteriosclerotic action of combinations of "risk fac- tors" enhanced by alloxan diabetes. The resultant of suppression by adrenal insufficiency and enhance- ment by hepatic insufficiency was the principal determinant of severity and distribution of arterio- sclerotic changes in rabbits with hyperlipemic hy- percholesteremia in the adult human range. Other support: U. S. Public Health Service, and the Schweppe Foundation. 168. Relation between metabolism of vitamin D and occurrence of calcitic arteriosclerosis in rabbits with cirrhosis R. Scott. R. Turner, M. Kedys. and Anne Lesak (G. Hass) Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center. Chicago, Illinois American Journal of Pathology 66:94a-95a. 1972 A regimen for production of severe chronic nodular cirrhosis in rabbits by subcutaneous in- jections of carbon tetrachloride was developed. These animals and controls were fed a diet contain- ing 0.25% cholesterol and given intramuscular in- jections of vitamin D at dosages varying from 780-200,000IU every fourth week. Cirrhotic animals within 5-7 months developed peripheral calcific atheroarteriosclerosis at serum cholesterol levels in the adult human range. This disease was comparable with that which occurred in controls at 100-fold greater dosages of vitamin D. In exploring the metabolic basis for increased sensitivity of cirrhotic animals to vitamin D, these animals and controls were anesthetized with nembutal. Vitamin D, 1-2-T (n) was injected into the inferior vena cava. At intervals up to 8-48 hours, the amounts of vitamin D and irts major active metabolite, 25-OH-cholecalcif- erol, were determined in samples of blood. Cirrhotic animals showed much less conversion of vitamin D to the metabolite than controls. Inasmuch as this compound is the major active metabolite formed from vitamin D, principally by the liver, the results offer no ready explanation for the enhanced sensitiv- ity of cirrhotic animals to vitamin D. Indeed, a few cirrhotic animals, otherwise in good health, de- veloped calcific arteriosclerosis without supplemen- tal vitamin D-a disorder which did not occur in controls. Other support: U. S. Public Health Service. 169. Inhibition of nicotine-induced calcific thromboarteritis in rabbits by adrenalectomy R. Scott, D. Henson, E. McClain, and A. Hemmens (G. M. Hass) Division of Pathology, Presbyterian-St. Luke's hospital, Chicago, Illinois Federation Proceedings 29:386 Abs, 1970 Rabbits on a nicotine-vitamin D-cholesterol reg- imen often develop calcific thromboarteritis of pe- ripheral arterial systems. This is due to enhancement TIMN 0115880 T200371 136

Cardiovascular St•stem prediction about how each animal is likelv to be affected bv the vasculotoxic action of the choles- tetol-vitamin D-nicotine regimen. Other support: U.S. Public Health Service. 190. Relations between metabolic increase of plasma free fatty acids and the occurrence of arteriosclerotic thromboarteritis in rabbits Richard A. Scott. Donald E. Henson. Anne Lesak. Robert J. Turner, Stanislava Malikova, and George M. Hass Department of Pathology. Rush-Presbyterian-St. Luke's Medical Center and Rush Medical College. Chicago. Illinois American Journal of Pathology 70:209-244. 1973 Rabbits maintained for several weeks on a regimen of modest amounts of vitamin D and dietary cholesterol were placed in three groups in ac- cordance with their response to repeated subcuta- neous injections of nicotine in mineral oil. The group that had the greatest increase in plasma FFA (free fatty acids) following nicotine injections grad- ually developed, over a period of about 12 weeks, severe calcific atheroarteriosclerosis with peripheral thromboarteritis. Those that had a moderate increase in plasma FFA following nicotine injections de- veloped calcific atheroarteriosclerosis but no throm- boarteritis. Those that had the least increase in plasma FFA following nicotine injections developed no arterial lesions. Comparable or much greater increases in plasma FFA occurred in rabbits on the vitamin D-cholesterol regimen when adrenalin, ACTH or heparin was injected rather than nicotine. These animals did not develop calcific atheroarterio- sclerotic thromboarteritis or any other lesions which could be correlated with the increased levels of plasma FFA. Inasmuch as nicotine, vitamin D or dietary cholesterol in the amounts used were in- nocuous when used alone, the interactions between the effects of at least these three factors need to be known in individual animals before the pathogene- sis of the calcific atheroarteriosclerotic lesions with thrombosis can eventually be understood. This study was the subject in part of preliminary reports appearing in American Journal of Pathology 59:72a, 1970 and Circulation 46(4) Supplement II:II-217, 1972. Other support: National Institutes of Health, and the Otho S. A. Sprague Memorial Institute. 144 F. Hemodvnamics 191. Cardiovascular hemodynamics: Determination from the distribution of pulmonary blood flow in seated patients Sheldon H. Steiner, and James M. Quinn III Departments of Medicine and Therapeutic Radiologc•. Chicago Wesley Memorial Hospital, Northcrestern University Medical Center, Chicago, Illinois Journal of the American Medical Association 203:850-856, 1968 lodinated I 131 serum albumin aggregated, when injected intravenously into seated patients, affords an accurate measurement of certain hemody- namic indices. In patients with mitral stenosis, the ratio of distribution of this drug in the upper and middle portions of the lungs correlates well with pulmonary arterial and left atrial pressures and inversely with cardiac output. The presence of idiopathic pulmonary hypertension, but not its se- verity, can be determined, since the flow distribu- tion pattern becomes relatively uniform despite a wide pressure range. The method has considerable advantage over the radioactive gas techniques with oxygen 15, krypton 85, and xenon 133, since the radioactive serum albumin is more easily handled and has only arterial distribution without an alveolar gas phase. Caution should be exercised in relying completely upon the results of the lung scans and ratios where emphysema, congestive heart failure, and pulmonary emboli concomitantly exist. Other support: U.S. Public Health Service, and the James R. Picker Foundation. 192. The effects of anesthesia with pentobarbital on hemodynamics and arterial blood gases in splenectomized dogs Sheldon H. Steiner, and James R. Calvin Section of Cardiology, Northwestern University School of Medicine, Chicago, Illinois, and the Department of Medicine and Heart Research Center, Indiana University School of Medicine, Indianapolis, Indiana Journal of Thoracic and Cardiovascular Surgery 54:592-598, 1967 The purpose of this investigation was to contrast the values obtained for commonly nieasured car- diopulmonary parameters between awake and pen- TIMN 0115888 T200379 i ~ , ! ~

r Cardiovascular System tions vvere complete provided no evidence for hydrolysis of 3-PAA-Glv. and added glycine did not alter FFA levels during the course of the ex- perirrients. The participation of a metabolite (3-PAA-Glv) of 3-PAA in suppression of lipolysis under the forego- ing experimental conditions is sufficient to suggest the active participation of 3-PAA-Glv under many conditions in vivo. 3-PAA is a natural compound in the sense that it is derived from the metabolism of nicotine in vivo. However, no present data permits one to conclude that concentrations of 3-PAA and 3- PAA-Gly arising from the metabolism of nicotine derived from the use of tobacco are sufficient to influence lipolysis to the extent observed in the present study. Other support: American Tobacco Company. I. Pharmacological Studies 210. Histamine release in the dog after leukocyte lysate injection Michael T. Kelly, Richard E. Brashear, R. Russell Martin, and Arthur White Infectious Disease and Pulmonary Divisions, Department of Medicine. Indiana University Medical Scbool. Indianapolis. Indiana Infection and Immunity 4:228-231, 1971 Mixed leukocyte suspensions, containing 106 leukocytes per ml, were prepared from dog periph- eral blood bv dextran sedimentation. Lvsates of these suspensions, prepared by sonification, were admin- istered intravenously to the autologous dogs. The plasma histamine levels were increased to an aver- age value 320% above the control values (P < 0.001). The individual elements in the mixed leukocyte suspensions were separated by sucrose gradient and glass bead column fractionation procedures, and purified suspensions of platelets (no other formed elements present), lymphocytes (95 to 100%), and granulocytes (90 to 98%) were obtained. Lysates of these suspensions all had significant histamine- releasing activity when administered intravenously to dogs. Although leukocytes and platelets from other species are known to harbor mediators of histamine release, this study provides the first dem- onstration of the histamine-releasing activity of dog leukocytes and platelets. Many of the studies of leukocvte and platelet histamine-releasing factors have employed in vitro systems leaving the in vivo role of these factors in doubt. The present studies provide evidence that leukocyte and platelet his- tamine-releasing factors have in vivo activity. Other support: U.S. Public Health Service, the India• na Heart Association, and the National Tuberculosis and Respiratory Disease Association. 211. Mediators of histamine release from human platelets, lymphocytes, and granulocytes Michael T. Kelly, R. Russell Martin, and Arthur k1'hite Infectious Disease Division. Department of Medicine, Indiana University Medical School, Indianapolis. Indiana /ournal of Clinical Investigation 50:1044-1049, 1971 Lysates of mixed human leukocyte suspensions released histamine from intact human leukocytes in vitro. Microgram quantities of leukocyte lysate pro- tein released up to 90% of the total available histamine. The mixed leukocyte lysates were sepa- rated by differential centrifugation into nuclear (800 g pellet), lysosomal (25,000g pellet), and postlysoso- mal supernatant (25,000 g supernatant) fractions. The degree of separation of the lysosomal from the other two fractions was assessed by measuring the relative activities of four lysosomal enzymes. The average distribution of enzyme activity was 11 :L- 2% (mean ± 1 SD), 72 ± 10% and 17 ± 8% for the nuclear, lysosomal, and supernatant fractions re- spectively. The histamine-releasing activity was equally distributed between the lysosomal and su- pernatant fractions, each of which had 5-fold greater activity than the nuclear fraction. Purified suspensions of platelets, lymphocytes, and granulocytes were prepared, and the lysates of these suspensions all had histamine-releasing activ- ity. Centrifugation at 100,000 g for 18 hr sedimented the histamine-releasing activity from all three types of lysate. After 20% ethanol fractionation for the preparation of cationic protein, only the activity from the platelet lysates was found in the 20% ethanol insoluble fraction. These mediators of histamine release from hu- man platelets, lymphocytes and granulocytes may play a role in the development of the vasodilation , TIMN 0115896 152 T200387 __~

_r Cardiovascular SYstem ''S uptake existed between AMPS-treated control mice (mice injected with normal sheep serum) and AMPS-treated stimulated mice (mice injected with serum from platelet-depleted sheep) than in normal mice treated with the same sera. Also, TSF in sheep sera was shown to lose activity after storage at -20°. Other support: American Heart Association, the Tennessee Heart Association, and the U. S. Atomic Energy Commission. 172. The hemagglutination-inhibition assay for thrombopoietin T. P. McDonald University of Tennessee Memorial Research Center and Hospital. Knoxville. Tennessee Blood. Journal of Hematology 41:219-223, 1973 A sensitive immunoassay procedure for the detection of the thrombopoietic-stimulating factor (TSF) has been developed. This procedure is similar to the hemogglutination-inhibition (HAI) technique that is available for erythropoietin. For the pro- duction of TSF, sheep were made thrombocytopenic by the injection of anti-platelet serum. TSF from sheep sera was partially purified by column chroma- tography and tested for biological activity in throm- bocythemic mice. A TSF-rich serum fraction was used to immunize rabbits, and the immune serum was tested for hemagglutinating antibodies. The hemagglutinating antisera were absorbed with nor- mal sheep serum to remove antibodies not specific to TSF. The absorbed antisera containing TSF-specific antibodies were then used to detect TSF in platelet- depleted sheep sera by use of the HAI assay pro- cedure. The data indicate that TSF can be detected and quantified in sheep serum by use of this technique. Other support: The Tennessee Heart Association, and the American Heart Association. 173. Effect of nicotine on dot retraction of rat blood platelets T. P. McDonald, Dolores Woodard, and Marilyn Cottrel l University of Tennessee Memorial Research Center and Hospital, Knoxville, Tennessee Pharmacology 9:357-366, 1973 Clot retraction in platelet-plasma preparations was enhanced by nicotine treatment of rats. Platelets 138 from nicotine-treated rats retracted to a greater degree if incubated in plasma from nicotine-treated rats than if incubated in control plasma. Further- more, platelets from nontreated rats retracted to a greater degree if they were incubated in plasma of nicotine treated rats, than in plasma of nontreated rats. The results indicate that the effects of nicotine are not directly on platelets. it is concluded that the enhanced clot retraction observed in the blood of nicotine-treated animals was due to a plasma factor and not to changes in the platelets themselves. This study was the subject of a preliminary report appearing in Blood 40:595, 1972. 174. The effect of carbon monoxide on diet- induced atherosclerosis in cynomolgus monkeys (Macaca fascicularis) M. R. Malinow, P. McLaughlin, D. S. Dhindsa, J. Metcalfe, A. J. Ochsner, III., J. Hill, and W. P. McNultv Oregon Regional Primate Research Center, Beaverton, Oregon, and University of Oregon Medical School, Portland, Oregon Unpublished report to the AMA-ERF Twenty-six adult female cynomolgus monkeys (Macaca fascicularis) were randomly assigned to groups receiving a standard laboratory diet or a semipurified diet containing cholesterol. Monkeys in each of these groups were exposed to CO in- termittently throughout the day for 14 months, while control animals breathed room air. No differences in plasma cholesterol or aortic and coronary atheroscle- rosis attributable to CO exposure were found. Other support: U. S. Public Health Service, and the Oregon Heart Association. 175. The effects of cigarette smoking on total body burden and excretion rates of carbon monoxide Stephen A. Landaw (John H. Lawrence) Donner Laboratory, University of California, Berkeley, California Journal of Occupational Medicine 15:231-235, 1973 Blood and breath CO content was studied in 31 adult college students with a smoking habit of '/2 to 21/2 packs of cigarettes per day. General conclusions reached are as follows: There is great variability in body CO burdens in subjects with similar smoking habits. Thus, it would TIMN 0115882 22~CJ31 3

Cardiovascular Sc•stem 204. Coronary disease in Staveley, Derbyshire with an international comparison with three ' towns in Marion County, West Virginia I. T. T. Higgins, M. D. Lockshin. J. C. Gilson, A. L. Cochrane. H. Campbell, W. E. 111`aters. B. G. Ferris, Jr., and M. «'. Higgins Department of Epidemiology. School of Public Health. University of Michigan. Ann Arbor. Michigan. U. S. A.. the Medical Research Council's Pneumoconiosis and Epidemiological Research Units, Cardiff. Glamorgan. England: and the Department of Physiology. School of Public Health. Harvard University, Boston. Massach usetts. U.S.A. Journal of Chronic Diseases 25:567-580, 1972 A survey of coronary disease has been carried out in a sample of men aged 35-74 living in Staveley, United Kingdom. The prevalence of car- diac pain based on standard questions and the frequency of certain electrocardiographic abnormali- ties was compared in miners and exminers, foundry and exfoundry workers, mixed dust and other ex- posure groups, and men who had never worked in dust or fumes. The findings have been compared with a similar survey conducted in three communi- ties in Marion County, West Virginia, United States. No striking occupational differences were found, though the prevalence of probable cardiac pain was slightly less in miners and exminers than in the other occupational groups. The prevalence of cardiac pain was twice as high in the United States as in the United Kingdom population. T wave inversion and left complete bundle-branch block occurred more frequently in the U.S. group. But abnormal Q waves, flat T waves, ST depression and other ECG changes were equally common in each countrv. The American men were nearly 5 cm taller and 5 kg heavier than the British men. Of the etiological factors examined, weight and smoking were associated to a small amount with coronary disease. But the most striking association was with blood pressure. Systolic blood pressure was roughly 20 mm Hg higher and diastolic blood pressure 10 mm Hg higher in the coronary compared with the other group. 205. Results and correlations of multistage exercise tests in a group of clinically normal business executives Alberto N. Goldbarg, John F. Moran, Roderick IV. Childers, and Henry T. Ricketts Department of Medicine. University of Chicago, Chicago, Illinois American Heart Journal 79:194-200, 1970 Multistage treadmill exercise tests were per- formed on 91 healthy business executives. Eleven of these subjects exhibited abnormal S-T-segment responses. Although no correlation was found be- tween abnormal exercise responses and the coronary risk factors [including cigarette smoking], the abnor- mal responders were older and had more abnormali- ties in the resting ECG. Other support: Chicago Heart Association; U.S. Pub- lic Health Service. 206. A new diagnostic test for coronary artery disease Alberto Cohen, Eide-Ditmar Luebs, Edward J. Zaleski, and Richard J. Bing Department of Medicine, Wayne State University School of Medicine, Detroit. Michigan Minnesota Medicine 49:17-21, 1966 Research-U.S.A. The results reported here confirm the findings of others that nitroglycerin increases coronary blood flow in normal individuals but not in patients with coronary artery disease. This is the basis for the test described here, which differentiates between these two groups. The procedure involves the use of a positron emitter (Rb-84) and the coincidence count- ing system. The advantages of this test are its technical simplicity and objectivity. Catheterization is not necessary and the test lasts no longer than 30 minutes, involving only intravenous infusion of the isotope and an arterial puncture. The data are then processed by a computer which calculates the coron- ary blood flow in ml/min, together with the standard deviations and the significance of the change in flow prior to and following the sublingual administration of nitroglycerin. The procedure does permit dif- ferentiation of patients with and without coronary artery disease with a reasonable degree of accu- racy. Other support: U.S. Public Health Service, the John A. Hartford Foundation, and the Council for Tobacco TIMN 0115894 150 T200385

of vitamin D action and some other effect of nicotine. Assuming that this might be due to catecholamine- releasing action of nicotine, repeated sublethal doses of adrenalin were given. This failed to reproduce effects of nicotine. Assuming that the effects might still be due to adrenergic action of nicotine, animals were maintained on reserpine. This failed to inhibit nicotine-induced calcific thromboarteritis. After par- tial adrenalectomy, animals were resistant to nico- tine action. Among 19 animals. 14 had less than 350 mg of residual adrenal. Among these, only 1 had calcific thromboarteritis and distal calcific arterio- sclerosis. The remaining 5 animals had more than 400 mg of residual adrenal. All had peripheral calcific arteriosclerosis and 2 had calcific throm- boarteritis. Of 31 control animals without adrenalec- tomv. 28 had peripheral calcific arteriosclerosis and 19 had calcific thromboarteritis. Hence, nicotine enhancement of vitamin D action with induction of calcific thromboarteritis seems mediated by adrenal cortical function. Other support: U. S. Public Health Service. 170. Effect of chronic nicotine administration on cholesterol metabolism of liver, serum, heart and brain Sigmundur Gudbjarnason Department of Medicine, Wayne State University School of Nledicine, Detroit, Michigan Journal of aharmacology and Experimental Therapeutics 161:47-54, 1968 A study was carried out to examine the effect of chronic nicotine administration on tissue content of, and incorporation of acetate-l-Ct* into, cholesterol, cholesterol esters, glycerides, phospholipids and free fatty acids in liver, serum, heart and brain of the dog. Nicotine pre-treatment consisted of subcuta- neous injection of 0.5mg/kg five times a day on week-days and 1 mg/kg in one injection per day on week-ends, for 6 months; the dogs were then sac- rificed for analyses 2 hours after injection of the acetate-1-C14. A significant diminution was observed in the rate of cholesterol synthesis in nicotine-treated animals, with the greatest reduction being in the synthesis of serum cholesterol (-68%), followed b-v liver (-6-1°°), brain (-50°o) and heart nlu5- cle (-37°0) cholesterol. A significant decrease was also observed in the incorporation of acetate-l-C" into the cholesterol moiety of cholesterol esters in liver(-57%) and serum (-69%) of nicotine-treated animals.The incorporation of acetate-l-C'a into fatty acids. free or esterified, remained unaltered in the experimental animals. The tissue content of lipids remained identical in control and nico- tine-treated animals, except for a small but statis- tically significant decrease in cholesterol con- tent of liver (-11%) and heart muscle (-7°o). The biologic half-life of exogenous cholesterol--1-C" was significantly longer in nicotine-treated animals than in control animals. The kinetic of acetate-1-C14 incorporation into serum cholesterol was also studied in dogs receiving nicotine for 12 months in the manner described above. Incorporation of the labeled acetate into cholesterol of nicotine-treated animals was lower than into cholesterol of control animals, but the time-incorporation relationship was similar in both groups. The consistent diminution in cholesterol svn- thesis in nicotine-treated animals without anv change in the tissue content of this lipid indicates that the rate of degradation has been reduced to the same extent as the rate of formation. Other support: Detroit General Hospital Research Corporation, and the U. S. Public Health Service. 171. Bioassay for thrombopoietin utilizing mice in rebound thrombocytosis T. P. McDonald University of Tennessee Memorial Research Center and Hospital, Knoxville, Tennessee Proceedings of the Society for Experimental Biology and Medicine 144:1006-1012, 1973 A bioassay for the detection of the thrombo- poietic stimulating factor (TSF or thrombopoietin) that utilizes thrombocythemic mice is presented. A single injection of anti-mouse platelet serum (AMPS) to mice produced a characteristic thrombo- cytopenia that was followed by rebound thrombocy- tosis. Two to three days later (7-8 days after the AMPS injection), a significantly (P < 0.05) de- pressed thrombopoiesis existed in response to the thrombocytosis. Normal and t:hrombocythemic mice were injected with normal sheep sera or sera from platelet-depleted sheep and Na235SO, incorporation into platelets was measured. A greater difference in TIlVII`+T 0115881 T2o0372 137

Cardiovascular System Overall, the findings demonstrate that the ,greater lipid and catecholamine response to cigarette smoke js due to the tendency to inhale cigarettes, with resultant greater nicotine absorption. Other support: National Institutes of Health, and the Council for Tobacco Research-U.S.A. 185. Effect of smoking and nicotine on the crystallization of cholesterol A. Kershbaum. D. J. Pappajohn, H. Osada. and S. Bellet Division of Cardiology. Philadelphia General Hospital. Philadelphia. Pa. .-lctn CardioloQicn 23:548-557. 1968 Serum lipids have previously been shown to influence the rapidity of crystallization of choles- terol from a standardized solution of cholesterol in trigJ~~ceride. In this stud~•, the effect of tobacco smoking and nicotine administration on the crystal- lization rate of cholesterol was investigated. A highly significant acceleration of cholesterol crvstallization was effected by the serum lipids of blood taken 30 minutes after heavy cigarette smok- ing, in normal smokers. It did not develop after cigar or pipe smoking. There was no change in cholesterol cr-,•stallization rate after coffee, but an increase occurred when coffee was followed by smoking. Similar free fattv acid elevations developed in both i nstances. In anesthetized dogs. there was a significant increase in crvstallization rate after intravenous nicotine. These observations suggest a possible contribut- ing mechanism in the higher incidence of severe coronary atherosclerosis in cigarette smokers. Other support: National Institutes of Health, and the Council for Tobacco Research-USA. 186. Cigarette, cigar, and pipe smoking. Some differences in biochemical effects Alfred Kershbaum, and Samuel Bellet Division of Cardiology. Philadelphia General Hospital, Philadelphia. Pennsylvania Geriatrics 23(3):126-134. 1968 See Kershbaum (Acta Cardiologica 23:317-329. 1968) for a summary of the studies described in this report. Other support: National Institutes of Health, and the Council for Tobacco Research-U.S.A. 187. Spontaneous occurrence of arteriosclerosis in rabbits with prolonged cirrhosis Robert E. Lee. Jr.. Richard A. Scott. and George M. Hass Department of Pathology. Rush-Presbyterian-St. Luke's .Wedical Center. Chicago. Illinois Circulation 48(Suppl 4):IV-248. 1973 Chronic cirrhosis was produced in rabbits to evaluate impairment of hepatic function as a factor in pathogenesis of arteriosclerosis. More than 100 control animals were kept on a regimen of dietarv cholesterol and i.m. injections of either 0. 390, 780, 1560. 12,500, 25,000 or 50.000 IU Ergosterol twice everv fourth week. An equal number in the ex- perimental group was placed on the same regimen plus periodic i.m. injections of carbon tetrachloride sufficient to produce subacute or chronic hepatic degeneration. Both groups had average serum cho- lesterol levels of 350-500 milligrams percent. Those with subacute hepatic disease and control animals developed no conspicuous arteriosclerosis. Those with progressive cirrhosis developed generalized medial calcific and intimal fibroatheromatous ar- teriosclerosis when dosage of vitamin D exceeded 1560 IU. Less severe disease occurred at dosages of 390 and 780 IU indicating a 100-fold decrease in tolerance of cirrhotic animals to vitamin D. Twelve cirrhotic animals on the regimen for 12-15 months with no supplement of vitamin D developed aortic and peripheral arteriosclerosis resembling senile arteriosclerosis in man. Perhaps, further search for causes of arteriosclerosis in man should not dis- regard unrecognized defects in hepatic function. 188. Influence of cirrhosis on production of atheroarteriosclerosis and thromboarteritis with vitamin D and dietary cholesterol George M. Hass, Donald E. Henson, Richard A. Scott, Eldon C. McClain, and Anne Hemmens Division of Pathology, Presbyterian-St. Luke's Hospital, Chicago, Illinois American Journal of Pathology 57:405-429, 1969 Groups of rabbits were kept for months on regimens productive of combinations of mild dietary hypercholesteremia, hypervitaminosis D, and car- TIMN 0115886 T200377 142

4 t ,v i- I s j I a 3 H. PhysiologicaJ Action of Nicotine Derivatives 207. Possible role of nicotine isomethonium ion in physiological responses to nicotine Robert C. Bost, and Herbert McKennis, Jr. Medical College of Virginia. Richmond, Virginia Federation Proceedings 31:541, 1972 Previous studies from the authors' laboratory have shown that the metabolism of nicotine in the dog leads to the urinary excretion of nicotine isome- thonium ion and cotinine methonium ion. The present study of the effects of nicotine, nicotine isomethonium ion, and cotinine methonium ion on blood flow in the surgically prepared forelimb of the dog has shown no increase in vascular resistance when cotinine methonium iodide is infused into the brachial artery at the rate of 2.3 µM per min for 2 min with pressure measured (as an indication of vascular resistance) at the ulnar artery. Increases in vascular resistance produced by nicotine (2.3 µM per min for 2 min) were delayed in onset, suggesting mediation via adrenal stimulation, and increases in vascular resistance from nicotine isomethonium iodide (2.3 µM per min for 2 min) were early in onset. The response to nicotine isomethonium iodide appears related to direct effects in the forelimb. The inactiv- ity of cotinine methonium ion and cotinine itself suggests that metabolism of nicotine to these two compounds assists in terminating vascular responses produced by the parent alkaloid. Other support: American Tobacco Company. 208. Adrenergic activity of 3-(2-methylaminoethyl) pyridine Robert G. Bost, Raymond S. L. Chang, C. T. Sprouse, Edward R. Bowman, and Herbert McKennis, Jr. Nfedical College of Virginia, Richmond, Virginia Federation Proceedings 32:705, 1973 Although Serc (2-(2-methylaminoethyl)pryrid- ine) has pronounced histaminic activity and the positional isomer (4-(2-methylaminoethyl)pyridine) has contrasting adrenergic activity, previous studies provide little information on the biological activity of 3-(2-methylaminoethyl)pyridine(3-2-MAEP). In current studies on the perfused partially isolated forelimb of the dog. 3-2-MAEP (12 µM/min) infused into the brachial artery produced a 47°i° increase in peripheral vascular resistance with carotid arterv pressure showing no significant change. On isolated aortic strips from the rabbit, contractions produced by 3-2-MAEP (5 x 10-4M) were blocked by phento- lamine (1.3 x 10-6M) and not by bretylium (4.8 x 10'6M) or cocaine (3.2 x 10-5M). Aortic strips removed from rabbits pharmacologically depleted of norepinephrine by reserpine showed contractile res- ponsivity to 3-2-MAEP, thus providing evidence that 3-2-MAEP can act directly on adrenergic receptors. Metabolic oxidation of 3-2 MAEP in the dog leads to the urinary excretion of 3-pyridylacetate. 3-Pyridyla- cetate itself was essentially inactive in the pharma- cologic preparation investigated. Othersupport: American Tobacco Company, and the U.S. Public Health Service. 209. Effects of 3-pyridylacetate and its glycine conjugate on free fatty acids in rabbit epididymal fat pads Faye J. Bowman, E. R. Bowman, and Herbert McKennis, Jr. Department of Pharmacology, Medical College of Virginia, Richmond, Virginia IRCS Journal of Medical Science 3:65, 1975 Extensive studies on 3-pyridylacetic acid (3- PAA) have led to the demonstration of numberous pharmacological properties, including inhibition of lipolysis in isolated fat cells, inhibition of free fatty acid (FFA) mobilization, and hypocholesterolemic activity. Although it has been asseted that 3-PAA is not metabolized and is excreted unchanged, recent evidence from the authors' laboratory for metabo- lism of 3-PAA in man, rabbit and dog, led to isolation of N-3-pyridylactylglycine (3-PAA-Gly) as a urinary metabolite of 3-PAA (Section I. Abs 24). In the present report, the comparative effects of 3-PAA and 3-PAA-Gly on lipolysis have been studied in incubated epididymal fat pad samples obtained from New Zealand rabbits. Both 3-PAA and 3-PAA-Gly suppressed lipolysis (FFA release) in the fat pad samples, with 3-PAA-Gly having a sig- nificantly greater effect (P < 0.025) than 3-PAA. The combination of 3-PAA and 3-PAA-Gly was not significantly different from 3-PAA-Gly alone. Chro- matography of aliquots of the media after incuba- TIMN 0115895 T200386 151

s Cardiovascular System similar values for end-diastolic variables in the three groups. The first derivative of left ventricular pres- sure (dP/dt) normalized for pre- and afterload was 2.4 `*_- 0.2 cm/sec-' in the control group, 1.41 t 0.12 in the cigarette-smoking group (P < 0.005) and 1.34 ± 0.08 in the nicotine group (P < 0.01). Although mean aortic pressure was significantly elevated in both the smoking (127 t 5 mm Hg) and nicotine (127 ± 10 mm Hg) groups, there was no significant correlation with the contractility indexes. Reduction of afterload to normal levels did not affect the abnormal ventricular performance. Hypertrophy, in- flammation and abnormalities of cell ultrastructures were not present, and myocardial lipid and cation composition were normal. Since interstitial fibrosis was evident in both experimental groups, an al- teraction of elastic elements may be operative. These cardiovascular abnormalities appear to be predomi- nantly dependent on the nicotine of cigarettes. Other support: Council for Tobacco Research, U.S.A. 195. Effect of mescaline on cardiopulmonary dynamics: Method for determination of right ventricular pressure in the guinea pig V incent de Paul Lynch, Emmett Clemente. and Steven Carson (Bernard L. Oser) Department of Pharmacognosy. Phannacology and Allied Sciences. College of Pharmacy. St. John's University. Jamaica, New York, and the Food and Drug Research Laboratories, Inc., Maspeth, New York Journal of Pharmaceutical Sciences 56:477-483, 1967 The effect of mescaline on respiratory dynamics, right ventricular pressure, and the ECG in the guinea pig is presented. A procedure for the acquisition of normal right ventricular pressure is described. Mes- caline in low dosages (1 to 20 mg/kg, i.p.) produced a slight increase in heart rate; higher dosages (50 to 240 mg/kg) induced bradycardia and conduction disturbances. Respiratory rate, resistance of the air- wavs, and minute volume increased significantly, while tidal volume and compliance decreased. The decrease in compliance was near maximum (-70 and -77 per cent), with high dosages (50 and 100 mg/kg). Right ventricular pressure increased, average 45 per cent, following moderate dosages of mescaline (25 mg/kg). Diphenydramine and chlorpheniramine (range 0.4 to 0.7 mg/kg) in sufficient concentration to block the effect of histamine (4mg/kg) Were utilized to observe possible mescaline antagonistic activity. However, the antihistamines were ineffec• tive against mescaline. It appears probable that mes- caline does not owe its activity on these parameters to histaminic receptor stimulation. 196. Determination of flow in a multicompartnment model by external detection of gamma radiation D. L. Bovd, and S. H. Steiner 14fedical Service. VA Hospital, and Indiana University School of Medicine. Indianapolis. Indiana Physiologist 8(3):120. 1965 An eight-compartment recirculating flow model was connected in series to a 150 ml compartment which served as a cardiac mixing chamber. All other compartments were in parallel. Total volume of the system was 14L and compartment volumes ranged from 0.5 to 3L. Compartment flow rates were varied from 120 to 2140 ml/min. Total system flow was maintained at 5.12 L/min by a calibrated flowmeter. Inflow to each chamber was measured without disturbing the system. Each compartment was ob- served in turn by a collimated scintillation probe which led to a linear ratemeter with a 1-second time constant. Rates of change of indicator (Cs137) input and washout were recorded. The difference between these mono-exponential functions was extrapolated to To semi-logarithmically. Counts per minute at Ta were converted to a fraction of the injected tracer by counting a known amount of injectate using identi- ca) volume and geometry. Systemic flow was cal- culated bv the Stewart-Hamilton indicator dilution method. Three methods were used to calculate compartment flow: (1) Extrapolation to To of indica- tor input and net rate of redistribution; (2) Ex- trapolation to To of change in organ indicator con- tent in the absence of recirculation; and (3) Using the "t '/2" of change in organ indicator content in the absence of recirculation. The To of the injected fraction (methods 1& 2) was multiplied by cal- culated systemic flow to determine compartment flow. Comparing actual and calculated flows gave the following mean percent errors and standard deviations: Method 1 (-0.15 ± 6.86); Method 2 (-0.65- 11.11); Method 3 (-2.07 ± 11.69). The TIMN 0115890 146 T200381 j

tobarbital-anesthetized dogs and to demonstrate in resting, awake. trained dogs the ease of obtaining these measurements by conventional catheterization techniques. The stability of these measurements were determined over a prolonged experimental period. An additional purpose was to attempt to clarify the problem concerning the wide range usually given for canine blood gases. Finally, the purpose was to emphasize the important vagal inhibitory properties of pentobarbital by comparing its effects to those of atropine and nerve cooling in the awake animal. Since previous investigation has shown that there is a significant decrease in circulat- ing red blood cell volume during pentobarbital anesthesia which is largely accounted for by splenic sequestration, to avoid introducing an additionaJ .•ariable. each animal was splenectomized at least 3 weeks before the training procedure. Hemodynamic measurements and calculations made included car- diac output, mean femoral and pulmonary artery pressures. stroke volume, and total pulmonary and srstemic resistances. Arterial blood gas analysis included measurement of pH. pCOZ, and pOz. The results demonstrate that routine serial ob- servations can be made by conventional cardiac catheterization techniques in trained resting, awake dogs. The measurements are stable, and provide a suitable base line for determining the response of the reasonably intact subject to a variety of stimuli. The administration of 25 mg/kg of pentobarbital to the awake dog abolishes the normal sinus arrhyth- mia and results in tachycardia even before complete lapse of consciousness. There is marked inhibition of normal vagal activity as evidenced by the fact that heart rate fails to respond to a stimulus which is completely effective in the awake animal. The re- sults for pentobarbital are indistinguishable from vagal cooling or atropine in this experimental situa- t ion. Other support: U.S. Public Health Service, and the t_'.S. Air Force. 193. Cardiovascular effects of cold pressor tests, 40° head-up tilt, and smoking on smokers and nonsmokers William J. Marshall, Jr., Edwin L. Stanley, and Paul Kezdi Cor Heart Institute, Kettering, OF.:o Diseases of the Chest 56:290-296, 1969 Cardiovascular responses to various stress tests were studied in 13 nonsmokers. 16 moderate (20 cigarettes per day) and 13 heavt• (40 cigarettes per day) smokers before and after cigarette smoking. Intra-arterial pressure, ECG, respiration. and skin temperature were continuously monitored and re- corded on tape. Dye dilution cardiac outputs were measured in some of the subjects. The analog data were analyzed on a digital computer using A-D conversion. Blood pressure, heart rate and cardiac output were not different in smokers and nonsmok- ers before and after smoking. Hyperreactor response to cold immersion by Hines' criteria was extremely frequent (30% to 58%) in all subjects with the use of continuous pressure measurement. A composite of maximal response, time of return and the area under the pressure curve is suggested for better definition of the cold pressor response. Presyncopal reactions during 40° head-up tilt were more frequent in heavN smokers after smoking. 194. Cardiovascular effects of long-term cigarette smoking and nicotine administration S. Sultan Ahmed, Christos B. Moschos, Michael M. Lyons, Henry A. Oldewurtel, Richard J. Coumbis, Timothy J. Regan and Bess Jenkins Departments of Medicine and Pathology, College of Medicine and Dentistry, New Jersey Medical School. Newark, New Jersey American Journal of Cardiology 37:33-40, 1976 The nature of the cardiovascular risk in cigarette smokers has not been characterized. To compare the relative effects of long-term smoking and nicotine administration on the cardiovascular system, 18 month old beagle littermates were prepared with a permanent tracheostomy. They were classified into three groups: I, seven control dogs; II, nine dogs that smoked seven cigarettes/day; and III, eight dogs that received an equivalent amount of nicotine. After a period of up to 22 months, the animals were cathe- terized under anesthesia for assessment of left ven- tricular function and volumes by indicator-dilution technique. Heart rate, stroke volume, left ventricular end-diastolic pressure and volume and intraventric- ular conduction times did not differ significantly in the three groups. Left ventricular ejection fraction was 44 ± 3 percent (mean ± standard error of the mean) in the control group, 35 ± 3 percent in the dogs that smoked cigarettes (P < 0.05) and 23 ± 3 percent in those given nicotine (P < 0.01) despite TIMN 0115889 T200380 145

t ; t ; i social situation during the last years before death in IHD even when genetic factors are kept under control. Other Support: Swedish National Association against Heart and Chest Diseases, and the Swedish Medical Research Council. 202. Psycho-social factors in relation to coronary heart disease and associated risk factors Birgitta Floderus (Lars Friberg) Department of Environmental Hygiene of the Karolinska Institute and the National Environmental Protection Board and the Department of Sociology. Stockholm University, Stockholm. Sweden \rordisk Hygienisk Tidskrift, supplement 6:1-148, 1974 A multifactorial analysis of psycho-social status, smoking and genetic factors in relation to "angina pectoris," CHD-death and gross mortality was per- formed. The subject group was comprised of around 8,000 same-sexed twin pairs, born 1901-1925. Signs of psycho-social maladjustment were as- sociated with smoking, CHD and death. The association between psycho-social maladjustment on the one hand and CHD and mortality on the other was valid irrespective of smoking habits. Moreover, psycho-social maladjustment was "important" enough in the development of CHD and death to discriminate subjects, to a large degree equivalent to genetic and early environmental predisposition. The firmest associations were found for unspecific symptoms, e.g. "nervous diseases", restlessness, i nsomnia. A psycho-social measuring instrument was tested for its suitability in epidemiological studies based on mailed questionnaires. Psycho-social status measured in accordance with the Eysenck Personal- ity Inventory (EPI) was related to CHD risk factors. The analvsis was carried out on 380 men born 1914-1925 and on 980 men and women born 1927-1957. The EPI was transformed into the EPI-Q, the latter adapted for mailed questionnaires. The EPI-Q was considered a valid instrument for the detection of subjects prone to CHD. The EPI-Q instability group comprised an excess number of subjects who smoked, showed excessive alcohol consumption, decreased physical activity, diastolic hypertension, elevated cholesterol and triglyceride levels, dia- betes, and severe chest pain lasting > 30 minutes. Other support: the Karolinska Institute, and the Research Secretariat of the National Swedish En- vironmental Protection Board. 203. Concordance for mortality with special reference to ischaemic heart disease and cerebrovascular disease. A study on the swedish twin registry Ulf de Faire, Lars Friberg, and Torbjorn Lundman Department of Medicine, Karolinska Institutet at Serafimerlasarettet. Department of Environmental Hygiene, Karolinska Institutet, and the Swedish National Environmental and Protection Board, Stockholm, Sweden Preventive Medicine 4:509-517, 1975 The Swedish Twin Registry contains about 11,000 same-sexed twin pairs born between 1886 and 1925 with both members alive when the registry was formed in 1961. During the years 1962 to 1973, 2780 deaths occurred. 727 deaths were due to ischaemic heart disease (IHD), 345 due to cerebro- vascular disease (CVD), and 727 due to cancer. The rate of concordance for the whole twin population revealed a significantly (p < 0.05) higher con- cordance rate for IHD among the male monozygotic (MZ) pairs as compared to the dizygotic (DZ) pairs (15.8% versus 8.0%). The corresponding figures for the female pairs were 11.0% (MZ) and 7.5% (DZ), respectively. With regard to death in CVD and cancer, the rates of concordance were about the same for MZ and DZ pairs in both males and females. When subgrouping was made for age groups, the difference in concordance rate for IHD in males was still more pronounced for the younger age group, born 1901-1925 (16.1% versus 5.4%). These data may indicate the existence of a genetic determina- tion on death in IHD, especially in males, whereas a genetic determination on death in CVD and cancer seems more uncertain. Other support: Swedish National Association against Heart and Chest Diseases. and the Swedish Medical Research Council. TIMN 0115893 T200384 149

1 (61.6 µL1) and nicotine (61.6 µ%1) were only partially inhibited by La---• Increases in residual'5Ca spaces (after a 120-minute washout) induced by K- or ACh were inhibited by La---, but responses obtained in the presence of nicotine were not altered. In the presence of La---. total §'Ca spaces were not changed after exposure to K-, nicotine or ACh. Either La--- or added nonradioactive Ca" produced both transient increases and smaller sustained in- creases in 4SCa efflux from muscles previously washed out in Ca"-free Ringer's solution. Thus, La--" can replace Ca" at saturable and superficial membrane sites and, in this manner, may specifi- cally inhibit cellular uptake of ;5 Ca. The Ca"- dependent action affected by La"' may be im- portant for the initial portion of the contractile responses to K-, nicotine or ACh. It would appear that the Ca-- sources utilized by nicotine to elicit contractile responses in frog rectus abdominis mus- cle differ in part from those used by K1 and ACh. 218. Modification of the "alarm" pattern by n icotine E. H. Rubinstein, and R. R. Sonnenschein Department of Physiology. UCLA School of Medicine. Los Angeles. California .`'europharmacol ogy 10:247-258. 1971 - The effect of nicotine on the behavioral and autonomic response pattern, "alarm", evoked in a%cake cats implanted with recording devices by stimulation of stereotaxically oriented hypothalamic electrodes was studied. Before stimulation, the low- est effective intravenous infusion rate of nicotine (10 p.g/min) induced EEG desynchronization, increased gastrointestinal motility and decreased heart rate. As the infusion rate was raised to 50 µg/min, the arterial pressure increased, and moderate iliac and mesen- teric vasoconstriction occurred. The effect of nico- tine on the response to short hypothalamic stimula- tion (100-500 msec) were: less tachycardia, a marked post-stimulus enhancement of gastrointestinal mo- tilitv, a decreased threshold for growling, and an increased number of somatic responses (head turn). The effects of nicotine on the response to long hypothalamic stimulation (1-5 sec) were, in ad- dition, a more marked pressor response and a reduction of the iliac vasodilatation. It is suggested that nicotine, bv altering the reactivity of both the CNS and peripheral effectors, could effectivelr mod- ifv the extent of behavioral and autonomic responses that ultimately may have some pathogenic role in the development of cardiovascular disease. 219. The effect of potassium on nicotine-induced contracture and Ca45 movements in frog sartorius muscle George B. Weiss Department of Pharmacology, Medical College of Virginia, Richmond. Virginia Journal of Pharmacology and Experimental Therapeutics 154:595-604, 1966 The experiments described in this study de- lineate the actions of nicotine upon ionic move- ments and tension in frog sartorious muscle and the inhibition by K- of some of these actions. The contracture and three-fold increase in Ca;~ uptake induced by nicotine (1.25-5.0 mivl) in frog sartorius muscle are inhibited by prior exposure of the muscle to Ringer's solution containing 80 m;~~1 K`.The inhibition of increased Ca;5 uptake by K-also occurs at concentrations of nicotine (0.415-0.83 mM) too low to elicit recordable contractures. The rate of Ca45 uptake is maintained for a full 10- minutes incubation period at lower nicotine con- centrations (0.415 and 0.83 mM), whereas more than five-sixths of the increased Ca45 uptake at 2.50 mM nicotine occurs during the first 5 minutes of a 10- minute incubation period. Resting Caa5 uptake is also inhibited by prior administration of K-. Pro- longed washout in O-Ca-Ringer's solution contain- ing 4 mM EDTA does not prevent a nicotine-induced contracture which is accompanied by an increased Ca4s efflux. Under these conditions, 80 mM K' inhibits the contracture but not the increased Ca4s efflux. These observations suggest that the inhibi- tion of nicotine-induced Ca4l uptake and contracture by 80 mM K' is a result of prior displacement by K- of that store of bound Ca+' normally released by nicotine. The actions of nicotine may occur at different calcium sites from those affected by other agents which induce contracture in frog sartorius muscle. This study was the subject in part of a pre- liminary report appearing in Federation Proceedings 25:717, 1966. TIMN 0115899 T200390 155

Cardiovascular System 231. Mechanism of secretion from the adrenal medulla. III. Studies of dopamine (3-hydroxylase I as a marker for catecholamine storage vesicle membeane in rabbit adrenal glands O. H. Viveros. L. Arqueros. R. 1. Connett. and N. Kirshner Department of Biochemistrv and Division of Pharmacology. Duke L'niversitl,%fedical Center. Durham. North Carol ina Molecular Pharmacology 5:60-68. 1969 The subcellular distribution of dopamine (3- hydroxylase in homogenates of rabbit adrenal glands has been measured to determine the possibility of using the enzyme as a marker to trace the fate of the catecholamine storage vesicle membranes following secretion of adrenaline. Optimal conditions for the assay of the enzyme in each of the subcellulqr fractions have been determined. These studies show that a large fraction of the total dopamine R-hy- droxylase activity of the adrenal glands is present in the 26.000 x g supernatant fraction. Most of the enzyme activity associated with the particulate frac- tion was retained in the particles when they were washed with sucrose. When the particles were washed with distilled water, however, a large pro- portion of the enzyme activity was obtained in the supernatant fraction but a significant amount re- mained in the particulate fraction, indicating that the enzyme was contained within vesicles in both a soluble form and a membrane-bound form. The distribution of adrenaline and dopamine (3-hvdroxy- lase in a sucrose density gradient showed a peak of dopamine (3-hydroxylase activity near the bottom of the gradient closely associated with the adrenaline storage vesicle fraction, and a second peak near the top of the gradient associated with a less dense particulate fraction. Other support: National Science Foundation, and the National Institutes of Health. 232. Storage and secretion of adrenal catecholamines Norman Kirshner Department of Biochemistry, Duke University Medical Center. Durham, North Carolina Advances in Biochemical Psychopharrnacology 1:71-89, 1969 The author presents a review of current knowl- edge on the storage and secretion of adrenal cate- cholamines. Conclusions reached are as follows: 1. Catecholamines are stored in vesicles which can be isolated from the adrenal glands and sym. pathetic nerves in an osmotically inactive, non- diffusible state. 2. Studies of the composition of the vesicles suggest that catecholamines. ATP and protein inter- act to form a stable complex. 3. Physical and chemical studies demonstrate that the amines and ATP, and the amines. ATP and Mg-- form complexes in solution, but the stability of these complexes cannot account for the storage mechanism. Binding of catecholamines to purified chromogranin A, in the presence or absence of Mg-- and ATP, also cannot account for the stability of binding in the isolated vesicles. 4. Catecholamines are secreted from the adfenal gland by exocytosis during which the soluble con- tents of the storage vesicles, including the amines, adenine nucleotides and protein, are simultaneouslv released directly to the exterior of the cell. The vesicle membrane remains within the cell. 5. A metabolic source of energy which can be obtained either by glycolysis or by oxidative phos- phorylation is required for secretion from the adre- nal gland. 6. The inhibition of both uptake and release of catecholamines by N-ethylmaleimide, prenylamine and P-286 suggests that the storage vesicles employ a common pathway for at least a portion of the respective processes. Other support: National Institutes of Health, and the National Science Foundation. 233. Mechanism of secretion from the adrenal medulla. V. Retention of storage vesicle membranes following release of adrenaline O. H. Viveros, L. Arqueros, and N. Kirshner Department of Biochemistry, Duke University Medical Center, Durham, North Carolina Molecular Pharmacology 5:342-349, 1969 Dopamine (3-hydroxylase (EC 1.14.2.1) was used as a marker to follow the fate of the adrenal storage vesicles following the release of adrenaline. Neu- rogenic secretion of adrenaline induced in the rabbit by insulin produced a decrease in the adrenaline and dopamine (3-hydroxylase contents of the gland. There was only a slight decrease in the enzyme , TIMN 0115904 ' 160 T200395

Cardiovascular System tration of insulin, caused decreases in both the dopamine-R-hvdroxvlase activity and the catechol- amine,content of the storage vesicle fraction. After sedimentation through a sucrose density gradient, the storage vesicles obtained from insulin-treated animals had the same density and the same ratio of dopamine-(3-hydroxylase to catecholamine as did vesicles from untreated animals. These and other data indicate that neurogenic secretion from the adrenal medulla occurs by an all-or-none release from the storage vesicles. Other support: U. S. Public Health Service. 237. Influence of nicotine on catecholamine metabolism in the rat T. C. Westfall, and G. P. Anderson Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia Archives Internationales de Pharmacodynamie et de Therapie 169:421-428, 1967 The effect of three different doses of nicotine on the 24-hour urinary excretion of epinephrine, nor- epinephrine. metanephrine, normetanephrine and 3-methoxy-4-hydroxymandelic acid was investigated in rats. All three doses (0.1, 0.5, and 1.0 mg/kg, intraperitoneal) produced a statistically significant increase in the excretion of epinephrine and its major o-methylated metabolite, metanephrine. There were no significant changes in the excretion of norepinephrine, normetanephrine, or 3-methoxy-4- hydroxymandelic acid. It is concluded that the release of epinephrine, from the adrenal medulla by nicotine, is much more important in producing the various pharmacological responses, than is the cor- responding release of norepinephrine. Other support: Council for Tobacco Research- U.S. A. 238. Effect of smoking and nicotine on adrenocortical secretion Alfred Kershbaum, Douglas J. Pappajohn, Samuel Bellet, vfasami Hirabayashi, and Hassan Shafiiha Division of Cardiology, Philadelphia General Hospital, Philadelphia, Pa. Journal of the American Medical Association 203:275-278, 1968 The effect of tobacco smoking and nicotine administration on the secretory activity of the adre- 162 nal cortex was studied in man and in animals. In eight human subjects, there was a 27% to 77% rise in plasma 11-hydroxycorticosteroid concentrations after heavy cigarette smoking, compared to a normal diurnal fall during control observations with no smoking. In anesthetized dogs, nicotine adminis- tered intravenously resulted in a 64% rise in plasma corticosteroids. In rats, plasma corticosteroids con- centrations increased 58% after intraperitoneal ad- ministration of nicotine, accompanied by an in- crease in the corticoid content of the adrenal glands and a decrease in their cholesterol content. It is suggested that this stimulation of adrenocortical activity is due to enhanced corticotropin release resulting from a nicotine-induced increase in sym- pathetic and catecholamine activity. Because of the physiological and pathological importance of adre- nocortical hormones, their broad use as therapeutic agents, and their relationship to lipoprotein syn- thesis, the effect of cigarette smoking on adrenocorti- cal secretion must be seriously regarded. Other support: National Institutes of Health, and the Council for Tobacco Research-USA. 239. Hypothalamic control of baroreceptor reflexes Gerard L. Gebber, and David W. Snyder Department of Pharmacology, Michigan State University, East Lansing, Michigan American Journal of Physiology 218:124-131, 1970 The effect of hypothalamic stimulation was studied on the cardiac and vascular efferent com- ponents of the baroreceptor reflexes. Bradycardia evoked by carotid sinus nerve stimulation by nor- epinephrine was blocked by hypothalamic stimula- tion in spinal cats. These data demonstrate the existence of a suprabulbar system which functions to inhibit vagal bradycardia induced by baroreceptor activation. In contrast, baroreceptor modulation of central sympathetic outflow was functionally im- portant during hypothalamic stimulation. The de- pressor response produced by carotid sinus stretch was not reduced by hypothalamic stimulation in vagotomized cats. The pressor response evoked by hypothalamic stimulation was markedly enhanced TIMN 0115906 T200397 T200397

activity of the particulate fraction obtained from 1ysed storage vesicles, but marked decrease in the actie•ity of-the soluble fraction. Twenty-four hours after the administration of reserpine (1 mg/kg) to rabbits, changes in the dopamine (3-hydroxylase were similar to those observed after insulin treat- ment, but the glands were more severely depleted of their catecholamines. After lower doses of reserpine (0.25 mg/kg), or if ganglionic transmission was blocked by chlorisondamine one hour prior to the administration of reserpine (1 mg/kg), there were significant decreases in the catecholamine content but no changes in the dopamine (3-hydroxylase activitv. These findings indicate that neurogenic secretion is characterized by the release of the soluble content of the storage vesicles directly to the exterior of the cell and retention of the storage vesicle membrane within the cell. Interference only with the uptake or storage mechanism by reserpine results in depletion of the catecholamine content. but not of the intravesicular protein. Other support: National Science Foundation, and the National Institutes of Health. 234. Release of catecholamines and dopamine-(3- oxidase from the adrenal medulla 0. H. Viveros. L. Arqueros, and N. Kirshner Department of Biochemistry and the Division of Pharmacology. Duke University Medical Center. Durham. North Carolina Life Sciences 7, part 1:609-618, 1968 It has been previously reported that during the acetvlcholine-stimulated secretion of catechol- amines in perfused isolated cow adrenal glands, a protein specifically found in the catecholamine storage vesicles was released into the perfusion fluid together with the catecholamines in the same rela- tive amounts as that present in the intact storage vesicles. The presence of dopamine-(3-oxidase (DBO), an enzyme which is also localized in the storage vesicles, could not be demonstrated in the perfusion fluid either by enzymatic or by immuno- logic assay. Recent studies in the authors laboratory indicated that during secretion of adrenaline from the adrenal gland "in vivo," DBO activity was decreased and suggested the possibility that the enzyme was secreted from the cell but in amounts too low to be detected by the methods previously employed. Using a sensitive radioisotope assay, it was possible in the present study to demonstrate that acetvlcholine as well as nicotine stimulates the release of DBO and that the release of the enzv-me parallels the release of the catecholamines. The data presented here add further support to the hypothesis that during secretion from the adrenal gland, the entire soluble content of the storage vesicle is discharged directly to the exterior of the cell. Other support: National Institutes of Health, and the National Science Foundation. 235. Endogenous inhibitor(s) in adrenal medulla of dopamine-¢-hydroxylase David S. Duch, Osvaldo H. Viveros, and Norman Kirshner Department of Biochemistry and the Division of Pharmacology. Duke University Medical Center, Durham, North Carolina Biochemical Pharmacology 17:255-264. 1968 The addition of Cu2+ ions to various subcellular fractions of the adrenal medulla caused marked increases in dopamine-(3-hydroxylase activity. Fur- ther studies with Cu2+ and other sulfhydryl-reactive reagents such as Hg2-, Ag'', p-hydroxy-mercuriben- zoate, and N-ethylmaleimide indicate that the increased enzymatic activity was due to the inactiva- tion of one or more endogenous sulfhydryl com- pounds. The addition of CazT, Mn2+, Zn2', Fe2', and Niz' to the various fractions of the medulla had no effect on dopamine-(3-hydroxylase activity. The do- pamine-(3-hydroxylase activity in the subcellular fractions measured with and without the addition of Cu2` confirms previous reports that the enzyme is largely localized in the catecholamine storage vesi- cles and also indicates that the enzyme is more easily solubilized than was previously thought. Other support: National Institutes of Health, and the National Science Foundation. 236. Quantal secretion from adrenal medulla: All- or-none release of storage veside content O. H. Viveros, L. Arqueros, and N. Kirshner Science 165:911-913, 1969 Neurogenic secretion of catecholamines from the adrenal medulla in rabbits, induced by adminis- TIMN 0115905 T200396 161

( 228. Nitroglycerin (glyceryl trinitrate) as a monoamine oxidase inhibitor Kouichi Ogacva. Sigmundur Gudbiarnason, and Richard J. Bing Department of Medicine. i'1'ayne State University School o0fedicine. Detroit. Micbigan Journal of Pharmacology and Experimental Therapeutics 155:-149-455. 1967 The biochemical effect of nitroglycerin on myo- cardial monoamine oxidase (MAO) activity was studied in vivo and in vitro. Nitroglycerin adminis- tered in vivo inhibited significantly the MAO activ- itc of rat heart mitochondria with tyramine as substrate. Nitroglycerin also inhibited MAO activity of rat heart mitochondria in vitro with both tyramine and tryptamine as substrates. The mechanism of the inhibition of the MAO activity by nitroglycerin was studied in vitro on a solubilized and partly purified mitochondria monoamine oxidase. The effect of nitroglycerin on the enzyme kinetics of MAO in- dicated that the drug acts as competitive inhibitor of %IAO. A significant inhibition of rat liver and brain .%IAO activity was also observed in the presence of nitroglycerin. The effect of nitroglycerin on MAO activity was compared to that of the other organic nitrates. but no correlation was observed between the number of nitrate groups per molecule and the molar inhibitorv action. A preliminary report of this work appeared in FPderation Proceedings 25:319, 1966. Other support: U. S. Public Health Service, the ',iichigan Heart Association, the Burroughs-Well- (ome Fund, and the Detroit General Hospital Re- search Corporation. J. Studies on Endocrine System, Induding Cate- cholamines 229. Effects of various agents on the Mgz+-ATP stimulated incorporation and release of catecholamines by isolated bovine adrenomedullary storage vesicles and on secretion from the adrenal medulla R. 'v1. Ferris, O. H. Viveros, and N. Kirshner Department of Biochemistry, Duke University Medical Center. Durham, North Carolina Biochemical Phannacology 19:505-514, 1970 The effects of several agents on the Mg2+-ATP stimulated incorporation and release of catechol- amines by isolated bovine adrenomedullart• storage vesicles suggest that both of these processes ma~• employ a similar or identical set of reactions for a portion of their respective pathways. N-eth~•lmal- eimide and N-N-di-isopropyl-N'-isoam~•1-N'-dieth,,•- laminoethylurea (P-286) inhibited equally both incorporation and release. Prenvlamine inhibited incorporation to a greater extent than it inhibited release. Reserpine and propranolol, at concentra- tions which almost completely blocked incorpora- tion, had little or no effect on release. N-eth~•lmal- eimide, P-286 and prenylamine, when present in the perfusion medium at concentrations which blocked release from isolated vesicles, also blocked the secretory response of the isolated perfused adrenal gland to acetylcholine. In isotonic salt solutions. Mgz+ plus ATP stimulates to an equal extent the release of both soluble dopamine-(3-hydroxylase and catecholamines. An hypothesis is proposed to ex- plain these observations and to serve as a model for additional experiments. Othersupport: National Science Foundation and the National Institutes of Health. 230. Mechanism of secretion from the adrenal medulla. IV. The fate of the storage vesicles following insulin and reserpine administration O. H. Viveros, L. Arqueros. R. J. Connett, and N. Kirshner Department of Biochemistry and Division of Pharmacology, Duke University Medical Center. Durham, North Carolina Molecular Pharmacology 5:69-82. 1969 Rabbits were treated with insulin and with reserpine to obtain information on the mechanism of secretion of catecholamines from the adrenal gland and on the fate of the catecholamine storage vesicles following secretion. The data obtained suggest that during neurogenic stimulation the entire soluble content of the storage vesicles, including dopamine- (3-hydroxylase, is secreted by exocytosis, leaving the vesicle membranes within the medullary cells. The depletion of catecholamines produced by low doses of reserpine (1 mg/kg) is due mainly to inhibition of the uptake mechanism rather than to neurogenic secretion. Higher doses of reserpine (5 mg/kg) pro- duced an effect which appeared to be a combination of neurogenic stimulation as induced by insulin and the depletion produced by lower doses of reser- pine. Other support: National Science Foundation, and the National Institutes of Health. TIMN 0115903 T200394 159

Cardiovascular Srstem thrombi and/or atheromatous plaques in cigarette smokers. 248. Effects of nicotine on mucopolysaccharides of rat blood platelets Ted P. McDonald, and Dolores Woodard C'niversitY of Tennessee !ldemorial Research Center. Knoxville. Tennessee Abstract reprinted from the Third Research Conference on Tobacco and Health held by the AMA-ERF. May 7-9, 1972, Newport Beach. California Previous studies have shown that blood plate- lets which contain high levels of mucopolysaccha- ride (MPS) show greater adhesive qualities than platelets with low MPS levels. Since it is now well documented that platelet adhesiveness is enhanced following smoking, it seems possible that nicotine might alter the MPS content of platelets. This alteration may account for the changes in functional capability of blood platelets from individuals who smoke. To test the hypothesis that nicotine may alter the MPS content of blood platelets, rats were in- jected intravenously with ten daily doses of 0.01, 0.04. 0.08, or 0.16 mg/kg of body weight of nicotine and measurements made of peripheral blood values, platelet MPS content, and platelet functional ca- pabilities. Following nicotine injection the platelet counts. WBC counts, RBC counts, hematocrit values, and body weights were not altered when compared to saline-injected controls. However, increased platelet hexosamine 'values (17.0 x 10-3 µM hexo- samine/mg protein following injections of 0.08 mg/kg of nicotine to 7.8 x 10-3 µM hexosamine/mg protein for controls) were found due to nicotine injection. In addition, altered platelet aggregation and clot retraction indices have been observed. In a typical experiment, clot retractions were 36.8% for saline-treated controls, 12.0% following 0.04 mg of nicotine/kg of body weight, 8.4% after 0.08 mg/kg and 4.9 :.% after 0.16 mg/kg. These data illustrate that platelets from nico- tine-treated animals are more functional than plate- lets from saline-treated controls. The studies of quantification of MPS show greater uptake of Na2"SO4 in platelets of nicotine-treated animals than control animal platelets without major alterations in the kinds of MPS. Resuits to date support the 166 contention that nicotine injection increases the amounts of MPS in rat blood platelets. These studies of the alterations of blood platelets MPS content and function due to nicotine treatment may provide the data to explain one role of smoking on coronary heart disease. 249. An investigation of the effects of tobacco smoke and nicotine on rat blood platelet functions Marshall E. Reese, Jr. (T. P. McDonald) University of Tennessee, Knoxville, Tennessee Doctoral Dissertation. 1974. i-ix - 1-88 pp Arterial thrombosis occurs with increased fre- quency among cigarette smokers. Since blood plate- lets are intimately involved in the formation of thrombi, this study was undertaken to determine the effects of tobacco smoke on certain blood platelet functions. Male Sprague-Dawley rats (225-325g) were exposed to the smoke from 2, 8. or 16 ciga- rettes/day for 10 days by means of a specially designed smoking machine. Other rats were given nicotine in their drinking water at concentrations of 12.5 mg of nicotine/liter of water or 25mg of nic- otine/liter of water. The cigarettes used for smoke exposure were the IRI standard reference cigarettes produced at the University of Kentucky Tobacco Research Institute. The results of this research indicated that smok- ing caused a significantly (P < 0.05) decreased food consumption and a decreased weight gain (P < 0.0005) when compared with control values. Nico- tine did not alter the weight gain, but rats exposed to 25 mg of nicotine/liter of water consumed sig- nificantly less food than the nontreated controls. Smoke-treated rats drank significantly (P < 0.05) more water than controls, whereas rats given nico- tine in their drinking water consumed significantly (P < 0.05) less water than controls. The peripheral platelet count and red blood cell counts were un- changed after either tobacco smoke or nicotine exposure, whereas the hematocrit values were sig- nificantly (P < 0.05) increased in the smoke-treated rats, but not in the nicotine-treated rats. Smoking also caused a significantly (P < 0.005) decreased white blood cell count but nicotine had no sig- nificant effect. Smoke exposure and nicotine treatment caused no change in the average size of the circulating platelets. The smoke of 8 and 16 cigarettes/day resulted in a significantly (P < 0.005) increased platelet. adhesiveness. Rats exposed to 8 cigarettes/ TIMN 0115910 T200401 T

during carotid occlusion or following section of the carotid sinus nerves. Also, peripheral sympathetic nerve discharges elicited by hypothalamic stimula- tion were reduced when arterial pressure was raised. it is concluded that tachycardia associated with the pressor response evoked by hypothalamic stimula- tion was the result not only of increased cardiac sympathetic nerve activity, but also of inhibition of baroreceptor-induced vagal activation. Other support: U. S. Public Health Service. 240. Effects of adrenergic agonists and antagonists on potassium metabolism Ravmond H. Lockwood, and Bert K. B. Lum Department of Pharmacology, University of Hawaii School of Medicine, Honolulu, Hawaii Journal of Pharmacology and Experimental Therapeutics 189:119-129. 1974 Epinephrine and isoproterenol were previously found to protect animals against an infusion of KCl which was lethal in most animals. The protection was correlated with a hypokalemic action of the amines (i.e., an ability to attenuate the hyperkalemia induced by the KC1 infusion). In the present study on cats, salbutamol and soterenol were found to exert a similar action. Norepinephrine (1µg/kg/min) was considerably less effective than an equal dose rate of epinephrine; the former produced some reduction of hyperkalemia but did not significantly decrease the mortality produced by KC1 infusion. The beta-1 stimulant, 1-isopropylamino-3-(2-thiazoloxy)-2-pro- panol. had no effect on the hyperkalemia or mortal- ity produced by KC1 infusion. The hypokalemic action of epinephrine was correlated with signifi- cantly higher levels of K* in cardiac and skeletal muscle. An infusion of glucose, which produced blood glucose levels higher than in the epinephrine series, did not have a hypokalemic or protective effect against KC1 infusion. The hypokalemic and protective effects of epinephrine were not modified by acute pancreatectomy. Pretreatment with non- selective beta blocking agents (propranolol or so- talol) and with beta-2 blocking agents (butoxamine and H35/25) reduced or abolished the hypokalemic and protective actions of epinephrine in cats given KC1. The alpha blocking agent, phenoxybenzamine, and the beta-1 agonist, practolol, did not sign9fi- cantly reduce these actions of epinephrine. The results indicate that beta-2 receptors subserve a hypokalemic action which is responsible for the protective effect of sympathomimetic amines against potassium intoxication. This hypokalemic action appears related to increased entry of K- into cells. Preliminary reports appeared in Federation Pro- ceedings 30:498, 1971; 31:509, 1972. Other support: Hawaii Heart Association. 241. Molecular interactions of nicotine Dan W. Urry Division of Molecular Biophysics, University of Alabama. Birmingham, Alabama Abstract reprinted from the Third Research Conference on Tobacco and Health held by the AMA-ERF, May 7-9. 1972, Newport Beach, California On addition of nicotine to solutions of chromo- gramin A (the catecholamine storage protein from the chromaffin granules of the adrenal medulla) a large decrease in absorption is observed but little effect is seen in the circular dichroism. The effect may be indicative of a molecule passing from a more polar to a less polar environment. Because this non- specific effect could occur with membranes as a general phenomenon, it is necessary to assess the interaction of nicotine with well characterized mem- brane fractions which are not known to contain receptor sites for biogenic amines before attempting to interpret effects of nicotine on membrane prepara- tions containing such receptor sites. Accordingly, the possible interaction of nicotine was studied on the (Na+ + K*) - ATPase membrane preparation from pig brain (which is considered not to have biogenic amine sites) and then on oxyntic cell membranes from the stomach (which, though not known to contain biogenic amine sites, may be involved in the secretion into the stomach of nicotine when the stomach vessels are perfused with nicotine contain- ing solutions). As with chromogramin A a decrease in absorption was observed but little or no evidence of interaction was seen in the circular dichroism. This work properly sets the stage for examining the effect of nicotine on synapses and other membrane preparations containing biogenic amine receptor or binding sites. Along a different line of investigation we have found that while nicotine does not exhibit specific TIMN 0115907 T200398 163

_r Cardiovascular SvStem With no alchohol. subjects had bradvcardia (mean less than 60 per minute). Alcoholic beverages blocked or reversed this. Effects were still present at 7 houis when blood alcohol concentrations were very small (mean = 13 mg%). Blood pressure effects were less clear cut, but still consistent with possible catecholamine release, modified bv vasodilatation. 215. Cardiovascular effects of intravenous acetaldehyde and propionaldehyde in the anesthetized rat John L. Egle, Jr., Patricia M. Hudgins, and Fong M. Lai Department of Pharmacology, Medical College of Vi binia. Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia Toxicology and Applied Pharmacology 24:636-644, 1973 Acetaldehyde and propionaldehyde have both been previously shown to possess sympathomimetic activity and to affect the cardiovascular system. The objectives of the present investigation were to study the dose-effect relationships of the two aldehydes and to establish the mechanisms of the pressor and depressor effects of the compounds. Low intra- venous doses of acetaldehyde and propionaldehyde produced a consistent dose-related rise in blood pressure. These responses were slightly reduced by adrenalectomy and more strongly antagonized by pretreatment with reserpine or phentolamine. This indicates that the pressor effects of these compounds are primarily due to vasoconstriction mediated by norepinephrine released from sympathetic nerve endings in vascular smooth muscle. Higher doses of the two aldehydes produced a sharp fall in blood pressure and a severe bradycardia. Atropine reduced the hypotensive and cardioinhibitory effects of the aldehvdes, suggesting that these actions are me- diated by the vagus nerve. This was confirmed by experiments • in vagotomized rats. After vagotomy, high doses of acetaldehyde and propionaldehyde produced an increase in blood pressure and a positive chronotropic response. The results of this study indicate that acetaldehyde and propionalde- hyde exert two opposing actions on the cardiovascu- lar system: (1) a sympathomimetic effect which predominates at doses below 20 mg/kg resulting in a rise in blood pressure; and (2) stimulation of higher centers resulting in bradycardia and hvpotension with higher doses. 216. Effects of intravenous acetaldehyde. acrolein, formaldehyde and propionaldehyde on arterial blood pressure following acute guanethidine treatment Mark A. Green, and John L. Egle, Jr. Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia Federation Proceedings 34(3):817, 1975 Guanethidine is currentlv used in the treatment of essential hypertension. This aldehyde series is a constituent of cigarette smoke and acetaldehyde is also an intermediate oxidative metabolite of ethanol. These aldehydes are also known to possess syrn- pathomimetic effects. These experiments were per- formed to show if an excess of these aldehydes alters guanethidine's antihypertensive effect. Dose re- sponse studies in the anesthetized rat have indicated that after acute guanethidine treatment (15 mg/kg), a dose of 3 mg/kg acetaldehyde will cause a sig- nificant pressor response. In contrast to this, doses of acrolein ranging from 0.05 to 0.5 mg/kg result in a depressor response with significance at the higher doses. Experiments are now in progress to determine the effects of propionaldehyde and formaldehyde on blood pressure following acute guanethidine treat- ment. Current work is also being done to elucidate the effects on blood pressure in the acute quanethi- dine treated rat of a combination of the above 4 aldehydes. 217. Inhibition by lanthanum of some calcium- related actions in frog rectus abdominis muscle George B. Weiss Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas Journal of Pharmacology and Experimental Therapeutics 185:551-559, 1973 The effects of lanthanum ion (La+++) on 4SCa movements and tension responses elicited with nicotine, acetylcholine (ACh) or high potassium ion (K+) were examined in frog rectus abdominis mus- cle. La'++ (1.0 mM) decreased 45Ca uptake and total Ca++ and Na+ contents, but total K+ contents were increased. Tension responses to K+(80 mM), ACh TIMN 0115898 T200389 154 ~

There were paired controls for those in group III and group IVE Adrenalectomized animals failed to sur- vive the carbon tetrachloride regimen longer than 4 weeks. Necropsies disclosed a severe myopathy restricted to animals on the combined programs in group III and group IV. The cardiomyopathy was most conspicuous throughout the inner half of the ventricular myocardium. though at times the full thickness of the auricular myocardium was in- volved. Atypical intestinal edema and stromal reac- tions were the earliest microscopic findings. Later, multiple diffuse foci resembling acute. myocardial infarcts of various ages in various stages of stromal repair were conspicuous. There were no significant vascular lesions. Skeletal muscle was usually also affected by severe degenerative and reparative changes. Similar though less severe myopathies occurred when thioacetamide was used as a hepato- toxin in place of carbon tetrachloride in adrenalec- tomized animals. These observations may bear on the pathogenesis of common puzzling skeletal and cardiac myopathies occurring in people with a historti' of alcoholism. Other support: Schweppe Foundation, and the Otho S. A. Sprague Memorial Institute. L. Hematology 245. Effect of tobacco smoking on binding of oxygen by hemoglobin Daniel Dupourque, Amoz I. Chernoff, and Carey A. Chambers Nfemorial Research Center and Hospital, University of Tennessee. Knoxville, Tennessee t_'npublished report to the AMA-ERF The effect of smoking on the interaction of hemoglobin with oxygen was investigated by studying two groups of young, healthy blood do- nors. one that was composed of 64 nonsmokers and the other of 75 cigarette smokers (one to two packs a day). The following parameters were calculated from hemoglobin-oxygen association curves:P,o, Pso. P9o1 and Hill's coefficient (concentration of carboxyhe- moglobin and 2, 3 DP6 diphosphoglyceric acid) and values of blood pressure are also reported. Oxygen- association curves are also reported as carried out on 44 blood samples of nonsmokers after the samples had been equilibrated in a tonometer with carbon monoxide. Variance analysis shows the smoking affected significantly the allosteric properties of hemoglobin but not its average affinity. Regression analysis shows that most of the effect observed in smokers can be explained by the increased con- centration of carboxyhemoglobin. 246. Linear regression analysis of the heat denaturation of hemoglobin A, F, and S Daniel Dupourque, Bennett F. Horton, Amoz I. Chernoff. and Carey A. Chambers Memorial Research Center and Hospital. University of Tennessee, Knoxville, Tennessee Unpublished report to the AMA-ERF The conditions for a pseudo-first order constant for the heat denaturation of ferrihemoglobin are described. This constant is a sensitive and accurate estimate of the stability of different hemoglobins under various conditions. Salts at high concentra- tion decrease heat stability. This effect is not due to a specific interaction of cation with the hemoglobins. At moderate ionic strength (0.1M), chloride anion has a protective effect on ferrihemoglobin. When a ligand is bound to the ferriheme, the protective effect of Cl is not observed. Anticoagulants used for collecting blood affect the heat stability of ferrihe- moglobin. There is no significant difference in the heat stability of hemoglobin obtained from blood donors who smoke and those who do not. 247. An acute effect of cigarette smoking on platelet function. A possible link between smoking and arterial thrombosis Peter H. Levine Blood Coagulation Laboratory of the New England Medical Center Hospital, and the Department of Medicine, Tufts University School of Medicine, Boston. Massachusetts Circulation 48:619-623, 1973 In a controlled, double blind study, the smoking of a single cigarette has been shown to increase the platelets response to a standard aggregating stimu- lus (adenosine diphosphate). The phenomenon appears to be specifically related to the inhaling of tobacco smoke; it does not follow the smoking of lettuce leaf filled cigarettes. The platelet effects seems independent of the rise in plasma free fatty acids which follows cigarette smoking. Smoking- inducqd potentiation of platelet aggregation may help to explain the increased incidence of arterial TIMN 0115909 T200400 165

s and increased vascular permeability which charac- terized the acute inflammatory response. Other support: U.S. Public Health Service, and the National Tuberculosis and Respiratory Disease Association. 212. In rifro effect of 6-hydroxydopamine on isolated rat atria M. Kuchii. and S. Shibata Department of Pharrnacology. School of Medicine, t'nirersih• of Hawaii. Honolulu, Hawaii British Journal of Pharmacology 44:583-585, 1972 In vitro treatment with 6-hvdroxvdopamine (6- OHDA), but not with oxidized 6-OHDA caused transient. positive inotropic and chronotropic responses in the isolated rat atria. In the presence of rat blood plasma the excitation effect of 6-OHDA persisted unless the drug was washed out by fresh medium. 6-OHDA failed to elicit the excitative response in the atria obtained from reserpinized rats or rats treated with 6-OHDA in vivo. In vitro treatment with 6-OHDA did not cause the develop- ment of supersensitivity to noradrenaline. Cocaine and desipramine, but not bretylium, inhibited the excitative action of 6-OHDA. No tachyphylaxis de- %•eloped after repeated exposure to 6-OHDA in vitro. Propranolol, but not phentolamine, blocked the excitatorv effect of 6-OHDA. It is thus concluded that in vitro treatment with 6-OHDA may cause atrial stimulation by an indirect action involving the release of catecholamines as a result of its dis- placement at the nerve ending. Other support: Hawaii Heart Association. 213. Effect of prolonged alcohol administration on calcium transport in heart muscle of the dog Richard 1. Bing, Harald Tillmanns, Jean-Marie Fauvel, Keith Seeler, and James C. Mao Huntington Vemorial Hospital, Pasadena, California, and the C'niversih• of Southern California, Los Angeles, California Circulation Research 35:33-38, 1974 The effect of prolonged administration of alco- hol on calcium binding and uptake by sarcoplasmic reticulum and mitochondria and on respirator}• function of mitochondria was investigated in heart muscle of dogs. Dogs were paired and maintained with and without alcohol for 6 months; alcohol was administered by adding it to drinking water and food with vitamin supplements. Measurements were made after alcohol had been temporarily withheld for 2 days. Prolonged alcohol ingestion resulted in a decline in calcium binding and uptake by sarcoplas- mic reticulum and mitochondria, suggesting a di- minished affinity of the reticular and mitochondrial membranes for calcium ions. The endogenous cal- cium content of mitochondria and sarcoplasmic reticulum decreased. Prolonged alcohol administra- tion failed to alter cardiac contractility, although contraction and relaxation tended to diminish fol- lowing the administration of angiotensin. The re- sults illustrate that one link in the regulation of the state of contraction or relaxation involving mvo- fibrillar calcium transport is weakened in dogs maintained on alchohol for prolonged periods of time. Other support: Council for Tobacco Research- U.S.A., the Herbert Hoover, Jr. Foundation, the Kenneth T. and Eileen L. Norris Foundation, and the U.S. Public Health Service. 214. Prolonged cardiovascular effects of alcoholic beverages M. B. Starr, H. B. Murphree, and R. E. Schultz Department of Psychiatry, Rutgers Medical School, and Center of Alcohol Studies, Rutgers University. ~\rew Brunswick, New Jersey Federation Proceedings 29:274, 1970 Earlier findings from this laboratory suggested that alcoholic beverages have cardiovascular effects which become maximal well after peak blood alco- hol concentrations. To test this, 9 men were given vodka, bourbon, or orange juice according to a latin square design, about 8:30 AM, one hour after a light, low-fat meal without caffeine. The doses of bourbon and vodka contained 1.00 ml/kg ethanol. Before dosage and hourly for 8 hours after, each subject's blood pressure and pulse rate were measured after 10 minutes of lying down on a tilt table. Then they were raised upright and the same measurements were made within 20 seconds. Blood alcohol cor.- centrations were determined with a Breathalyzer. TIMN 0115897 T200388 153

day resulted in a significantly (P < 0.05) increased percent platelet aggregation after challenge with ~DP; however, smoke from 2 or 16 cigarettes/day or to nicotine showed no effect. Aggregation of platelet initiated by collagen was not different between tobacco-smoke-treated and nicotine-treated rats. In addition. the rates of aggregation were unaltered by either ADP or collagen challenge. The platelets from smoke-treated rats also had significantly (P < 0.005) increased clot retraction at each level of cigarette exposure (2. 8 and 16 cigarettes/day). These findings strongly suggest that smoking caused an increase in platelet function. This altered platelet function may contribute to the increased incidence of heart attacks among smokers as compared to nonsmokers. Other support: Tennessee Heart Association. 250. Immunoassay and bioassay for thrombopoietin Ted P. McDonald t'niversitv of Tennessee Memorial Research Center. Knoxville. Tennessee In Platelets: Production. Function, Transfusion, and Storage. M. G. Baldini and S. Ebbe. Editors. Grune & Stratton. Inc.. New York, 1974. pp 81-92 Both a bioassay and an immunoassay procedure for the measurement of TSF [thrombopoietin-stimu- lating factor] have been reported. The bioassay procedure utilizes thrombocythemic mice after a single injection of anti-mouse platelet serum. This antiserum caused a characteristic thrombocytopenia in mice that was followed by rebound thrombocyto- sis. Thrombocythemic mice were injected with nor- mal or TSF-containing serum or plasma fractions and the Na,35SO., incorporation into platelets was measured 3 days later. For the immunologic assay, a TSF-rich serum fraction was used to immunize rabbits and the immune serum was found to contain hemagglutinating antibodies. These antisera were absorbed with normal sheep serum, normal sheep plasma, or fractions of normal serum to remove antibodies not specific to TSF. The specificity of the remaining antibodies to a chemical found only in platelet-depleted sheep serum was apparent since these HA antibodies could be removed only by absorption with a TSF-rich fraction. The absorbed antisera containing TSF-specific antibodies were then used in the HAI hemagglutination-inhibition procedures to detectand quantify TSF in thrombocy- topenic sheep sera and fractions of plasma from platelet-depleted sheep. The results indicate that TSF can be partially purified by both ethanol pre- cipitation methods and DEAE-phosphate cellulose column chromatographic procedures and that TSF can be detected quantified by both assay procedures in whole serum or plasma fractions. Other support: American Heart Association. the Tennessee Heart Association, and the U. S. Atomic Energy Commission. 251. Regulation of thrombopoiesis T. P. McDonald University of Tennessee Memorial Research Center and Hospital, Knoxville, Tennessee Medicina 33:459-466, 1973 In this presentation an attempt is made to: 1) present a model of thrombopoiesis based on cur- rently available data; 2) consider available informa- tion on methods for assay and the chemistry of the thrombopoiesis stimulating factor (TSF); and 3) report progress on further development of both a bioassay and an immunoassay for TSF. Other support: American Heart Association, and the Tennessee Heart Association. 252. Cigarette smoke induced formation of methemoglobin (Met-Hb) in blood A. J. Ingenito, and L. Procita Department of Pharmacology, Albany Medical College, Albany, New York Federation Proceedings 32(3):805, 1973 Estimates of nitric oxide (NO) in tobacco smoke vary from 100 to 1000 ppm. Oxides of nitrogeti can form Met-Hb in blood which may contribute to the toxicity of these gases. Data as to whether Met-Hb can be formed at NO concentrations attainable in smokers lungs is presently lacking. To investigate this, ot-chloralose-anesthetized cats were made to inhale smoke from 1-to 3 research cigarettes as a series of 10 ml puffs blown directly into the trachea. Femoral arterial and venous blood samples were taken immediately after each cigarette and pO2, pC02, % oxy-, carboxy- (COHb) and hfet-Hb were determined, the latter by an established spectropho- tometric method. No (< 0.5%) Met-Hb was detected in any §ample, even though as much as 30% COHb was present in some. pO2, and pCO2 were un- TIMN 0115911 T200402 167

r Cardiovascular System 220. On the site of action of nicotine on contracture in frog sartorius muscle George B. Weiss Department of Pharmacology. Medical College of t'i binia. Richmond. Virginia - Journal of Pharmacology and Experimental Therapeutics 163:43-53. 1968 Several experimental approaches were utilized to obtain information concerning the mechanism by which nicotine acts to alter CaiS movements and induce contracture in frog sartorius muscle. Nicotine elicited an increased Ca4S efflux only when EDTA was present in a Ca-free Ringer's bathing solution. Substitution of nitrate anion for chloride resulted in a potentiation of nicotine contracture but no ad- ditional Ca;1 uptake as measured by Ca4S spaces after 120-minute washout. Increasing the potassium con- centration to 80 mM markedly increased nicotine-C" efflux but did not significantly alter the nicotine-C" tissue space occupied. Neither nitrate anion nor 80 mM K- altered intracellular pH as measured by DMO-C" spaces. Also, high concentrations of d-tubocurarine chloride (0.3 mg/ml) did not prevent a nicotine contracture and associated increases in Ca4s uptake. These observations provide further indica- tion that nicotine acts at sites in the transverse tubule membrane to alter Ca'' movements and in- duce contracture. Furthermore, these actions of nico- tine mav occur at calcium sites different from those at which corresponding effects are elicited by ex- posure to 80 mM potassium. 221. On the site of action of nicotine in frog rectus abdominis muscle George B. Weiss Departments of Pharmacology, Medical College of L'i binia. Virginia Commonwealth University, Richmond. i'irsinia and University of Texas Southwestern Medical School. Dallas. Texas Journal of Pharmacology and Experimental Therapeutics 183:169-181. 1972 Experiments were designed to elicit information about the cellular site of action of nicotine in frog rectus abdominis muscle. Nicotine (6.16 and 61.6 µM) increased muscle 45Ca retention after a sub- sequent 120-minute -wash.out, decreased total Ca-' content and did not alter 45Ca loss when added during the slow component phase of the washout. Increasing pH from 6.80 to 9.40 increased nicotine- induced }5Ca retention and decreased the rate of development (but not the magnitude) of the con- tractile response. Procaine (3.67 mM), high K- (80 mM) and d-tubocurarine (25 µM) but not acetvlcho- line (ACh 2.3 µ:vf) blocked contractions and in- creased ;1 Ca retention induced by nicotine. whereas nicotine blocked contractions elicited bv ACh but not those obtained with high K-. Hemicholinium-3 partially inhibited contractions and the increased {SCa retention elicited by nicotine but did not affect similar actions of ACh. These results indicate that nicotine (1) acts at receptors at the neuromuscular junction and only a portion of these may be ACh receptors, (2) is active in both ionized and non- ionized molecular forms and (3) does not exert these actions at sites beyond the neuromuscular junction. The varied responses obtained with either the ion- ized or nonionized forrps of nicotine occur at similar extracellular concentrations. The importance of each of these stimulatory mechanisms in the evaluation of actions of nicotinic agents on frog rectus abdominis muscle is uncertain. This study was the subject of a preliminary report appearing in The Pharmacologist 13:264, 1971. 222. The effect of pH on nicotine-induced contracture and Ca4S movements in frog sartorius muscle George B. Weiss Department of Pharmacology, Medical College of Virginia, Richmond, Virginia Journal of Pharmacology and Experimental Therapeutics 154:605-612, 1966 The contracture and three-fold increase in Caas uptake induced by 2.5 mM nicotine in frog sartorius muscle can be inhibited by lowering the pH of the Ringer's bathing solution from 8.4 to 7.4. The nicotine induced contracture and Ca;s efflux ob- tained at pH 8.4 after prolonged washout in O-Ca- Ringer's solution containing 4 mM EDTA are also inhibited by decreasing the pH to 7.4. If the amount of nicotine in pH 7.4 Ringer's solution is raised to 10 mM, the concentration of nonionized nicotine is approximately equal to that in pH 8.4 Ringer's solution containing 2.5 mM nicotine. The contrac- ture and increased Ca45 uptake induced by 10 mM nicotine in the pH 7.4 Ringer's solution does not i differ significantly from that obtained using pH 8.4 T11VIN 0115900 156 T200391 1

55. Brain Area Nicotine Levels in Male and Female Rats with Different Levels of Spontaneous Activity ...........................:...................................... 198 56. Conditional Aversion to a Preferred Solution Following Methamphetamine Injections ....... 199 57. Impairment of Alternation Learning in Rats Following Microinjection of Carbachol into the Hippocampus ............................................................. 199 58. Visceral and Behavioral Responses to Intraduodenal Fat ............................. 199 Neuropharmacology a. .%,fodel Veuronal Svstems 59. Evoked Surface-Positive Potentials in Isolated Mammalian Olfactory Cortex ............... 199 60. Action of Nicotine on Identified Cells of the Snail Brain ............................. 200 61. The Separation of Two Cholinergic Systems in the Brain Stem ........................ 201 62. Behavioral Evidence for Two Types of Cholinergic Receptors in the C.N.S . ............... 201 63. Estimation of Ionic Concentrations and Intracellular pH in Slices from Different Areas of Rat Brain .................................................................... 201 64. On the Measurement of Extracellular Space in Slices Prepared from Different Rat Brain Areas . 201 65. Drug-Induced Alterations in Respiration of Rat Brain Cortex and Striatum Slices in a Carbon Dioxide-B icarbonate-Buffered Medium ........................................... 202 66. Dependence of 14C-Nicotine Accumulation on Intracellular K' in Rat Brain Area Slices ...... 202 67. Ionic Basis for Intracellular 14C-Nicotine Accumulation in Slices from Different Rat Brain Areas .................................................................... 202 68. Effects of Pharmacological Agents on [14C]-Nicotine Distribution and Movements in Slices from Different Rat Brain Areas ..................................................... 203 69. On "C-Nicotine Distribution and Movements in Slices from Monkey Cerebral Cortex ........ 203 70. Characterization of [14C]-Nicotine Accumulation and Movements in Slices from Different Rat Brain Areas ............................................................... 203 b. Sensory Svstems 71. Effect of Nicotine on Cochlear Function and Noise-Induced Hair Cell Loss ............... 204 72. Spontaneous Vertical Eye Movements on Closing the Lids ............................ 204 73. Horizontal Nystagmus in Routine Subjects by Electronystagmography ................... 204 74. Acute Effects of Smoking on the Labyrinth ....................................... 204 c. Motor Srstems 75. Characteristics of Sustained Tremor Induced by Nicotine in Pilocarpine-Treated Animals .... 205 d. Temperature Regulation 76. Cholinergic and Adrenergic Interactions in the Thermoregulatory Centers of the Rat ........ 205 77. Drugs and Body Temperature ......................................... ....... 205 78. Temperature Changes Following lontophoretic Injection of Acetylcholine into the Rostral Hypothalamus of the Rat ..................................................... 205 TIlyIN 0115917 T200408 173

Cardiovascular Svstem and systolic blood pressure during exercise (P < 0.001). The capacity of these patients to perform a multistage exercise test, however, was not improved significantly, and the ischemic ST-segment changes were not altered by the treatments. Thus, proprano- lol appeared to be effective on the symptoms of angina pectoris, but it did not significantly improve exercise performance, and it did not prevent the ischemic patterns in the exercise ECG in this group of patients. Oral isosorbide dinitrate, alone or in combination with propranolol, was ineffective in this study. Other support: U. S. Public Health Service, and the Chicago Heart Association. 259. Continuous prehospitalization monitoring of cardiac rhythm Gary J. Anderson, Suzanne B. Knoebel, and Charles Fisch (Kalman Greenspan) Krannert Institute of Cardiology, Marion County General Hospital, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana American Heart journal 82:642-646, 1971 A continuous electrocardiographic telemetry system is described which permits simultaneous voice communications. The data observed suggest that continuous ECG telemetry is a useful tool in the prehospitalization detection of arrhythmias in pa- tients with coronary heart disease. The instituting of earlier therapy for arrhythmias may reduce the incidence of early death. Other support: U. S. Public Health Service, the Herman C. Krannert Fund, and the Indiana Heart Association. 260. Hemodynamic consequences of atrial fibrillation Robert E. Edmands, and Kalman Greenspan Department of Medicine, Indiana University School of Medicine. Indianapolis, Indiana Geriatrics 26(1):99-107, 1971 Hemodynamic consequences of atrial fibrilla- tion is discussed in relation to two principal factors. The first of these stems from the loss of a coordinated atrial systole. A second area of concern lies in the 170 effect of an irregularly irregular ventricular response upon myocardial function. Other support: Herman C. Krannert Fund, U. S. Public Health Service, and the Indiana Heart As- sociation. 261. A qualitative platelet defect in severe vitamin 13, deficiency: Response, hyperresponse, and thrombosis after vitamin B12 therapy Peter H. Levine Blood coagulation and Blood Research Lahoratory, New England Medical Center Hospitals; and the Department of Medicine, Tufts University School of Medicine, Boston, Massach usetts Annals of Internal Medicine 78:533-539, 1973 Three patients with vitamin B,Z deficiency had profound qualitative platelet abnormalities: lack of secondary aggregation (release reaction) with ADP stimulation, total lack of aggregation in response to standard amounts of epinephrine or collagen, and, in two patients, bleeding times of more than 20 minutes. Vitamin B12 therapy corrected all these defects. Serial studies, after vitamin B12 administra- tion, showed increased platelet aggregation re- sponse in two of the patients, when compared with 60 healthy controls. All three developed increased platelet factor 3 availability. One patient developed evidence of arterial and venous thrombi at this time, with no thrombocytosis. A retrospective chart re- view suggests that thrombotic episodes during re- covery from vitamin B,Z deficiency are not rare. The platelet apparently is one of the cells of the body that are susceptible to metabolic abnormality in severe vitamin B12 deficiency. Platelet hyper-function may be important in the pathogenesis of thrombosis. 262. A cigarette smoking machine design for cardiovascular research Steven B. Cliff (T. P. McDonald) Department of Engineering Science and Mechanics, University of Tennessee, Knoxville, Tennessee Tennessee Engineering, Summer, 1973, pp 24-27 A device for the exposure of rats to cigarette smoke is described. This device follows stringent standard smoking conditions while providing the full degree of freedom which those standards allow. TIMN 0115914 T200405 i s

Cardiovascular System tic mast cells. Alkaline conditions were required to convert the releasing agents to the form of their free base thus making available the 2s-electron pair of the molecule's alict•clic nitrogen atom for interaction with the cell's trigger sites. The potency of the compounds as releasing agents was directly related to their basicitv. The elevated pH was also needed to convert the cryptic or conformationally unreactive trigger site to an available or reactive form that permitted the interaction with the releasing agent to occur. Other support: Connecticut Heart Association. 226. Further evidence for nicotinic and muscarinic receptors and their interaction in dog adrenal medulla Akira Tsujimoto, and Takashige Nishikawa Department of Pharmacology, Hiroshima University School of Dentistry, Hiroshima, Japan European Journal of Pharmacology 34:337-344, 1975 Isolated adrenal glands of dogs were perfused through the adrenolumbar vein with Krebs-Ringer phosphate solution. Nicotine or acetylcholine (Ach) significantly increased the proportion of norepi- nephrine in the effluent whereas muscarine did not alter the relative proportions of epinephrine and norepinephrine. d-Tubocurarine and hexametho- nium (C6) inhibited the response to nicotine com- pletely but scarcely affected the response to Ach and significantly potentiated the response to muscarine. Atropine inhibited the response to muscarine com- pletely, that to Ach partially and that to nicotine slightly. Preinfusion of physostigmine potentiated the secretory response to Ach but not that to nicotine and muscarine. When nicotine and muscarine were infused simultaneously, catecholamine (CA) release was greater than the sum of the responses to nicotine and muscarine separately. Continuous infusion of nicotine for 60 min caused block of the adrenal medulla but potentiated CA release in response to Ach and more especially to muscarine. This po- tentiated release of CA was completely blocked by preinfusion of atropine. Continuous infusion of muscarine for 60 min also blocked CA release and significantly potentiated the response to nicotine, but slightly inhibited the response to Ach. These potentiated and inhibited responses were also com- pletely blocked by preinfusion of d-tubocurarine or C6. On the contrary, during the blockade phase caused by Ach (in combination with physostig- mine), nicotine or muscarine did not cause release of CA. In addition, the continuous infusion of nicotine plus muscarine completely blocked the response to Ac h. From these results, it is concluded that there are nicotinic and muscarinic receptors for acetylcholine in the adrenal medulla and that cholinergic trans- mission is possible via both mechanisms in isolated adrenal glands. When one type of receptors is blocked by continuous contact with an agonist or by d-tubocurarine or C6, the sensitivity of the other type is increased; inactivation of the one is thus com- pensated by the increased response due to potentia- tion of the other. 227. A correlation between the inhibition of monoamine oxidase activity and the relief of angina pain by organic nitrates K. Ogawa, and S. Gudbjarnason Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan Archives Internationales de Pharmacodynamie et de Therapie 172:172-182. 1968 The effect of glyceryl trinitrate, erythrytol tetra- nitrate, mannitol hexanitrate and isosorbide dini- trate on rat heart monoamine oxidase (MAO) was investigated in vitro. MAO activity was assayed by the isotopic method, C'4-tryptamine as substrate. All organic nitrates tested inhibited MAO activity of rat heart mitochondria significantly. The effect of the organic nitrates on the enzyme kinetics of MAO of rat heart mitochondria indicated that all these drugs acted as competitive inhibitors for MAO. The inhibi- tor constants (Ki) of glyceryl trinitrate, erythrytol tetranitrate, mannitol hexanitrate and isosorbide dinitrate were 0.64 mM, 1.83 mM, 3.85 mM, and 61.6 mM respectively. A relationship between Ki values of these organic nitrates and the dose of these drugs recommended to relieve anginal pain in patients was observed. There was a linear correlation between the mean human doses of the organic nitrates and the logarithmus of the respective Ki values. Other support: U. S. Public Health Service, the Michigan Heart Association, and the Detroit General Hospital Research Corporation. TIMN 0115902 I t 158 T200393

5 m~i nicotine Th 2 i t i . ng . e a n Rin,ger"s soluti.on con uptake of C"-labeled nicotine into frog sartorius muscles is inore than doubled by increasing the pH of the Ringer's solution from 7.4 to 8.4. These obsen•ations indicate that the magnitude of the nicotine-induced contracture and Caa5 movements is dependent upon the concentration of nonionized nicotine in the bathing solution. Thus, the non- ionized form of nicotine appears to be the molecule ()f importance not only for penetration of the cell membrane. but for alterations in Ca;' movements and contracture tension in frog sartorius muscle. 223. Actions of procaine on nicotine-induced effects in frog sartorius muscle George B. Weiss Department of Pharmacology. Wedical College of I'irginia. Richmond. Virginia /ournal of Pharmacology and Experimental Therapeutics 160:148-158. 1968 The effects of 3.67 mM procaine on nicotine- induced tension and Ca'S movements and on nico- tine-C'} movements were ascertained in frog sar- torius muscles at pH 7.40 and 8.40. The nicotine contracture is sustained by procaine at pH 8.40 and inhibited by procaine at pH 7.40. Contractures also occur at pH 8.40 when muscles are exposed to procaine alone. Residual Cai5 uptake (after a 120- minute washout) is significantly increased when muscles at pH 8.40 are treated with procaine alone or when procaine is added to Ringer's solution contain- ing 0.415 mM nicotine, but not when procaine is added to solutions containing 2.50 mM nicotine. Residual Ca{5 uptake is inhibited at pH 7.40 when procaine is added to solutions containing 10 mM nicotine. Caa' efflux into Ca-free Ringer's solution containing 4 mM ethylenediaminetetraacetic acid is increased by procaine at pH 8.40 but not at pH 7.40. Procaine decreases uptake and increases efflux of nicotine-C'} whereas iodoacetic acid increases up- take and decreases efflux of nicotine-C'&. Both of these agents alter the DMO-C14 space and the re- sultant calculated intracellular pH values in a man- ner which indicates that this action could account for their effects upon nicotine-C'¢ distribution. The ionized form of procaine appears to be important for inhibition of nicotine-induced contracture and re- sidual Ca45 uptake as well as for the increased intracellular pH levels. The presence of nonionized procaine molecule can be correlated with contrac- ture and increased residual Caa' uptake in a manner similar to that of nonionized nicotine. 224. Nicotine and pyrrolidine-induced release of 5-hydroxytryptamine and histamine from neoplastic mast cells Herbert Felsenfeld, and Lynn A. Corrado Department of Pharmacologv. UniversiM of Connecticut. Schools of Medicine and Dental Medicine. Farmington. Connecticut Biochemical Pharmacology 22:2381-2390. 1973 Nicotine stimulates the secretion of biogenic amine-containing granules from the adrenal me- dulla, the gastrointestinal tract, from blood platelets, and from rat peritoneal mast cells. Using mouse neoplastic mast cells, the present paper describes the conditions under which the nicotine-induced re- lease of biogenic amines from these cells can occur; it suggests the reason for the occurrence of release under these conditions; and it characterizes the structural subunit of the nicotine molecule that triggers and maintains the release process. In brief, nicotine and other pyrrolidines induce the release of the components of the multivesicular granules of mouse neoplastic mast cells, including 5-HT(serotonin), histamine and heparin. The ele- vation of the pH required for the compounds to show activity makes a mast cell site available for the chemical initiation and the maintenance of the release process. At the optimum pH, only the pyr- rolidine ring of the nicotine molecule is needed for activity. This study was the subject of a preliminary report appearing in Federation Proceedings 32(3):806, abs, 1973. Other support: Connecticut Heart Association. 225. Structural requirements for nicotine and cydic imine-induced amine release from neoplastic mast cells Herbert Felsenfeld, Joseph Kubilus, and Lynn Corrado Department of Pharmacology, University of Connecticut, Schools of Medicine and Dental Medicine, Farmington, Connecticu t Biochemical Pharmacology 23:2235-2246, 1974 Nicotine, pyrrolidines and cyclic imines induce the relegse of biogenic amines from mouse neoplas- TIMN 0115901 T200392 157

79. The Effect of Nicotine on Thermosensitive Units in the Rostral Hypothalamus ............. 206 80. The Effects of Acetylcholine and Nicotine on Unit Activity in the Hypothalamic Thermoregula- tory Centers of the Rat ....................................................... . 206 81. Temperature Changes Following Microinjection of Histamine into the Thermoregulatory Centers of the Rat ............................--................................... 207 82. Temperature Changes Induced by Chlorpromazine and N-Methyl Chlorpromazine in the Rat .. 207 83. The Sites and Mechanisms of Action of Drugs Affecting Thermoregulation ............... 207 e. :Veurotransmitter Interaction 84. A Combined Procedure for the Histochemical Fluorescence Demonstration of Monoamines and Microautoradiography of Water-Soluble Drugs ..................................... 207 85. Brain Amine Changes in Stressed and Normal Rats Pretreated with Various Drugs .......... 208 86. Effects of Acute Stress on Forebrain 5-Hydroxytryptamine Metabolism and Pituitary Adrenal Function .................................................................. 208 87. Differences in Brain Area 5-Hydroxvtrvptamine Turnover and Rearing Behavior in Rats and :~~Iice of Both Sexes ............................................................... 208 88. Forebrain Biogenic Amine Function in High and Low Active Female Rats ................ 208 89. Effects of Nicotine on Behavioral Arousal and Brain 5-Hvdroxytrvptamine Function in Female Rats Selected for Differences in Activity ......................................... 209 90. Effects of Nicotine on Brain Area 5-Hydroxytryptamine Function in Male and Female Rats Separated for Differences of Activity ............................................ 209 91. Brain 5-Hydroxytryptamine Correlates of Behavior in Rats: Strain and Sex Variability ....... 210 92. Effects of Chronic Nicotine Administration and Age on Various Neurotransmitters and Associated Enzymes in Male Fischer-344 Rats ..................................... 210 93. Effect of Nicotine and Related Substances Upon Amine Levels in the Brain ............... 211 94. Alteration of 5-Hydroxytryptamine S4C Efflux by Nicotine in Rat Brain Area Slices ......... 211 95. Effects of Nicotine on Distribution and Release of "C-Norepinephrine and 14C-Dopamine in Rat BrainStriatumandHypothalamusSlices ......................................... 211 96. Effects of Different Potassium Ion Concentrations and of Procaine and Pentobarbital on ['IC] Glutamate Fluxes in Rat Brain-Cortex Slices ...................................... 212 97. Effect of Central Catecholamine Depletions by 6-Hydroxydopamine on Morphine Antinocicep- tion in Rats ............................................................... 212 98. Morphine and the Electrophysiology of the Cingulum: A Preliminary Study .............. 212 f. Neuroendocrine 99. Hypothalamus: Anterior Pituitary Gland ......................................... 213 100. Brain Serotonin and Pituitary-Adrenal Function in Rats of Different Emotionalities ......... 213 101. Blood Glucose, Growth Hormone, and Cortisol Levels after Hypothalamic Stimulation ....... _2 13 102. Ammunoassay of Plasma Growth Hormone in Cats Following Fasting and Administration of Insulin, Arginine, 2-Deoxyglucose and Hypothalamic Extract .......................... 214 T1MN 0115918 T200409 174 --- -- ~

Abstracts (145) A. Central Nervous System ......................................................... 182 Human Behavior 1. Cigarettes and the College Freshman ............................................ 182 2. Smoking-Academically Speaking! ............................................. 182 3. Cigarettes and the College Senior ............................................... 182 4. Cigarette Smoking: Health Knowledge and Practices ................................ 183 5. Cigarettes: College Freshmen in 1964 and 1969 .................................... 183 6. The Role of Nicotine as a Determinant of Cigarette Smoking Frequency in Man with Observations of Certain Cardiovascular Effects Associated with the Tobacco Alkaloid .................. 183 7. Trends Observed in the Time Estimation of Three Stimulus Intervals Within and Across Sessions ................................................................. 184 8. Electroencephalographic Changes in Man Following Smoking ......................... 184 9. Computer Time-Series Analysis of the Effects of Some Analgesics Upon Human EEGs ...... 184 10. Multilead Time-Series Frequency Analysis of the EEG Effects of Thiopental in Psychotic and Nonpsvchotic Men ........................................................... 184 11. Comparative Study of EEG Effects of Antihistamines in Normal Volunteers ............... 185 12. Effects of High Congener Intake by Human Subjects on the EEG ...................... 185 13. Electroencephalographic Effects of Caffeine. Nicotine. Tobacco ........................ 186 14. Computer Time-Series Frequency Analysis of the Electroencephalograms of Male Nonpsvchotic and Chronic Schizophrenic Subjects ............................................. 186 15. Effects of Intravenous Nicotine in Smokers and Nonsmokers .......................... 186 16. Abstinence Effects in Smokers .... . ............................................ 187 17. EEG Study of Caffeine and Amphetamine as Possible Surrogates for Smoking in Humans .... 187 18. Effect of Acute Deprivation of Smoking on Aggression and Hostility .................... 187 19. Effects of Selection on Mortality ............................................... 187 Animal Behavior 20. Nicotine Self-Administration in Monkeys ........................................ 188 21. Effect of Propranolol, d-Amphetamine and Caffeine on Ethanol as a Discriminative Cue ..... 188 22. C.N.S. Effect of Nicotine as the Discriminative Stimulus for the Rat in a T-Maze ........... 189 23. Effect of Mecamvlamine on Discrimination Between Nicotine and Arecoline-Produced Cues .. 189 24. Nicotine as a Discriminative Stimulus in Rats Depleted of Norepinephrine or Hydroxytrypta- mine .................................................................... 189 25. Nicotine as a Discriminative Cue in Rats: Inability of Related Drugs to Produce Nicotine-Like Cueing Effect ......................... . .................................... 189 26. Studies on the Time Course and the Effect of Cholinergic and Adrenergic Receptor Blockers on the Stimulus Effect of Nicotine .................................................. 190 TIlVIN 0115915 T200406 171

with right ventricular failure 3-127 days after chronic partial pulmonary artery obstruction. For the majority of failed muscles (at 36°C, 30 stimuli/ min). resting potential. action potential overshoot, action potential maximum rate of rise, isometric active force at optimal length (Po), and dP/dt were decreased compared with the same parameters in normal muscles. Time to peak force and duration of contraction were unaltered. Action potential con- figuration was changed and action potential du- ration was lengthened in failed muscles. Epineph- rine (10-6M) markedly increased Po and resting potential in severely depolarized failed muscles. These data suggest that electrical depression might exacerbate the contractile deficit in experimental chronic right ventricular failure. Other support: U. S. Public Health Service. 256. A new genetic polymorphism of human serum: a2 macroglobulin (AL-M) juhani Leikola, H. Hugh Fudenberg, Reiji Kasukawa, and Felix Milgrom i'nirersih• of California School of Medicine, San Francisco, California, and State University of New York. Buffalo. New York American Journal of Human Genetics 24:134-144. 1972 Sera from 60 Japanese families were tested against two serologically identical human sera (each of which contained antibodies to globulin); a poly- morphism in human a globulin was demonstrated. The a globulin appeared to be inherited as an autosomal Mendelian trait and was not linked to Cm. Am. or haptoglobulin serum groups. When isolated from a single individual, the antigen was shown to be associated with a2 macroglobulin. 257. Anionic sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization P. Pinto Da Silva. P. S. Moss, and H. H. Fudenberg Department of Xfedicine, University of California School of .~fedicine. San Francisco, California, and Department of Zoology. University of California, Berkeley, Cali,f_ornia __ Experimental Cell Research 81:127-138, 1973 Freeze-fracture and freeze-etching techniques disclose exclusive association of a ferritin derivative (with high isoelectric point, used as a marker for anionic sites) with the regions at the outer and inner surface of the membrane of human erythrocyte ghosts which correspond to the membrane-interca- lated particles. At the outer surface the sites include sialoglycoprotein. Exclusive association of anionic sites and membrane particles, and comparison of the number of sialic acid residues and intercalated particles implies clustering of acidic groups over discrete sites at the surface. Association of the label with the outer and inner surface regions which correspond to the membrane intercalated particles. provides further support for the concept of protein- containing structures which are intercalated and traverse the hydrophobic matrix of membrane re- gions with bilayer organization. Other support: U. S. Public Health Service, and the Council for Tobacco Research-U.S.A. 258. Therapy of angina pectoris with propranolol and long-acting nitrates Alberto N. Goldbarg, John F. Moran, Thomas K. Butterfield, Rimgaudaus Nemickas, and Gustavo A. Bermudez Department of Medicine. University of Chicago. Chicago. Illinois Circulation 40:847-853, 1969 A double-blind study of the effects of isosorbide dinitrate, 10 mg given orally four times a day, propranolol, 40 mg four times a day, and the combination of these two drugs was performed on 21 patients with angina pectoris. Each patient received placebo, isosorbide dinitrate, propranolol, and the combination of the two drugs for one month each in a random sequence over four months. The number of anginal pains and nitroglycerin tablets used were recorded, and a multistage treadmill ECG exercise test was performed after each treatment period. Frequency of anginal pains was reduced sig- nificantly with propranolol (7.0 -_* 2.5 anginal pains/ week) and the combination of propranolol and isosorbide dinitrate (9.9 -_* 2.9 as compared with placebo (21.0 ± 6.4). Similarly, the number of nitroglycerin tablets was reduced with propranolol- containing regimens; isosorbide dinitrate was not significantly better than the placebo. Symptomatic improvement with propranolol could be related to a reduction in heart rate, and the product of heart rate TIMN 0115913 T200404 169

c 126. Uptake of 'H-Norepinephrine by Fluorescent Nerves of the Heart ....................... 223 127. Blockade of Conduction in Vagal Fibers by Nicotinic Drugs ........................... 223 Skeletal Neuromuscular 128. Comparison of the Effects of Procaine, Chlorpromazine and Their Quaternary Derivatives on Nerve Action Potentials ...................................................... b b 224 a 129. Nicotine-Induced Depolarization and Stimulation of Potassium Efflux in Striated Muscle .... 224 130. Antinicotine Action of Nicotine and Lobeline on Frog Sartorius Muscle ................. 225 131. Modification of the Nicotine-Induced Depolarization in Striated Muscle by Altered [H']o, [Ca-1oand[Cl-]o .......................................................... 225 132. A Comparison of Lobeline and Nicotine at the Frog Neuromuscular Junction .............. 225 133. Receptor Desensitization by Lobeline and Nicotine ................................. 226 134. Blockade by Lobeline of Potassium Exchange in Skeletal Muscle: Relationship to Receptor Desensitization at the Endplate ................................................ 226 135. On the Site of Action of Lanthanum in Frog Sartorius Muscle ......................... 226 C. Nicotine Derivatives ............................................................ 227 136. Quaternization Products of S-(-)-Nicotine ........................................ 227 137. Quaternization Products of S-(-)-Nicotine ........................................ 227 138. The Biochemorphology of Nicotine ............................................. 227 D. Viral Chemotherapeutic ......................................................... 228 139. Antiviral Activity of Tobacco Leaf on Encephalomyocarditis (EMC) Virus Infection ......... 228 140. Antiviral Activity of Tobacco Smoke Condensate on Encephalomyocarditis Infection in Mice . 228 141. Effects of Nicotine upon Cortical and Subcortical Electrical Activity of the Rabbit Brain: Quantitative Analysis ....................................................... 228 142. The Modification of Central Nervous System (CNS) Function by Autonomic Drugs. Amine Shift Responses Differentiate Between CNS Nicotinic and Muscarinic Effects .................. 229 143. Effect of Nicotine Response to Frustrative Non-Reward in the Rat ...................... 229 144. Pharmacological Effects of Nicotine and Smoking .................................. 229 145. Army Rank and Subsequent Mortality by Cause: 23-Year Follow-Up .................... 230 176 h S' a F, '1'IlVIN 0115920 T200411

?0, Cardiovascular Svstem interactions with a-elastin (a solubilized protein from elastic fibrils), nicotine does specifically bind a-elastin which has previously bound calcium ion at neutral sites. The result is of interest in connection with a recent proposal that the binding of calcium ion to the elastin protein matrix is an initial step in the chronic process of arteriosclerosis. The interac- tion of nicotine with elastin after, but not before, calcium binding is of interest in relation to the pathogenesis of atherosclerosis. 242. Sympathomimetic amines and heart lactic dehydrogenase isoenzyrnes Duane G. Wenzel, and James P. Lyon Department of Pharmacology and Toxicology, School of Pharmacy. University of Kansas, Lawrence, Kansas Toxicology and Applied Pharmacology 11:215-228, 1967 The duration of the effect of a 100 mg/kg dose of isoproterenol on total and isoenzyme lactic dehy- drogenase (LDH) activities in the rat heart was determined. Maximum reduction of total heart LDH activity occurred between the first and third day following isoproterenol. Heart LDH isoenzymes 3, 4, and 5 increased to a maximum on the second day and gradually returned to the control level by the tenth day. The LDH 1 and 2 isoenzyme activities were reduced over this same period. Changes in total heart LDH and in the ratios of LDH-H and LDH-M subunits were used as measures of the potential blocking effects of pronethalol and phentolamine on the activity, of isoproterenol, epi- nephrine, norepinephrine, and phenylephrine. Re- sults were essentially the same whether expressed as total LDH activity or as H/M ratios. Phentolamine blocked the effects of epinephrine, norepinephrine, and phenylephrine, whereas pronethalol was inef- fective with all the sympathomimetics. The effects of phentolamine and pronethalol were also determined by measuring gross myo- cardial lesions produced by norepinephrine or iso- proterenol. With the exception of a trend toward protection by pronethalol against the effects of larger doses of isoproterenol, the results obtained by using lesion grades were essentially the same as those obtained with total LDH or LDH isoenzyme mea- surements. 243. Effect of beta adrenergic blocking agents on the noradrenaline content of rat heart before and after noradrenaline infusion T. C. Westfall Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia European Journal of Pharmacology 2:163-168, 1967 Four beta adrenergic blocking agents, :viJ 1998, MJ 1999, Ko 592 and the stereo isomers of INPEA [structural formulae and sources in text], have been studied for their ability to influence endogenous rat heart noradrenaline levels. In addition, the amount of noradrenaline recovered in the heart and spleen following the infusion of this catecholamine before and after MJ 1998, MJ 1999 and Ko 592 has been studied. MJ 1999, Ko and L(-) and D(-)INPEA did not produce any significant changes in the noradren- aline content of the rat heart in a dosage range of 0.2-10 mg/kg when the tissue was measured at 1 or 6 hr following a single injection or following daily administration for 7 days. MJ 1998 produced a significant decrease in the noradrenaline content 6 hr after a single injection of 10 mg/kg and also after the single daily administration of 10 mg/kg for 7 days. MJ 1998 (10 mg/kg) also produced a 50% decrease in the amount of noradrenaline measured in the heart following an infusion of this amine (1 µg/kg/min for 20 min). Neither MJ 1999 nor Ko 592 had any effect on the noradrenaline content of the heart due to infusion of noradrenaline. Other support: Council for Tobacco Research- U.S.A. 244. Production of severe cardiac and skeletal myopathy by interference with hepato-adrenal function G. M. Hass, R. E. Lee, Jr., and J. A. Kaiser Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois American Journal of Pathology 78:33a, 1975 Young male New Zealand albino rabbits were placed in four groups. Group I was given carbon terachloride subcutaneously in mineral oil on a regimen standardized for production of nodular cirrhosis within 6 to 8 months. Group II was adrenal- ectorrLized and maintained for several months with dexamethasone, DOCA and sodium chloride. Group III and Group IV, respectively, consisted of animals of group I placed on the group II program and animals of group II placed on the group I program. 164 TIMN 0115908 T200399

27. Transfer of State-Dependent Control of Discriminative Behaviour Between Subcutaneously and Intraventricularlv Administered Nicotine and Saline ................................ 190 28. Atropine Antagonism of Arecoline-C_ued Behavior in the Rat .......................... 190 29. Lrsergic Acid Dieth~rlamide (LSD) as a Discriminative Cue: Drugs with Similar Stimulus Properties .................:............................................... 191 30. d-Amphetamine as a Discriminative Cue: Drugs with Similar Stimulus Properties .......... 191 31. Morphine as a Discriminative Cue: Effects of Amine Depletors and Naloxone ............. 191 32. A Comparison of the Stimulus Effects of Morphine and Lysergic Acid Diethylamide (LSD) ... 191 33. Discriminative Control of Behavior by Eiectrical Stimulation of the Dorsal Raphe Nucleus: Generalization to Lysergic Acid Diethylamide (LSD) ............... .................. 192 34. Morphine and D-D9-Tetrahydrocannabinol: Tolerance to the Stimulus Effects .............. 192 35. Generalization of IMorphine and Lysergic Acid Diethylamide (LSD) Stimulus Properties to Narcotic Analgesics ......................................................... 192 36. Generalization of Morphine and Lysergic Acid Diethylamide (LSD) Stimulus Properties to Narcotic Analgesics ......................................................... 193 37. Ethanol as a Discriminative Cue: Reduction Following Depletion of Brain Serotonin ........ 193 38. Behavioral Tolerance to an Effect of Nicotine in the Rat .............................. 193 39. Azione Della Nicotina Sul Condizionamento Di Salvaguardia Di Ratti Di Un Mese .......... 194 40. Differente Action De La Nicotine Au Cours De La Journee Et De La Nuit Dans L'Activite Pontanee (Running Activity) Du Rat .................................................... 194 41. Effects of Nicotine on Avoidance Conditioning of Inbred Strains of Mice ................. 194 42. Facilitation of Simultaneous Visual Discrimination by Nicotine in the Rat ................ 194 43. Action of Nicotine on Spontaneous and Acquired Behavior in Rats and Mice ............. 195 44. Effetto Antifatica Dell'Amfetamina E Di Farmaci Stimolanti Centrali Sulle Prestazioni Di Topi Durante Sedute Di Condizionamento Prolungate ................................... 195 45. Anak•sis of the "Anti-Fatigue" Activity of Amphetamine. Role of Central Adrenergic Mech- anisms ................................................................... 195 46. %lemon• and Consolidation Mechanisms in Avoidance Learning of Inbred Mice ........... 195 47. Effects of Nicotine and Strychnine on Transfer of Avoidance Learning in the Mouse ........ 196 48. Effects of Scopolamine on Avoidance Conditioning and Habituation of Mice .............. 196 49. Facilitation of Simultaneous Visual Discrimination b_v Nicotine in Four "Inbred" Strains of `fice .................................................................... 196 50. Genetic Aspects of Learning and Memory in Mice .................................. 197 51. Comparison of Behavioral Effects of Nicotine, d-Amphetamine, Caffeine and Dimethylheptyl Tetrahydrocannabinol in Squirrel Monkeys ....................................... 197 52. Effects of Nicotine, Nicotine Monomethiodide. Lobeline, Chlordiazepoxide, Meprobamate and Caffeine on a Discrimination Task in Laboratory Rats ................................ 197 53. Effects of Nicotine on the Exploratory Behavior of Female Rats ........................ 198 54. Brain Area Nicotine Levels in Male and Female Rats of Two Strains .................... 198 172 T'IlVIN 0115916 T200407

`'Pow"10" Introduction-The effects of tobacco smoking and nicotine on the central and peripheral nervous system are widespread. Knowledge in this area has been expanded considerably. It is obvious that tobacco smoking produces a number of effects which are reinforcing to the organism. This may explain ,,•hv the tobacco habit is so entrenched in some indiriduals. Abrupt withdrawal leads to a number of important signs and symptoms which are promptly relieved with tobacco and nicotine ingestion. The following are cited contributions which help to explain in part the complex effects of tobacco smoking on the nervous system. A. Central Nervous Svstem Human Behavior Why people smoke tobacco is still a mystery. but some light has been shed on the subject. The smoking patterns and socioeconomic characteristics of college students have been described in a series of studies which began in 1964 (1-5). Differences in smokers and nonsmokers were highly statistically significant with regard to approval of teen-age smoking. academic achievement, organization mem- bership. use of leisure time, belief in the incidence of cancer in smokers, and belief in smoking as a public health problem. Many of the differences of opinion are expected among those who do and do not smoke. It appears that association of nonsmokers with smokers represents a significant health risk since most smokers are more careless of their health habits. One factor, among many, in tobacco smoking appears to be nicotine itself. It has been shown that nicotine given intravenously in relatively large amounts to normal volunteer smokers causes a small but statistically significant decrease in the number of cigarettes smoked (6). The physiological alterations in blood pressure, heart rate and EKG induced by tobacco smoking can be reproduced by parenteral nicotine. During this study the subjects engaged in a six-hour experiment in which time estimation was a test. A decreasing trend in time estimations was observed. perhaps related to a lack of feedback (7). `icotine infusions had no effect on this behavior. The effects of tobacco smoking on human brain waves (EEG) appear to be stimulant rather than tranquilizing (8). Quantitative EEG studies are very useful in classifying different categories of psv- choactive drugs (9. 10. 11. 12. 13) as well as characterizing the brain wave patterns of psychotic patients (14). Intravenous nicotine produces similar EEG changes in smokers and nonsmokers (15). However, the EEG effects of intravenous nicotine differed from that of tobacco smoking. probably because of the different dosages used. SurprisinglY. smokers did not demonstrate greater tolerance than nonsmokers to intravenous nicotine. Perhaps the most significant finding is that tobacco smokers deprived of cigarettes for six hours showed a re- duction in blood pressure and pulse rate which returned or overshot the baseline on smoking. Sim- ilarly. marked EEG changes were observed in de- prived subjects allowed to resume smoking. %iepro- bamate, caffeine, and placebo had no antidoting effects while d-amphetamine was doubtfully ef- fective in reversing the EEG depression (16. 17). No relationship was found between the degree of tobacco deprivation and the anxiety level. Never- theless, tobacco deprivation in chronic smokers produced considerable behavioral distress. For example, acute deprivation of smoking in chronic smokers caused a significant increase in aggression scores. This may be a factor in the continuance of the smoking habit (18). One study which has relevance to statistics dealing with mortality of humans is the effect of bias of medical selection (19). The selection process of 84,491 white male World War II army veterans had a profound effect on mortality rate statistics. Generally the mortalitv rates were well below those of the general population within the first few years of discharge and thereafter gradually approached those of the general population (145). Animal Behavior A very impressive fact is that monkeys will self- administer nicotine even in relatively large amounts (20). This indicates that nicotine is positively re- warding and hence nicotine content probably is related to the tobacco smoking habit in man. The intake of nicotine markedly increased when the animals were exposed to mild but stressful electric shocks (21). Nicotine itself is a discriminative stimu- lus in various animal behaviors (22-27) as are other psychotropic drugs (28-37). Significant behavioral tolerance to those effects of nicotine occurredd within four days of daily administration (38). Vicotine in TIMN 0115921 T200412 177

Cardiovascular Srstem changed. Cat blood equilibrated with cigarette smoke in vitro gave 50-90% COHb and 3-6% Met- Hb. Si;nilar in vitro equilibrations of cat blood with 200-2000 ppm NO in air gave Met-Hb levels of 10- 100%. Cats inspiring 200 ppm NO in air for 30 min gave about 8% blood Met-Hb. These findings sug- gest that oxides of nitrogen do not occur in cigarette smoke in sufficient concentrations to form Met-Hb in vivo. 253. Effects of chronic smoking upon the clotting mechanism T. J. Regan, C. B. :vloschos, and S. S. Ahmed Department of Wedicine. College of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark. New Jersey Unpublished report to the AMA-ERF Several studies have been conducted in the past in an effort to elucidate the effect of smoking upon the clotting mechanism. The hypothesis for these studies has been that enhancement of the thrombotic process by smoke may underlie the pathogenetic mechanism of increased cardiovascular disease in the smoker via the process of atherosclerosis which was found prevalent among smokers. Litter mate beagles 12-18 months of age were prepared by permanent tracheostomy and subsequently were submitted to chronic smoking equivalent to two and one-half packs of cigarettes per day smoked by humans with an average weight of 70 kilograms. The cigarettes used were without filters and contained 1.35 mg nicotine/cigarette, manufactured by the Tobacco and Health Research Institute of the Uni- versity of Kentucky. The dogs were smoking in rotation in two sessions, morning and afternoon, each session lasting approximately three hours. The duration of smoking was for 18 months. Clotting and fibrinolvtic tests were carried out in 11 smoking and 5 control dogs at control time, before the initiation of smoking, and was repeated at 6, 12, and 18 months. The conducted tests included plastic tube clotting time, fibrinogen levels, platelet counts, partial thromboplastin time (PTT) and euglobin lysis in duplicate using the double syringe method for sampling. At the end of the smoking period of 18 months, 100 µCi of canine fibrinogen 90-99% clotta- bility was injected in all animals to determine fibrinogen decay patterns. Also, at the end of the smoking period platelet rich plasma was collected from smoking and control dogs for platelet aggrega- tion by the optical density method. The turnover rate of fibrinogen was found significantly increased (P<.01) in chronic smokers. In addition, clotting times as well as PTT were found to accelerate in the smoking group when compared to controls, at the end of the 18 month period. There was a gradual decrease in platelet counts in the smoking group significant at the end of the study (P<.01) which might indicate increase turnover rate of platelet in the smokers, verifying similar suggestions made in previous studies. In addition, there was significant enhancement of platelet aggregation in these ani- mals which would indicate "sensitization" of the platelet population as a result of chronic smoking. In conclusion, the data obtained prospectively from this small group of animals would suggest sig- nificant enhancement of the coagulation mechanism in the smoking group. The role of such changes in the pathogenesis or acceleration of cardiovascular disease in the smoker remains to be further defined. Other support: Council for Tobacco Research- U.S.A. Abstracts not Cited in Summary Text 254. Neuronal and extraneuronal uptake of adrenergic transmitter in the blood vessel O. A. Nedergaard, and J. A. Bevan Department of Pharmacology, U. C. L. A. School of Medicine, Los Angeles, California Proceedings of the Symposium on the Physiology and Pharmacology of Vascular Neuroeffector Systems, Interlaken, 1969, pp 22-34 (Karger, Basel 1971) The authors present a review of current knowl- edge on the above subject, including studies from their laboratory. Other support: U. S. Public Health Service. 255. Depressed transmembrane potentials during experimentally induced ventricular failure in cats Henry Gelband, and Arthur L. Bassett Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York Circulation Research 32:625-634, 1973 Transmembrane potentials and isometric force were recorded in right ventricular muscles from cats , I TIMN 0115912 T200403 168

Nervous System comparison to other psvchotropic drugs had marked effects on the rate of acquisition of conditioned behavior and locomotor activity in both mice and rats (39-50). Depending upon genetic and age fac- tors, nicotine may produce a marked enhancement of conditioned behavior. In general, poor perfor- mance was facilitated and high level performance depressed by nicotine in reasonable dosage. Six of nine inbred mouse strains showed a facilitating effect on avoidance conditioning following nicotine. Learning differences in inbred strains of mice appear to involve a multitrace view of memory storage mechanisms which are affected by nicotine. The temporal pattern of responding under different oper- ant procedures in squirrel monkeys is the dominant factor determining the behavioral effects of many psychotropic drugs including nicotine (51). Rats trained in a discrimination task in order to obtain a food reward showed an improvement following nicotine, lobeline, and two antianxiety drugs, mep- robamate and chlordiazepoxide (52). Interestingly, caffeine and nicotine monomethiodide were without effect on this task. In rats subject to a frustrative non- reward task, nicotine decreased the magnitude of the frustration (143). This finding using rats is con- sistent with other studies reporting a decline in aggression after nicotine. It is interesting to note that nicotine-treated rats habituate to a novel environ- ment more readily than control animals (53). High' activity female rats appeared to be more responsive to these effects of nicotine (54. 55). An interesting technique has been described in which rats show conditional aversion to a preferred saccharin solution following methamphetamine in- jections (56). The technique has an application with drugs such as nicotine. Another technique in which nicotine should be studied is spontaneous alterna- tion in rats. This behavior involved cholinergic neurons in the dorsal hippocampus (57), and, hence, should be affected by nicotine. The local instillation of milk or corn oil into the cat duodenum causes local gastrointestinal changes, as might be expected. However, behavioral sedation is also observed which is mimicked by cholecystokinin-pancreozy- min (58). One wonders what the possible interaction of tobacco smoking and nicotine may have on this svstem and its relation to their known alerting and hunger depressing effects. In the rat nicotine exerts behavioral effects resembling amphetamine on some tasks and diazepam-like on others (144). Self-ad- ministration of nicotine is increased in monkevs exposed to unavoidable (and hence very stressful) electrostimulation. Neuropharmacology a. Model Neuronal Systems-Thin slices of brain are the major means of studying intact mam- malian neurons with their synaptic connections in vitro. Surface negative and positive potentials can be recorded in isolated olfactory guinea pig cortex following electrical stimulation of the lateral olfac- tory tract (59). This technique will be important in studying the actions of drugs on the central nervous system. Another technique is to isolate well identi- ffed neurons of model neuronal systems such as the brain of the snail. These nerve cells are relatively large, compared to mammalian cells, and hence are very easy to study. There are two kinds of cells that respond to acetylcholine. One type is excited or depolarized (D-cell) and the other is inhibited or hyperpolarized (H-cell). Nicotine acts on both types, just like acetylcholine. The excitatory and inhibitory effects of nicotine on receptors of these two cell types were blocked by tubocurarine and atropine. However, hexamethonium reduced the excitatorv effects of nicotine on D-cells, but did not affect its inhibitory effects on H-cells. The results on D-cells of the snail brain resemble those of the cholinergic receptors of frog sartorius muscle (60). In the de- cerbrate cat. there also is evidence of two kinds of receptors for acetylcholine. The spino-bulbo-spinal inhibitory system has a muscarinic cholinergic com- ponent which is blocked by atropine. The choliner- gic system involved with REM (rapid eye movement) sleep has a nicotinic cholinergic and possibly a muscarinic cholinergic component (61). There is also behavioral evidence for two types of cholinergic receptors in the rat central nervous system, one of which is muscarinic and the other nicotinic (62). Interestingly, atropine had no effect on the rat's abilitv to discriminate the central cueing effect of nicotine in contrast to its ability to block the actions of nicotine on snail neurons. Similar differences in nicotine and muscarinic cholinergic agonists have been reported using the EEG (142). Complex re- ductions in mutual involvement of the neocortex and important subcortical brain areas are observed using the EEG in nicotine treatment animals (141). Tissue slices from different areas of the rat brain can be used to obtain comparative estimates of tissue compartment size, cation contents and distribution (63, 64). In addition, the rates of oxygen utilization c i c r 1. 178 TIMN 0115922 T200413

103. The Effects of Intravenous Nicotine on Blood Glucose Plasma Corticosterone and Growth Hormone Levels in Rats and Rabbits ............................................ 214 104. Effect of Intracerebral Injection of Nicotine and Carbachol on Corticosterone Secretion in the Rat ........................_............................................ 214 105. Thyroid Activity Following Intracerebral Injection of Morphine in the Rat ................ 215 B. Peripheral Nervous System ...................................................... 215 Autonomic - 106. Uptake of Nicotine and Extracellular Space Markers by Isolated Rat Gangeia in Relation to Receptor Activation ......................................................... 215 107. Evidence Against a Presynaptic Action of Acetylcholine During Ganglionic Transmission .... 215 108. A Note on the Effect of Dithiothreitol (DTT) on the Depolarization of Isolated Sympathetic Ganglia by Carbachol and Bromo-Acetylcholine .................................... 216 109. Origin of the After-Hyperpolarization that Follows Removal of Depolarizing Agents from the Isolated Superior Cervical Ganglion of the Rat ..................................... 216 110. Nicotine Washout Rates from Isolated Rat Ganglia in Relation to Recovery from Nicotine Depolarization ............................................................. 217 111. Intracellular pH in Rat Isolated Superior Cervical Ganglia in Relation to Nicotine-Depolarization and Nicotine-Uptake ........................................................ 217 112. Changes of Intracellular Sodium and Potassium Ion Concentrations in Isolated Rat Superior Cervical Ganglia Induced by Depolarizing Agents .................................. 218 113. Prolonged Ganglionic Facilitation and the Positive Afterpotential ...................... 218 114. Dissociation of Depolarization and Ganglionic Blockade Induced by Nicotine ............. 219 115. Observations on Drug-Induced Activation of Cholinoceptive Sites in a Sympathetic Ganglion . 219 116. Comparison of Differential Secretion of Adrenal Catecholamines by Splanchnic Nerve Stimula- tion and Cholinergic Agents .................................................. 219 117. Antagonism of Ganglionic Stimulants by a, a'-Bis-(Dimethylammoniumacetaldehyde Diethyl- acetal)-p.p'-Diacetylbiphenyl Bromide (DMAE) . ................................... 220 118. Autoradiographic Localization of Sites of ('H]-t-Aminobutyric Acid Accumulation in Peripheral Autonomic Ganglia ......................................................... 220 119. Electrophysiological and Pharmacological Evaluation of Possible Interactions Between Thiocya- nate and Nicotine in Mammalian Autonomic Ganglia ............................... 221 120. Biogenic Amines and Enzymes Following Immunosympathectomy in Mice ............... 221 121. Effects of Drugs on the Behaviour of Immunosvmpathectomized Mice ................... 221 122. The Effect of Nicotine upon Ureteral Peristalsis .................................... 222 123. Ureteral Dynamics: Pathophysiology. Drugs, and Surgical Implications .................. 222 124. Effects of Acetaldehyde. Propionaldehyde. Formaldehyde and Acrolein on Contractility, "C- Norepinephrine and 45Calciurn Binding in Isolated Smooth Muscle ..................... 222 125. Histochemical Fluorescence Microscopy and Microautoradiography Techniques Combined for Localization Studies 223 ........................................................ TIMN 0115919 T200410 175

t L i , t r [ tIf the in Ph,i~t= [I. 75 percent usuallt- ,nu7(,ed mure than throt• ~tuarter of a cigarette. and ,hproximately half this number smoked 90 percent ,,f the tobacco portion. The number who smoked fiiter cigarettes had increased from : 5 percent in phase I to 85 percent in Phase II. Over one third (36.3%) of the seniors who smoked had started since Phase I. Of the women in Phase II who smoked. t-wo in five began after Phase I ,ind of the men. one in three. Among additional findings. of the 1964 fresh- nien kcho had smoked one hundred or more ; i~arettes, one in five had discontinued smoking in 1967. Twice as many men as ~vomen had discon- tinued smoking. The author finallc• stresses the need to protect the rights of the nonsmoker b~• restricting smoking. 4. Cigarette smoking: Health knowledge and p ractices Dorothy F. Dunn School of Public Health. University of lllinois at the Wedical Center. Chicago, Illinois lnurnal of the American College Health Association 22;12-1-125. 1973 Smoking patterns and socioeconomic character- istics were studied in 1964 of freshmen at the L'niversitv of Illinois, Urbana campus and of those same students again in 1967 to learn what changes in smoking patterns occurred during these years: Both smokers and nonsmokers considered cigarette smoking to be a major health hazard. Smokers felt thev were overweight. failed to eat daily breakfasts, drank coffee or tea. had a cough, needed dental care, had been absent from classes seven or more days, and more of their absences were due to illness or injury. Nonsmokers snacked between meals, used milk and carbonated beverages, and were absent trom class due to personal or family reasons or to ~:omplete an assignment for another course. During the period. many students had discontinued smok- ing. The descriptive statistics reported in this study are said to suggest that administrators in institutions of higher education should give considerable thought to the special part that universities play in considering the health risk factors which involve the nonsmoker's association with the smoker. 5. Cigarettes: College freshmen in 1964 and 1969 Dorothy F. Dunn School of Public Health. C'nirersitv of tllinois nt the Medical Center. Chicago. Illinois lournal of the .-lmerican College Health .-lssorirrtinn 22:126-127. 1973 Five vears after the first collection of data on freshmen enrolled at the [:niversitv of Illinois, Urbana. data were again collected on the Urbana Campus from freshmen entering college in 1969. A comparative study is reported on differences be- tween the two freshmen classes. -kmong the findings, in 1969 more freshmen smoked a pack a day than in 1964 and more of them smoked a larger portion of the tobacco in the cigarette. Ltore 1969 freshmen smoked filter ciga- rettes than did their 1964 counterparts and fewer had fathers and mothers who smoked. 6. The role of nicotine as a determinant of cigarette smoking frequency in man with observations of certain cardiovascular effects associated with the tobacco alkaloid B. R. Lucchesi, C. R. Schuster, and G. S. Emley Department of Pharmacology. University of Michigan Medical School. Ann Arbor. Nfichigan Clinical Pharmacology and Therapeutics 8:789-796. 1967 The effect of intravenously administered nico- tine (over 6-hour periods) upon smoking behavior was studied in smokers who were unaware of the nature of the administered drug and the true purpose of the study. Smoking behavior was not altered significantly when nicotine was administered in a dose of 1 mg/hour for 6 hours. A significant decrease in smoking frequency was obtained when nicotine was administered at the rate of 2 mg for the first hour and at 4 mg/hour for the succeeding 5 hours. Mean numbers of cigarettes consumed during 6 hours under saline control versus 6 hours with nicotine infusion for 5 subjects were: 17.9 vs 14.4, 7.0 vs. 4.4, 7.3 vs 4.8, 11.4 vs 8.2, and 6.7 vs 4.7. Additionally, there was a decrease in the amount of each cigarette smoked during nicotine treatments, suggesting de- crease in motivation to smoke. C TIMN 0115927 T200418 183

! I : r f J I>0lated rat ~upt°rinr + E•rvic tl ganglia recover ,iu,re slrn%'l}' from the tlr-lu+l<iririn, action of nicotine than from Choline deriVdtik,es. This slow recovPrv is rc•lated to a slow efflux of intracellular nicotine (1 it)1. During nicotine induced depolarization, there is a fall in intracellular pH related too metabolic ;timulation following sodium ion entry (111). The ,ifterh}'perpolarization of aanalion cells is related to tht• F°Iec trogenic extrusion of accumulated sodium i„n ciuring the depolarization (112). There is evi- ,l+.rxe• that the positive afterpotential is involved in t}1,. ,enesis of ganglionic facilitation (113). .~lthouQh +,,in"lionic transmission blockade and depolariza- tic+n ec-oked by nicotine are generally considered related events. new data indicates a dissociation l t 1-tl. The site at which nicotine evokes depolar- imtion appears distinct from that which blocks ri,•uronally released acetx-lcholine. The mode of ,cdministration of drug-induced activation of cholin- +,cc•ptire sites in the cat superior cervical ganglion is t ritiral (115). Postganglionic discharge evoked be' nirotine ~ras abolished b}' hexamethonium and un<rltered bv atropine. .VdrPnal medullarx ' cells are derived from ner- %+,us tissue embrc'ologicall~•. and hence. resemble t,++•t,'anglionic adrenergic neurons. Nicotine re- It a>rs equivalent amounts of epinephrine and nort•pinephrine from the adrenal medulla. but ace- ttjc holine and dimethy)phenrlpiperazinium prefer- .•ntiallv release epinephrine (116). Low frequency ~t+l<rnc hnic nerve stimulation preferentially released n++rf•E,inephrine. whereas high frequencv stimula- ri++n released epinephrine. There is additional evi- i+ nc t> that nicotine and other ganglionic stimulants it t,iate receptors that differ from those activated by ++etr(rholine released from preganglionic nerve •t irncrl<ction (117). .\Ithough Qamma-aminobutcTic acid does not +t+f)"dr to function as a ganglionic inhibitory trans- 'Irittc•r. it iS taken up by isolated sympathetic and ganglia. Autoradiographs indicate that the ..,b+•lecl c:ompound is taken up by glial cells. rather 111,111 neurons (118). Inasmuch as thiocyanate is formed from cvano- ,t•ns inhaled in tobacco smoke, it was of interest to !.nmt• if this agent altered ganglionic transmission rnvdiated through nicotinic receptors. Concentra- tions of thiocyanate equivalent to those seen in the t)l,rsma of smokers had no effect on nicotinic gan- ,lionic transmission (119). Immunological svmpathectomv is produced by injPCtions of nerve growth factor antiserum. This f results in destruction of much of the peripheral sympathetic system. Mice injected with this antise- rum show reduced heart and gastrointestinal ncir- epinPphrine. while serotonin was increased and histamine levels were unaffected (120). Such mice behave differently from normal animals, particularl}' in tests that include aversive stimuli. Ganglionic stimulants including nicotine improve shuttle box performance in normal mice. while ganglionic blockers like mecamylamine and pentolinium dis- rupt such behavior. Immunost•mpathectomized mice are adversely affected by both classes of drugs (121). One prominent theory of neurogenic control of ureteral peristalsis implicates the sympathetic ner- vous svstem. In dogs. both topical and intravenous nicotine. and inhalation of cigar smoke all cause hvperperistalsis of the ureter (122. 123). The smooth muscle of the rat vas deferens is under norepineph- rine regulation by the sympathetic nervous system. Some of the constituents of tobacco smoke including acetaldehyde. propionaldehyde. formaldehyde and acrolein have been said to have indirect svmpatho- mimetic activity. Hence. the effects of these al- deh-%•des were determined on the rat vas deferens. All four aldehvdes were shown to interact with tissue norepinephrine stores and affect nonspecific membrane calcium binding sites (124). Formalde- hvde and acrolein were potentially more toxic. Both of these aldehvdes reduced I;C-norepinephrine and }'Ca accumulation. A combined autoradiography with formaldehvde-induced histochemical fluores- cence technique has been developed (125). This method has demonstrated that'H-norepinephrine is taken up only by those nerve structures in the heart which show a specific fluorescence reaction due to endogenous catecholamines (126). Conduction of vagal nerve "C" fibers in the cat is readily blocked by nicotine-like drugs injected into the arterial blood supply of the nodose ganglion (127). The blockade lasted onlv a few minutes and was prevented by hexamethonium. Skel etal Murom uscul ar The isolated frog sciatic nerve is a useful system to study the effects of drugs on neuronal conduction. It can be used to distinguish the mechanisms by which drugs including local anesthetics block trans- mission (128). The isolated frog sartorius muscle is another useful preparation to study the effects of TIMN 0115925 T200416 181

can be measured with and without various drugs. Nicotine had no effect on control or potassium ion stimulated, oxygen consumption in either rat brain cortex or striatum slices in contrast to other drugs (65). Radioactive nicotine has been shown to ac- cumulate in rat brain slices (66). The intracellular accumulation of "C-nicotine appears to depend upon the potassium ion concentration, and may result from potassium-induced alterations in the binding capacity of a subcellular depot (66, 67). Various drugs i.ncluding lobeline. physostigmine and to a lesser extent. metanephrine compete with : aC-nicotine for cellular binding sites• or in the case of procaine apparently prevent access of nicotine to the intracellular sites (68). The uptake and dis- tribution of 'aC-nicotine in capuchin monkev brain slices is quantitatively and qualitatively similar to those of rat brain slices (69). This appears to be due to an energy dependent process for it cannot be attributed solely to diffusion (70). b. Sensorv Systems-As might be expected. very large doses of nicotine had very little effect on guinea pig cochlear potentials (71). No evidence of nicotine ototoxicitv was demonstrated. The acute effects of smoking on auditorv and vestibular func- tion were studied in 100 subjects using electrical recording techniques (72, 73, 74). There was no depression of hearing or any consistent effects on the function of the lab«inth. c. Motor Sc•stems-Nicotine-pilocarpine in combination have been shown to produce a sus- tained tremor in mice and rats (75). A central c.holinergic mechanism is involved. This animal model of tremor is blocked by atropine, various n icotinic antagonists. promethazine, phenobarbital, and propranolol but not L-DOPA. This would in- dicate this is a unique tremor model. d. Temperature Regulation-There is evidence in animals. as «•ell.as in humans, that the regulation 4if body temperature involves a cholinergic link. Animal data suggests this is in the rostral hypothala- mus (76). Both a muscarinic and a nicotinic mecha- nism is involved (77-80). In addition, catechol- <Imines. as well as histamine, are implicated (76. 81). The h~.perthermic actions of drugs may be related to blockade of cholinergic receptors in the thermoregu- latory centers of the brain (82, 83). e. ,\'er.irotransmitterinteraction-The actions of nicotine on receptors of the cholinergic system_ are tivell known and have been elaborated further bv the grantees as described above. In addition, nicotine and/or tobacco smoking result in the release of other chemical messengers including the catecholamines and 5-hvdroxytryptamine (serotonin). A procedure for combining microautoradiographv and histo- chemical fluorescence for aromatic monoamine chemical messengers or neurotransmitters has been described (84). Following acute horizontal oscilla- tion stress, norepinephrine neurons in the rat brain were activated, while serotonin neurons were con- currently depressed. Furthermore• brain areas varied in their response to stress (85). In rats exposed to passive avoidance stress, forebrain serotonin me- tabolism was reduced in learners and stimulated in non-learners (86). Data in rats and mice indicate that the serotonin brain system normally depresses the ability of an animal to habituate to a novel en- vironment (87). Female rats selected for low or high activity were studied behaviorally and neurochemi- cally using inhibitors of either norepinephrine or serotonin synthesis (88). The serotonin svstem was more functional in the forebrain of high activitv rats. while the norepinephrine system appeared more functional in low activitv rats. Nicotine variable effects on behavior of rats of different temperaments is related to its effects on the forebrain serotonergic system (89). Furthermore, female rats are more sensitive both behaviorally and neurochemicallv in the brain serotonin than similarly treated males (90). Female rats also accumulated more nicotine in their brains and were more sensitive to its behavioral stimulating effects (54. 55). In addition. female rats of two different strains were generally more active and brain serotonin metabolism correlated posi- tively to the behavioral findings (91). The effects of acute and chronic nicotine administration and age were studied on various neurotransmitters and as- sociated enzymes in male rats (92). Nicotine pre- treatment did not alter significantly serotonin or norepinephrine in a variety of tissues, nor whole brain choline, acetylcholine and acetylcholinester- ese activitv. Histamine tissue concentrations were variably affected, but gamma-aminobutyric acid lev- els were elevated throughout various brain areas. In an independent but related study acute and chronic nicotine administration was studied on total and regional brain levels of dopamine and norepineph- rine (93). "Smoking doses" of nicotine had no effect on rat brain norepinephrine levels, but very large doses of nicotine did lower mouse whole brain and rat diencephalic norepinephrine in a time-response related nianner. Of three nicotine analogs studied. 179 TIMN 0115923 T200414

Nervous Sti,stem pine. did not affc°t t nuirphine ()r LSD discrimination. The narcotic antagonist. nalo.\one. blocked__the stim-__ . ulus effect of morphine, but did not alter LSD discrimination. The results indicate that the mor- phine and LSD stimuli are dissimilar and that the integrity of 5-HT or ACh nervous systems is not essential for morphine or LSD to serve as a dis- criminative stimulus. Other support: U. S. Public Health Service. 33. Discriminative control of behavior by electrical stimulation of the dorsal raphe nucleus: Generalization to lysergic acid diethylamide (LSD) tra D. Hirschhorn. Ronald L. Hac•es, and John A. Rosecrans _ Medical College of Virginia. Richmond. Virginia Brain Research 86:13-1-138.1975 The parikarva of 5-hydroxytryptamine contain- ing neurons in the brain are located in the raphe nucleus (Dahlstrom and Fuxe, Acta Pht•siol Scand 62:1, 1965). The present experiments sought to 1) determine whether electrical stimulation of the dor- sal raphe nucleus could serve as a discriminative stimulus and 2) investigate the interaction of certain drugs with this stimulus. Bipolar electrodes were implanted in the dorsal raphe nucleus of 10 rats. Following recovery from surgery, the rats were trained to press both bars of an operant chamber and discrimination training was begun. Depression of one of the levers resulted in food reinforcement in the presence of brain stimulation (biphasic rectangu- lar pulse pairs. 300-600 µamp) and responses on the opposite lever were reinforced in the absence of brain stimulation. A high degree of discriminated responding rapidly occurred. Injections of morphine sulfate (2.5-10.0 mg/kg) or LSD tartrate (25-100 µg/ kg) did not alter responding during stimulation. In the absence of stimulation. LSD, but not morphine produced responses appropriate to electrical stimu- lation. These results indicate that electrical stimula- tion of the dorsal raphe nucleus and LSD have similar stimulus properties. Other support: U. S. Public Health Service 34. Morphine and A9-tetrahydrocannabinoL• Tolerance to the stimulus effects Ira D. I-lirschhorn. and John A. Rosecrans Department of Pharmacologt•. Medical College of ['irginia. Richmond. Virginia Psrchopharmacologia 36:243-253. 1974 Morphine and saline served as discriminative stimuli for one group of rats in a 2-lever discrimina- tion task. A9THC and vehicle were discriminative stimuli for a second group. Depression of one lever resulted in reinforcement following the administra- tion of morphine or X9THC and the opposite lever was reinforced after vehicle. A high degree of discriminated responding occurred with both drugs. During daily supplemental injections of morphine nr 19THC up to several times the training dose.for a period of 2 months, subjects still discriminati-d morphine or A9THC from vehicle. However, the degree of discrimination was reduced indicating a limited tolerance to either drug. Naloxone precipi- tated narcotic-like withdrawal symptoms in rats chronically treated with either morphine or A9THC, indicating that there is an interaction between chronic administration of A9-THC and the narcotic antagonist, naloxone. Other support: U. S. Public Health Service. 35. Generalization of morphine and lysergic acid diethylamide (LSD) stimulus properties to narcotic analgesics Ira D. Hirschhorn, and John A. Rosecrans Medical College of Virginia. Richmond, Virginia Federation Proceedings 34(3):787, 1975 Morphine (7.5 mg/kg) and LSD (0.1 mg/kg) served as discriminative stimuli in a two lever operant task. Rats were trained on an FR-4 schedule in which one lever was reinforced under the drug state and the opposite lever reinforced under non- drug conditions (saline). No responses were rein- forced during the first 2.5 minutes of a daily 15 minute session. Following the establish{nent of the discrimination, rats were tested (test sessions were 2.5 min with no reinforcement) for generalization to several narcotic analgesics methadone (1-6 mg/kg), meperidine (5-15 mg/kg), and pentazocine (5-20 mgl TIMN 0115936 T200427 192

\rn Ous Scstem itt cif alpha assc~c.iated with drowsiness is similar to that found with other beverages but the 8.5 per sec activity in the parietals is a new finding. 13. Electroencephalographic effects of caffeine. nicotine, tobacco smoking, and alcohol Henry B. Murphree Rutgers Medical School of the College of Medicine and Dentistn• of \'ew ferser. Piscatacray. \'eit- Ierser Psrrhotropic Drugs and the Human EEG. Modern Problems of Pharmacopscchiatn•. vol 8:22-36. ed. Turan M. Itil. \ew York. \. Y. (Karger. Basel 1974) This paper concerns the quantitative and quali- tative EEG effects of caffeine, nicotine and tobacco smoking, and alcohol. It is limited mainly to the effects of amounts ordinarilc• consumed. Many psy- chotropic drugs have effects on sleep cycles. and some data of this type are also included. The presentation is based on 70 literature references. the author's research being presented in context in this rec•iew. 14. Computer time-series frequency analysis of the electroencephalograms of male nonpsychotic and chronic schizophrenic subjects H. B. %turphree, and R. E. Schultz Rutgers t'nirersitl .%fedical School. New Brunswick. .\"ew Jersec• In: Schizophrenia: Current Concepts and Research. D. V. Siva Sankar. Editor. PJD Publication Ltd, HicksviJle. \e«• York. 1969. pp. 242-252 Electroencephalographic recordings from non- -Ychotic subjects were compared with those from c.hronic schizophrenic patients. The comparisons were made bc• means of integrative determinations of total electrical energy in one lead of the electroen- cephalogram and simultaneous determinations of energy in discrete portions of the spectrum from 1.0 to 36 cc•cles per second in the same lead. From these studies. it is concluded that: 1. The previous observations that male chronic schizophrenic patients tend to have smaller than normal variability in the mean electrical energy of their electroencephalograms are confirmed. 2. The observed differences in variabilit\ r,f electroencephalographic energies between schizo. phrenic patients and nonpsychotic: subjects is due in large part to differences in the variability uf alpha activity. Patients and nonpsychotic subjects who have low alpha activity do not differ from each other. Patients and nonpscchotic subjects who have greater alpha activity do differ from each other. The dif- ferences in alpha activity cannot be reliabl}• detected by visual inspection of the records. 3. The observed differences may he clue to a tendencv for the nonpatients to become dro«•sl• while the patients remain fixedly awake. 15. Effects of intravenous nicotine in smokers and nonsmokers L. P. Kenig. and H. B. Murphree Hahnemann Medical College. Philadelphia. Pennsl•lvania. and CNiDNJ-Rut;ers Medical School. PiscatawaY. New Jerser Federation Proceedings 32(3):805. 1973 After a baseline periods, 8 smoker and 6 non- smoker men were given nicotine tartrate by remote control timed intravenous (IV) infusion 6 µg per sec, to an effective criterion of 15 beats per min increase in heart rate (HR) or a maximum dose of 3 mg. EKG. HR, respiratory rate, blood pressure (BP) and EEG were recorded by polygraph. EEGs were also re- corded on FM magnetic tape for computer analysis. A mood adjective check list (Nowlis) was given before and after each trial. Both groups showed highly significant increases in HR and BP, with no significant difference between the means, but with greater variance in the smokers. No EKG abnormali- ties. occurred. Respirations showed irregularity of rate and tidal volume rather than changes in rate. EEG effects were similar in both groups, transient slow activity. There were no significant differences within or between groups on the Nowlis. The conclusions are that the EEG effects of IV nicotine were unlike those of smoking, probably because of different dosages, and that the smokers did not demonstrate greater tolerance than honsmokers to IV nicotine. TIMN 0115930 T200421 186

Nervous System iru luclin1, '-6011int•. \-(a-pvridrlmethvl) piperidine and \-(3-ptiridvlmt,thvO pyrrolidine. only the latter had Any effect on brain norepinephrine. Chronic nicntint- injections had no effect on brain norepi- nephrine or epinephrine but did decrease brain serotunin after seven days with a return to normal hrain serntonin in fourteen dat•s. Nicotine has been shown in slices of different areas of the rat brain to affect "C-serotonin move- ment bx- affecting sodium ion dependent efflux (94). \icotine also releases "C-norepinephrine from rat hYpothalamus slices by a specific action at amine binding sites or stores (95). In contrast. labeled dopamine content in rat striatum slices was not affected by nicotine. In addition, nicotine does not alter 1'C-nlutamate fluxes in rat brain cortical slices (96). As described above. nicotine has subtle effects on the brain catecholamines. a neurotransmitter svstem also affected by morphine. The adrenervic neuronal destroyer 6-hc•droxydopamine depletes both brain dopamine and norepinephrine. When combined with desmethvlimipramine. this agent preferentially depletes brain dopamine which is correlated with reduced antinociceptit•e effects of morphine (97). Interestinbl~•. morphine can modify the neuronal patterns of the cinoulate gyTUs of the rat (98). The cingulate gyTus is part of the limbic or emotional system of the brain. - f. `'euroendocrine-Pituitarv function is under brain control. particularly by the hypothalamus and other areas of the limbic system (99). Lots of different emotionalities would be expected to have differ- ences in brain control of pituitary function. Rats of low or high emotionalitv have similar endocrine function. hut forebrain serotonin turnover was shown to, be depressed in highly emotional rats whit h had elevated plasma and adrenal corticoste- ronw '.ot pl~ ( 100). Chronic electrical stimulation of thi, h\pt,thalamus in conscious cats produces chan;Y; in blood ;lucose and cortisol lerels. but not in -rr~wth hormone (101). Growth hormone does not play an ic.tive role in short-term homeostasis to altered blood glucose in cats (102). Intravenous nicotine in large. convulsant doses elevates plasma glucose and corticosterone levels. These effects are not mediated via the srmpathetic-adrenal system (103). Low doses of nicotine had no effect. In- tracerebral injections of nicotine and carbachol were made in the rat to localize the central sites of nicotine on corticosterone secretion. Focal injections of nicotine into the ventromedial and pc~sterior hypothalamic areas, as well as the amvQdala and midbrain reticular formation. produced a sNnificant rise in plasma corticosterone levels. Injections of nicotine into the anterior hypothalamus. septum, and dorsal hippocampus were without effect. (n contrast, all brain areas injected with c:arbachol produced sibnificant increases in plasma c:ortir.oste- rone (104). Injections of microquantities of morphine into the hypothalamus of rats depresses the release of radioiodine from the thV-roid gland (105). A dual action seems to be involved: stimulation of neurons in the caudal hypothalamus that inhibit th',Toid stimulating hormone release and depression of neu- rons in the rostral hypothalamus that activate the pituitary-thyroid axis. B. Peripheral Nervous St•stem Autonomic Nicotine is a classic ganglionic stimulant and depressant. depending upon its concentration. Hence. it is of interest that both the isolated rat superior cervical sympathetic and nodose ganglia accumulate 'H-nicotine (106). Nicotine depolarized the former but not the latter ganglia. It was sho«•n that nicotine may be concentrated within neurons. Such intracellular accumulation is enhanced during depolarization. It has been suggested that acety1- choline acts presynaptically during ganglionic transmission to release greater amounts of itself. the so called "percussion cap" theory of acetylcholine release. However. acetylcholine, carbachol. nicotine. tetraethylammonium. and methacholine were all ineffective in releasing 'H-aceh•lcholine followino incubation (107). There is pharmacological evidence that the postsynaptic ganglionic nicotinic receptors are quite similar to those in leech dorsal muscle (108). Nicotine causes ganglionic depolarization which is not followed by hyperpolarization unless hexamethonium is added. Ganglionic depolarization produced by carbachol is followed by pronounced ganglionic hyperpolarization. This after-hyperpolar- ization is due to the electrogenic extrusion of so- dium ion accumulated in ganglion cells during the preceding depolarization. with no involvement of specific muscarinic receptors (109). TIlqN 0115924 T200415 180

Nervous System ,iminP sulf<itt-. anil 0.''i-2.0 mg;kg arecoline hc•- c(rc~bromide produced responses which resembled the established saline effect. Pretreatment with 10.0 m-/k- lobeline (used as a smoking deterrant agent in dishabituation clinics) had no significant effect on the nicotine cued response. The inability of these structurallc• and/or functionally similar drugs to produce a nicotine-like discrimination effect is dis- cussed. 26. Studies on the time course and the effect of cholinergic and adrenergic receptor blockers on the stimulus effect of nicotine 1. D. Hirschhorn. and J. A. Rosecrans Department of Pharmacology. Medical College of Virginia. Richmond. Virginia PsYchopharmacolo;ia 40:109-120. 1974 This studv inrestigated the stimulus property of nicotine in the rat. The primary objectives of the study were 1. to determine the time course of the nicotine stimulus and its relationship to brain levels of the drug and 2. to determine whether the nicotine stimulus is dependent upon the integrity of specific neurotransmitter s_vstems. A lever choice discrimi- nation was used. After injection of nicotine, de- pression of one lever in an operant test chamber resulted in food reinforcement according to a vari- able interval schedule of 15 sec. When saline was administered. the opposite lever was reinforced. A high degree of discriminated responding was ob- served when either 400 µg/kg or 200 µg/kg of nicotine was used as a discriminative stimulus. The degree of discrimination decreased as the length of the time period between the injection of nicotine and the test of discrimination was increased. This de- cline in discrimination was similar to the decline in brain levels of nicotine suggesting that nicotine discrimination is directly related to the concentra- tion of nicotine in the brain. Atropine, mecamyla- mine, dibenamine. propranolol and a-methyl-para- tyrosine (ANIPT) were all tested, in a range of doses, for effects upon nicotine discrimination. Of these, only mecamvlamine antagonized the nicotine stimu- lus. These results indicate that the stimulus effect of nicotine is mediated specifically through nicotinic- cholinergic receptors and not muscarinic-choliner• gic or adrenergic receptors. 27. Transfer of state-dependent control of discriminative behaviour between subcutaneously and intraventricularly administered nicotine and saline Martin D. Schechter Department of Pharmacology. University of Mel6ourne. Parkville. Victoria. Australia Psrchopharmaco(ogia 32:327-335. 1973 Rats were trained to choose between the side compartments of a three-chambered shock-escape apparatus according to whether they were injected with nicotine or saline. Half of the rats learned to discriminate between 0.4 mg/kg nicotine and saline administered subcutaneously, whereas the other half learned to differentiate between 644 ng nicotine or. saline administered intraventricularly. The rats trained bv the subcutaneous route of administration had the ability to discriminate between nicotine and saline given intraventricularly and the rats trained by the intraventricular route could differentiate when the two substances were injected subcuta- neously. This transfer of state-dependent control of discriminative behavior between subcutaneouslv and intraventricularly administered nicotine and saline is presented as evidence for the central origin of the nicotine-produced interoceptive cue. 28. Atropine antagonism of arecoline-cued behavior in the rat Martin D. Schechter, and John A. Rosecrans Department of Pharmacology, :14edical College of Vi oinia. Richmond. Virginia Life Sciences 11, Part 1:517-523, 1972 Related to the preceding studies, rats were trained to make a specific behavioral response in a two-levered Skinner apparatus conditional upon whether they were injected subcutaneously with 0.5 mg/kg of arecoline or 0.9% saline. Pretreatment with 0.5 mg/kg atropine sulfate signiffcantly inhibited the arecoline-cued behavior. The results suggest that the arecoline-produced interoceptive cue is of central origin, and the possible existence of specific mus- carinic and nicotinic cholinergic receptors in the CNS. TIMN 0115934 T200425 190

29. Ly'sergic acid diethylamide (LSD) as a discriminative cue: Drugs with similar stimulus p roperties ,,(artin D. Schechter. and John A. Rosecrans ,nf.partment of Pharmacologt•. Medical College of 1•frs-,inia. Richmond. t'irginia /'ci•rhopharmacologia 26:313-316. 1972 Rats were trained to choose between the arms of ,, T-maze apparatus according to whether they were injec:ted i.p. with 0-1 µmol/kg LSD or 0.9% saline. The LSD drug-state acquired the properties of a dist riminatire stimulus, possibly by producing in- tercuceptice cues. Doses of 9.0 µmolJkg psilocybin <uid 90 and 120 µmol/kg mescaline produced cueing ~~ffec.ts which were not significantly different from the cueing effect of LSD. However. d-amphetamine 114.8 and 29.6 p.mol/kg) did not appear to produce ,1n LSD-like cue. These results suggest that LSD. mescaline and psilocybin, when administered in functionally equivalent doses, produce qualitatively yimilar interoceptive cues in the rat. (Jther support: U. S. Public Health Service. 30. d-Amphetamine as a discriminative cue: Drugs with similar stimulus properties %Lirtin D. Schechter. and John A. Rosecrans nopartment of Pharmacology. Medical College of tYrgoinia. Richmond. Virginia Furopean Journal of Pharmacology 21:212-216, 1973 Rats were trained to choose between the side ..ompartments of a 3-chambered shock-escape ap- traratus according to whether they were injected I intraperitoneally) with 4 mg/kg d-amphetamine or t).9"o saline. The d-amphetamine drug state acquired ,ill of the properties of a discriminative stimulus by producing interoceptive cues. Doses of 0.4 mg/kg nicotine. 7.4-29.7 mg/kg mescaline, 4.0-8.0 mg/kg fenfluramine and 0.048 mg/kg LSD failed to produce an amphetamine-like cue. However, 1-amphet- amine. 8.0 mg/kg, produced responses shown to be statistically similar to the 4.0 mg/kg training dose of cl-amphetamine. 31. Morphine as a discriminative cue: Effects of amine depletors and naloxone John A. Rosecrans, `tax H. Goodloe. Jr.. Garv 1. Bennett. and Ira D. Hirschhorn Department of Pharmacoloar. Medical Cnllege of Virginia. Richmond. Virginia European Journal of Pharmacolog[• 21:252-256. 1973 Nfale Wistar rats were trained to escape a painful shock in a 3-compartment chamber contingent upon their ability to discriminate between 2 drug states. viz., morphine, 20 mg/kg and saline. Maintenance of this discrimination task (State-dependent behavior) was tested after brain 5-hydroxytryptamine and brain norepinephrine levels were reduced by the amine synthesis inhibitors, p-chlorophenc•lalanine and a-methyl-p-tyrrosine. The results of this experi- ment showed that discrimination was dependent upon the integrity of brain 5-hydroxytrvptamine systems, but not upon brain norepinephrine svs- tems. It was also observed that morphine discrimina- tion could also be antagonized by naloxone. a narcotic antagonist. Other support: U. S. Public Health Service. 32. A comparison of the stimulus effects of morphine and lysergic acid diethylamide (LSD) Ira D. Hirschhorn, and John A. Rosecrans Department of Pharmacology, Medical College of Virginia. Richmond. Virginia Pharmacology Biochemistry and Behavior 2:361-366. 1974 Morphine and lysergic acid diethylamide (LSD) each was used as a discriminative stimulus for rats. After the injection of drug (morphine or LSD), depression of one lever of an operant test chamber resulted in positive reinforcement according to a variable interval schedule of 15 sec (VI-15 sec). When saline was given, responses on the opposite lever were reinforced. Discriminated responding occurred when either morphine or LSD served as the discriminative stimulus. When animals which were trained to discriminate morphine frotri saline ,were given LSD, they pressed predominantly the saline- correct lever. Similarly, LSD discrimination did not generalize to morphine. Two 5-hydroxytryptamine (5-HT) antagonists, cyproheptadine and methylser- gide, and one acetylcholine (ACh) antagonist, atro- TIMN 0115935 T200426 191

i t I I r kol produced responses appropriate for morphine in animals trained to discriminate morphine which Was dose related. Generalization to nalorphine (1-40 malko) or cyclazocine (0.25-1.0 mg/kg) was not e% ident. Of the five drugs tested. only cvclazocine produced LSD-appropriate responses in animals trained to discriminate LSD from saline. These results suggest that narcotic drugs with primarilv antagonist effects have stimulus properties similar to those of morphine, but narcotics with strong an- tagonist effects produce dissimilar effects. Further- moreo the similarity of LSD and cvclazocine stimulus effects is also congruous with the observation that cvclazocine frequently produces psc•chotomimetic eEfects in man. Nalorphine. which also produces ps}•chotomimetic effects in man has stimulus prop- erties in the rat which are discriminable from those of LSD. Other support: U. S. Public Health Service. 36. Generalization of morphine and lysergic acid diethylamide (LSD) stimulus properties to narcotic analgesics t. D. Hirschhorn. and J. A. Rosecrans Department of Pharmacology. Nfedical College of c~irefnia. Richmond. ('irginia Ps}•chopharmacology 47:65-69. 1976 The present investigation sought to determine ivhether the stimulus properties of morphine and lysergic acid diethylamide (LSD) would generalize to several narcotic analgesics which var_v in their subjective effects. Morphine and saline served as discriminative stimuli for one group of rats in a 2- lever discrimination task. LSD and saline were discriminative stimuli for a second group. Depres= sion of one lever in an operant chamber resulted in reinforcement following the administration of mor- phine or LSD and the opposite lever was reinforced after saline. After discriminated responding was stable. stimulus generalization tests with narcotic analgesics and antagonists showed that the stimulus properties of morphine generalized to methadone and meperidine, and partially to pentazocine, all of 1 which produce morphine-like subjective effects in humans. Morphine stimulus properties did not gen- eralize to nalorphine or cvclazocine. which produce dissimilar subjective effects. The stimulus properties of LSD generalized partially to cyclazocine. but not to nalorphine. In humans cvclazocine and nalor- phine produce a high incidence of psychotomimetic effects, but the subjective effects of cvclazocine arP differentiable from those of LSD. Other support: Special Action Office for Drug Abuse Prevention. and the U. S. Public Health Service. 37. Ethanol as a discriminative cue: Reduction following depletion of brain serotonin Martin D. Schechter Department of Pharmacology. University of Melbourne. Parkville. Victoria, Australia European Journal of Pharmacology 24:278-281. 1973 Administration of p-chlorophenvlalanine, a trvptophan hydroxylase inhibitor that selectively depletes brain serotonin, to rats conditioned to discriminate between ethanol and saline, produced complete inhibition of discriminative behavior. These findings provide evidence for a serotonergic mediation in the discrimination of interoceptive cues produced by ethanol in the central rtervous svstem. 38. Behavioral tolerance to an effect of nicotine in the rat M. D. Schechter, and J. A. Rosecrans Department of Pharmacology. Medical College of Virginia, Richmond, Virginia Archives Internationales de Pharrnacodynamie et de Therapie 195:52-56, 1972 Rats were trained to make a specific behavioral response for food reward in a T-maze apparatus conditional upon whether they were injected with 0.4 mg/kg nicotine or saline. After attaining a criterion of 8 out of 10 consecutive correct first choice responses, according to the substance in- jected, the rats received four daily injections of nicotine over a five day period, to investigate if tolerance to the behavioral task would develop. The results indicate that a significant tolerance did occur TIMN 0115937 T200428 193

Nervous System when discriminative responses of the first nicotine administration of each day are compared with the fourth dailv administration. The need for investiga- tions regarding the development of tolerance as a primary cause in the human smoking habit, and, for caution in the conducting of behavioral testing with nicotine, are discussed. 39. Azione della nicotina sul condizionamento di salvaguardia di ratti di un mese Francesco Robustelli (D. Bovet) Istituto di Farmacologia dell Universitci di Sassari, Italy Academia Nazionale dei Lincei 40:490-497. 1966 The effects of nicotine on rate of acquisition of escape-avoidance conditioning by 30-day-old Wistar Albino rats in a shuttle-box were investigated. with appearance of a light preceding by 5 seconds an electric shock from the grid floor. Performances of the untreated animals were lower in young than in adult rats of the same strain and the enhancement of rate of avoidance condition- ing by nicotine was proportionately much greater in the young animals. The split-litter technique was used, treated rats receiving 0.2 mg/kg of nicotine subcutaneouslv 15 minutes before trials, and the controls were injected with saline. 40. Differente action de la nicotine au cours de la journee et de la nuit dans 1'activite pontanee (running activity) du rat Filomena Bovet-Nitti, et Daniel Bovet Istituto di Farrnacologia dell'Universita di Sassari, Italie Comptes Rendus de L'Academie des Sciences, Serie D 262:316-320. 1966 Computerized activity wheels were used to record running activity of Wistar SM rats, aged three months when introduced to the apparatus and four- plus months at the start of pharmacological tests. The existence of a rhythm of voluntary activity was confirmed; locomotor activity which during the day averaged about 150 m/hr could attain at night even 30 times this value. The effect of nicotine, 0.2 and 1.0 mg/kg injected subcutaneously at 10 a.m. or 10 p.m., on voluntary activity was studied. Per- formances over a three hour period following ad- ministration of nicotine were compared with those over the same hours on preceding days. The dose of 1.0 mg/kg significantly stimulated (p = 0.02) activitv in the day tests and showed a depressant (sedative) effect in the evening tests (p = 0.05). Effects of 0.2 mg/kg were in the same opposite directions, stimu- lation in the day tests (p = 0.01) and depression in the evening tests (p = 0.1). 41. Effects of nicotine on avoidance conditioning of inbred strains of mice Daniel Bovet, Filomena Bovet-Nitti, and Alberto Oliverio Departments of Physiology and Pharmacology, UCLA School of Medicine, Los Angeles, California Psychopharmacologia 10:1-5, 1966 Inbred male mice of 9 different strains (Jackson Laboratory) and a common Swiss Albino strain wete used. Avoidance conditioning was studied using an automatic programming and recording "shuttle box", with light as the conditioned stimulus and electric shock as the unconditioned stimulus. The mice were given 100 trials a day for 5 consecutive days. Experimental animals received 0.5 mg/kg of nicotine intraperitoneally 15 minutes before the test; controls were injected with saline. The results indicate that the effects of nicotine are strain dependent. A facilitating effect on avoid- ance conditioning was observed in 6 of the 9 inbred strains, the strains characterized by a low per- formance showing generally the highest degree of facilitation. In two strains with a very high level of performance there was an impairing effect of nico- tine on avoidance performance. Other support: General research support grant from the University of California, School of Medicine, Los Angeles. 42. Facilitation of simultaneous visual discrimination by nicotine in the rat F. Bovet-Nitti (D. Bovet) Istituto di Farmacologia dell' Universitd di Sassari, Italy, and Department of Pharmacology, School of Medicine, University of California, Los Angeles, California Psychopharmacologia 10:59-66, 1966 Male Wistar rats were used in a study of the effect of nicotine on two choice and on five choice visual discrimination. The animals could avoid an electric shock by choosing the right pattern of the 43 act D. Ist ttu t 'C ,lt ,t rt .\c c: u ab: inr ph in Ot I It: t'C 44 stil du A. De, Mr• 11 F v ic as of arr ant Nit Ot, Gr Ca TIMN 0115938 T200429 194

Nervous SZ-stem three strains characterized bt• a homogeneous per- formance and bc• a low level of emotivity shows that C3HiHe mice reach the best performance when the trials are massed. DB A/2J mice perform better when the trials are spaced. while the strain BALB/c is char- acterized bv an intermediate type of performance which is independent of the training schedule. The results suggest that learning differences of inbred strains of mice can be interpreted on the basis of a multitrace view of memory storage mechanisms. Other support: Italian Consiglio Nazionale delle Ricerche. Gruppo Nazionale di :vfedicina Sperimen- tale, and from a U. S. Public Health Service General Research Support Grant to the U.C.L.A. School of Medicine, Los Angeles. California. 47. Effects of nicotine and strychnine on transfer of avoidance learning in the mouse Alberto Oliverio (D. Bovet) Department of Pharmacology. University of Sassari. Sassari. Italv Life Sciences 7:1163-1167, 1968 Transfer of avoidance (shuttle-box) learning was shown to occur when mice previously trained to respond to a given stimulus (tone) were subjected to a session in which a new stimulus (light) was used. The magnitude of the transfer phenomenon was strictly dependent on the length of a training session during which the animals were given an experience of a compound stimulus formed by the already known and by the new stimulus together. Both pre- and post-trial (T + L) administration of nicotine (0.5 mg/kg intraperitoneally) and post-trial (T + L) ad- ministration of strychnine (0.03 mg/kg intraperito- neallv) enhanced the transfer of learning (p < 0.01), as measured 24 hours later during the light session, in comparison to a control group. 48. Effects of scopolamine on avoidance conditioning and habituation of mice Alberto Oliverio (D. Bovet) Department of Pharmacology, University of Sassari, Sassari, Italy, and Centro di Psicofarmacologia, Consiglio Nazionale delle Ricerche, Sassari Psychopharmacologia 12:214-226, 1968 Effects of scopolamine on avoidance condition- ing of naive and previously trained mice and on habituation to the to-be-conditioned stimulus was studied by using a shuttle-box avoidance technique and a light as the conditioned stimulus. The results show that the action of the drug depends on four major phenomena: 1. A drug state in which learning is dissociated from learning under non-drug state. 2. A so-called "amnestic" effect which disap- pears once the effect of the drug is over. This effect is evident as an impairment of the performance of previously trained animals. 3. A photophobia due to the mydriatic effect of the drug, responsible for an enhanced sensitivity of the animals to luminous stimuli. 4. A facilitatory effect of the drug on arousal regulating mechanisms in naive animals. In accompanying studies with nicotine, find- ings supported previous studies showing that nico- tine facilitates learning by acting on memory pro- cesses. The action of nicotine was not state dependent (following withdrawal of the drug, the level of responding did not change)., Other support: Italian Consiglio Nazionale delle Ricerche. 49. Facilitation of simultaneous visual discrimination by nicotine in four "inbred" strains of mice F. Bovet-Nitti (Daniel Bovet) Istituto di Farmacologia dell'Universitd di Sassari, Italia Psychopharmacologia 14:193-199, 1969 Five-choice visual discrimination learning was determined in four different strains of inbred mice: DBA/2J, C3H/He, A/J and BALB/c (The Jackson Laboratory). The task was to discriminate between four locked doors carrying the same type of pattern and a swinging door (characterized by the positive pattern) leading to a goal box in which the animal avoids the electric shock which is delivered in the rest of the apparatus. All strains were able to learn this task in a small number of sessions. By distribut- ing or massing the practice further evidence was obtained suggesting that efficient consolidation mechanisms characterize the strain DBA/2J while short-term memory characterizes C3H/He mice. Nicotine (0.5 mg/kg injected intraperitoneally 15 minutes before each session) exerted a facilitating effect in three of the four strains (A/J, C3H/He, DBA/ 2J) while an impairing effect was evident at the same dose in the strain reaching the highest performance under control conditions (BALB/c). Withdrawal of % TIMN 0115940 196 T200431

\ervous System .-lnimal Behnc inr 20. Nicotine self-administration in monkeys G. A. Deneau. and Reizo Inoki Department of Pharmacology. L'nic•ersitr n_f .ltichi;an. Ann .-lrbor. Xlichigan Annals of the New York Academy of Sciences 142, article 1:277-279. 1967 Rhesus monkeys (Macaca mulatta) were pre- pared for intravenous injections via a mechanical injector which could be activated by an electric timer or bv the monkey if it pressed an appropriate lever. At initial single doses of 10 µg/kg nicotine. none of seven monkeys tested initiated self-administra- tion even after a two-month period of automatic hourly injections. On raising the single dose to 25 µg/kg, two of the seven monkeys began to self- administer nicotine spontaneously. After periods of priming ranging from 2 to 10 days, each of the other five monkevs began to self-administer supplemen- tarv doses between the scheduled hourly injections. When the self-administration began, the automatic injections were discontinued and all five monkeys continued to self administer the drug. At this dose level. average total daily doses ranged from 0.7 to 1.7 mg/kg with as much as 100% change on consecutive days for a giren monkey, the tendency being to take a large dose one day followed by a small dose on the succeeding day. At intervals of approximately one month, the individual doses were raised to 50, 100, 200, 500, 1000. and finally 2000 µg/kg. With each increment, all monkevs took fewer doses per day, but the total daily dose increased until a step was reached at which a given monkey refused to self-administer the next higher dose. One monkey died accidentally at the 25 µg/kg dose level. Of the six survivors, one refused to self-administer the 50 µg/kg dose, two stopped at 100 µg/kg and one each at 500 and 1000 µg/kg; one continued to self administer at the 2 mg/ kg level at an average daily intake of 9.6 mg/kg and a maximum of 14 mg/kg. Objective physical signs following injection of n icotine were not observed at doses up to 100 µg/kg. Doses of 200 µg/kg through 2.0 mg/kg produced signs of increasing sec•eritv which included ya«•n. ing. piloerection. flushing followed by pallor. m%-driasis. followed bv miosis. dyspnea. retching, vomiting, and muscular weakness to the point of prostration. Even at doses of 1.0 and 2.0 maik®, the major signs of drug effect persisted only for period of 5 to 10 minutes. and all signs disappeared after 20 minutes. During the course of the studv. the monkeys rarely self-administered nicotine during the hours of midnight to 8:00 AAtL when the laboratory lights were out. There was no evidence of an increased fre- quency of dosing either in the morning or late in the evening. It is pointed out that the circumstances of the study in no way resemble the natural habitat of monkeys, nor does the intravenous route of ad- ministration of nicotine resemble closely the manner in which man self-administers this drug. However, the fact that monkeys will self-administer nicotine at all is considered to suggest that this alkaloid may be one of the substances in tobacco smoke which is responsible for man's use of tobacco. But, it is cautioned, experiments with other pharmacologi- callv active substances in tobacco smoke should be made and other routes of self-administration should be tested before any conclusions can be drawn concerning the relationship of self-administration of nicotine by animals and smoking of tobacco by man. Other support: U. S. Public Health Service. 21. Effect of propranolol, d-amphetamine and caffeine on ethanol as a discriminative cue Martin D. Schechter Department of Pharmacology, University of Nfelbourne. Parkville, Victoria, Australia European Journal of Pharmacology 29:52-57. 1974 Rats were trained to escape a shock in a three- compartment chamber contingent upon their ability to discriminate between two drug states. namely,1.5 mg/kg ethanol and 0.9% saline. This discriminative ability was found to be dose responsive and was tested after pretreatment with 1, 5, 10 and 20 mg/kg propranolol, 4 mg/kg d-amphetamine sulfate and 100 mg/kg caffeine. The results of the pretreatment experiments showed that, of the three possible antagonists, only amphetamine significantly de- TIMN 0115932 188 T200423

,)n1v mot•able door adjacent to the goal box. The facilitating effect of nicotine 10.2 mg/kg, subcuta- [leously) on visual discrimination learning appeared evident when the animals chose between two pat- terns and less evident in the five-pattern choice. ~everthelesse nicotine facilitated stronglv the rec•er- ;al of program (order of door patterns). Uther support: General research support from the t'niversitc of California. School of Medicine. Los An ge les. .13. Action of nicotine on spontaneous and acquired behavior in rats and mice D. Bovet, F. Bovet-Nitti, and A. Oliverio lstituto di Farrnacologia della Universita de Sassari. ttalc. and Departments of Physiology and Pharmacology. i'CLA School of Medicine. Los Angeles. California .lnnals of the New York Academy of Sciences 142, .i rt ic.le 1:261-267. 1967 As part of a symposium held at the New York At:ademv of Sciences, the authors present and dis- cuss two aspects of their studies on nicotine [both abstracted elsewhere in this Section]: (1) the seem- inglr,• opposite effects of nicotine on the various phases of the nocturnal cycle of spontaneous activity in the rat: (2) the importance of genetic factors. Other support: Consiglio Nazionale delle Ricerche (Ital}•): USPHS General Research Support Grant to the ('CLA School of Medicine, Los Angeles, California. 44. Effetto antifatica dell'amfetatnina e di farmaci stimolanti centrali sulle prestazioni di topi durante sedute di condizionamento prolungate l. Oliverio (D. Bovet) t)epnrtment of Pharmacology, UCLA School of NlHdicine. Los Angeles, California It Furmaco Ed Sci 22:159-171, 1967 A decrement in performance appeared in pre- riously trained and naive DBA/2J inbred mice given a session of 2500 trials in a shuttle box over a period Of 20 hours and 50 minutes. Injection of amphet- amine counteracted the decrement in both instances, aiid in the case of naive mice increased performance. Nicotine, on the other hand, proved ineffective. Other support: USPHS General Research Support Grant to UCLA School of Medicine, Los Angeles, California. '45. Analysis of the "anti-fatigue" activity of amphetamine. Role of central adrenergic mechanisms A. Oliverio (D. Bovet) Istituto di Farmacologia dell'C'niversitb di Sassari Il Farrnaco Ed Sci 22:441-449, 1967 Naive DBA/2J mice given a prolonged avoid- ance session [shuttle-box] performed at a very poor level. Amphetamine, injected at the end of the session, enhanced the performance of the animals. This effect was antagonized by a previous injection of a-methyltyrosine. A long lasting enhancement of the performance was obtained through a pre- treatment with a monoamine oxidase inhibitor, pargyline. Pargyline and nicotine improved the performance also during short avoidance sessions intervaled by 24 hours. However, the action of pargyline consisted in a plain effect on the per- formance, since the daily increments of the respond- ing were not retained. The action of nicotine con- sisted instead in an enhancement of the retention confirming previous results showing a facilitating effect of this drug on learning. The results are discussed in relation to the role exerted by catecholamines and adrenergic mech- anisms in antagonizing the inhibitory reactions occurring during uninterrupted and even short avoidance sessions. Other support: Consiglio Nazionale delle Richerche (Italy). 46. Memory and consolidation mechanisms in avoidance learning of inbred mice Daniel Bovet, Filomena Bovet-Nitti, and Alberto Oliverio Department of Pharmacology, University of Sassari, Sassari (Italy) Brain Research 10:168-182, 1968 Avoidance learning of different strains of mice was assessed by using an automatic shuttle-box apparatus. The results show that avoidance learning is strongly determined genetically and that there is no correlation between this acquired behavior and the spontaneous motor activity of the animals as mea- sured in the shuttle-box. The responses within the same strain were rather homogeneous. An analysis of time variables shows that for a given type of training there is an optimal schedule depending on the strain considered. The study of TIMN 0115939 T200430 195

Ner-Vous System tion of the lateral olfactory tract. In agreement with other investigators. stimulation of slices incubated at 37°C evoked a surface negative wave in prepyriform cortex. olfactory tubercle and periamygdaloid cor- tex, but the surface positive wave seen in vivo was absent. Variations in slice thickness indicated that the surface negative wave was generated superfi- cially since it could be evoked in slices as thin as 0.28 mm and that the surface-positive wave was generated from deeper lavers since it could not be seen in slices less than 0.46 mm thick. To provide adequate oxygenation, the thicker slices were in- cubated at ambient temperatures. At these tempera- tures, 0.46-0.78 mm thick slices exhibited surface negative waves over the entire olfactory cortex, including entorinal cortex, as well as sequential surface-negative and surface-positive waves over the periamygdaloid cortex. Optimum conditions were obtained with slices 0.52 mm thick, incubated in Krebs solution containing 2.5 mM CaZ` at 23-27°C. Under these conditions evoked potentials were well maintained up to 1-2 days. Such slices demonstrated intracellular \Ta-. K- and water contents similar to those of thinner slices incubated at 37°C. The surface potentials were sensitive to changes in calcium, maonesium and stimulus-frequency in a manner suggesting a synaptic potential. Available evidence accorded with the view that the surface negative wave represented post-synaptic excitatory potentials of superficial elements and that the surface positive wave resulted from inhibitory hyperpolarizations mediated through deep lying multisynaptic path- ways. Other support: U. S. Public Health Service. 60. Action of nicotine on identified cells of the snail brain John C. Hancock Department of Pharmacology, Louisiana State University, School of Nfedicine, New Orleans, Louisiana .\aunvn-Schmiedeberg's Archives of Pharmacology 280:275-294. 1973 The present experiments were undertaken to characterize the action of nicotine on single identi- fied neurons and to study the development of desensitization of these neurons to nicotine. The brain of the snail, Helix pornatia, is well suited for this study since the cells are large and peripherally placed. This makes it possible to work on a known neuron from preparation to preparation. ' Evidence is presented to show that nicotine causes desensitization of certain cells of the snail brain. On cholinoceptive cells where the action of nicotine was excitatory (D-cells), nicotine (10-5 to 10 'M) caused a dose-dependent depolarization and a decrease in input resistance. Firing rate was increased in pacemaker cells. On cholinoceptive cells where the action of nicotine was inhibitorv (H- cells), nicotine (10-6 to 10''M) caused a dose- dependent hyperpolarization and a decrease in in- put resistance. Firing rate was decreased in pacemaker cells. Both the excitatory and inhibitory effects of nicotine were blocked by d-tubocurarine (10-' tn 10-;M). Hexamethonium (10-"`) reduced the r,• sponse to nicotine on D-cells but not on H-cells. The membrane potential of both D- and H-cells returned to control in the presence of nicotine. Thi• duration of the nicotine-induced depolarization of cell V7 was 15 -* 0.8 min (n = 10). The duration of the hyperpolarization of cell V5 was 5-* 0.7 min (n = 4). The duration of the nicotine-induced hyperpolarization of H cells was consistently shorter than the duration of the nicotine-induced depolar- ization of D cells. The time course of changes in input resistance and firing rate paralleled the time course of changes in membrane potential in both cell types. The duration of the nicotine-induced change in membrane potential, input resistance and firing rate was not related to the concentration of nicotine. Similarly, the duration of the depolarization caused by acetylcholine, carbachol, lobeline or DMPP was not related to the drug concentration. At the time of repolarization after nicotine, the application of a second dose of nicotine had no effect or caused a depolarization that was low in amplitude and brief in duration. d-Tubocurarine (10-g to 10-5M) reduced the amplitude of the nicotine-induced change in mem- brane potential but did not affect the duration. Increasing the extracellular calcium boncentra- tion from 7 mM to 14 mM decreased the duration of the nicotine-induced changes. Also, increasing the pH from 8.0 to 10.0 decreased the duration. TIMN 0115944 T200435 200

NPr%',tlls Sl stem Rc•cnrdin_., f~t~ti~lic hlnr)d pressure. heart rate dnd the EKG. uwtle during the studies. indicated that the physiological alterations in these parameters produced by smoking can be reproduced b~ pa- rental nicotine. The results are interpreted to suggest that nic- otine plays a small but significant role in the smoking habit and that part of the crac•ing for a cigarette can be satisfied bc• the intravenous ad- ministration of the alkaloid. 7. Trends observed in the time estimation of three stimulus intervals within and across sessions G. S. Emley. C. R. Schuster, and B. R. Lucchesi Department of Pharmacolo-}•. C'nirersitr of M_ichigan .tledical School. Ann Arbor. Michigan Perceptual and .\totor Skills 26:391-398. 1968 Five subjects participated in a 6-hr experiment of which the time estimation procedure was a part. The production method with no feedback was used. Subjects produced 30 trials at each of 5-. 15- and 30- sec interc•als. There were two sessions (AM and P~t) on each of 9 experimental days. An increasing gradient over the first 30 trials was observed in the group data for the 5- and 15-sec inten-als. There was a descend- inQ jradient for the 30-sec interval. These gradients do not occur on the second 30 trials or in subsequent sessions. A decreasing trend in the estimations over davs was observed. %fean A.m estimations was sig- nific antly higher than nlean_ a%t estimations. The reQularitv and stability of the data may possibly be attributed to the environmental constancy. 8. Electroencephalographic changes in man following smoking Henry B. Murphree, Carl C. Pfeiffer. and Lillys M. Price Bureau of Research in Neurology and Psychiatn•. \'. J.. .`"turnpse•chiatric Institute. Princeton. ``.J. .lnnals nt the Nefr York .lcademy of Sciences 142. .i rt icle i: 2-t5-260. 1967 A progress report on EEG changes in man following smoking is presented leading to the fol- lowing conclusions: 1. The effects of drugs upon the CNS as re- flected in the EEG are dependent upon the state of the subject before dosage. 2. There may be a very rapid, reflex effect upon the EEG of smoke inhalation occurring before any blood-borne pharmacological effect. 3. The effects of smoking. as reflected in the EEG. appear to be stimulant rather than tranquiliz- ing, although subjects may exhibit individual dif. ferences. 4. Verv slight stimulant effects maybe reflected in a reduction in variance of the EEG before any reduction of alpha or total energy. Other support: U. S. Public Health Service: Ges- chickter Fund for Medical Research. 9. Computer time-series analysis of the effects of some analgesics upon human EEGs H. B. Murphree and C. C. Pfeiffer Bureau of Research. Netc Jerset \'europst•chiatric Institute. Princeton. Nert• Jerset• Archivos de Biologia c• Medicina E.eperimentales 4:29-35. 1967 The following drugs were tested in 1.0 gram doses: aspirin. acetaminophen. phenacetin. salicyl- amide. and buffered aspirin. Sodium salicylate wes given in 1.2 gram doses. The authors' previous finding that aspirin has effects upon the quantitaffi-e electroencephalogram like those of drugs having minor tranquilizing or antianxiety behavioral effects cvas confirmed. None of the other drugs had any significant similar effect. The present study shocvs, however, that individual subjects mac• react quite differently, depending upon their initial behavioral state before the drug is given. It is possible that these differences may provide a useful basis for classifying people according to their initial states and their electroencephalographic reactions to drugs. It is also quite likely that failure to take the biasing effects of initial states into account has contributed greatly to the production of conflicting and confusing reports in the field of clinical psychopharmacology. Other support: U. S. Public Health Service. 10. Multilead time-series frequency analysis of the EEG effects of thiopental in psychotic and nonpsychotic men H. B. Murphree and R. E. Schultz i Neuropharmacology Section, New Jersey Bureau of Research in Neurology and Psychiatry, Princeton, New Jersey Pharmacologist 9:212, 1967 Psychotic and nonpsychotic male subjects were given thiopental, 1.5 mg/kg by rapid intravenous TIMN 0115928 T200419 184 a

S r 41 ,- s if ,f ~f e I the drug induced a decrement of performance in the strains in which a facilitating effect was evident. 50. Genetic aspects of learning and memory in mice Daniel Bovet. Filomena Bovet-Nitti, and Alberto Oliverio DNpartment of Pharmacology. University of Sassari. Itoh•, and the University of California School of Atedicine. Los Angeles. California ti, ;ence 163:139-149. 1969 One of the most controversial issues in biology >ince the time of Locke and Leibniz has been that of the role exerted by heredity and environment-what is inborn and what is acquired. The authors ex- tensively review their own studies on mice as they relate to this problem. The findings reported until now and in the present paper are said to suggest that there can be no clear-cut determination of the role of the environmental factor modifying learning and retention aptitudes. In a general way their findings show that adaptive behavior, less that emotional behavior, is subject to the influences of environment. As an answer to a question which Hull raised con- cerning the role exerted by the genetic factors in the determinationof the individual variability in adaptive behaeior. the findings reported here illuminate the important role exerted by heredity in the deter- mination of learning and retention mechanisms. Uther support: Italian Consiglio Nazionale delle Ricerche and a USPHS general research support grant to the University of California, Los Angeles. 51. Comparison of behavioral effects of nicotine, (l-amphetamine, caffeine and dimethylheptyl tetrahydrocannabinol in squirrel monkeys t' R. A. Davis. C. J. Kensler, and P. B. Dews t.ife Sciences Division. Arthur D. Little, Inc., Cambridge, ?Inssachusetts. and Karvard Medical School, Boston, Wassach usetts PsYchopharmacologia 32:51-65, 1973 The effects in squirrel monkeys of nicotine, d- amphetamine, caffeine and dimethylheptyl tetrahy- drocannabinol were examined in three standard behavioral procedure: Fixed Interval, Fixed Ratio and Continuous Shock Avoidance, and two pro- cedures developed to test neuromuscular perfor- mance: Physical Activity and Steadiness. Nicotine increased responding in the first half of Fixed Intervals and in Continuous Avoidance; d-amphet- amine increased responding under all procedures except Physical Activity; caffeine increased re- sponding under all procedures except Fixed Ratio and dimethvlheptvl tetrahydrocannabinol increased responding under all procedures except Continuous Avoidance, where responding was reduced. Nico- tine and d-amphetamine caused disruption in the Physical Activity procedure. Thus the different pro- cedures revealed different aspects of the behavioral effects of the drugs. Findings are consistent with interpretation that it is the temporal pattern of the responding under the different procedures that is the dominant factor in determining the behavioral ef- fects of the drugs. 52. Effects of nicotine, nicotine monomethiodide, lobeline, chlordiazepoxide, meprobamate and caffeine on a discrimination task in laboratory rats I. Geller, R. Hartmann. and K. Blum Department of Experimental Pharmacology. Southwest Foundation for Research and Education. San Antonio. Texas Psychopharmacologia 20:355-365, 1971 Hungry rats were trained on a discrimination task in order to obtain food rewards. During each experimental session, discrete stimuli of 1 minute duration were delivered through a small speaker in the experimental chamber at random intervals on the average of once every 2 minutes. Lever press responses in the presence of a light and tone were correct and produced food rewards. Lever responses in the presence of the light stimulus were incorrect and were punished by total inactivation of the experimental chamber. Rats were selected for this experiment based on their inability to acquire the discrimination task even after six months of training. Administration of nicotine, lobeline, chlordiazepox- ide and meprobamate produced an improvement in discrimination performance through a reduction of responses to incorrect stimuli. Caffeine and nicotine monomethiodide, a quaternary salt of nicotine, were without effect on the discrimination. ' This study was presented in part at the 77th Annual Convention of the American Psychological Association, 1969, and appears in the Proceedings, pp. 867-868. TIMN 0115941 T200432 197

; te ,re us :njet.tiun- anti their FEGs were recorded with fre- ,uen<<• modulatiun „n cna,netic tape for later analv- .is from the frontal, parietal. and occipital areas of :,oth hemispheres (monopolar) for 15 min before and 'U min after dosage. The methodologies for record- in, and analysis have been described previously i tn1t .\'I' Acad Sci 142:245. 1967). The results (unfirm the differences observed earlier between patients and nonpsychotic subjects. Also the various arNas of the cortex were affected in different cvays by the drug. Fast activity ("barbiturate buzz") occurred snost prominently in the frontal areas: alpha. when l>rE-,ent. was more prominent in the occipital areas: :ltnw activity occurred diffusely over the entire (.tartex. This amounts to quantitative confirmation of ,ome earlier clinical and semiquantitative observa- tions of the actions of thiopental. In addition. some interhemispheric differences in the amount of elec- trical energy in the alpha band after the drug were noted. which may be related to cerebral domi- nance. Other support: U. S. Public Health Service. 11. Comparative study of EEG effects of antihistamines in normal volunteers i.-mi,it• Goldstein. Henry B. Murphree. and Carl C. l'friffer \',uropharmacologc• Section. New fersel- Bureau of h,•.rnrch in \'eurologr and Psychiatry. Princeton. Vew L•n?t- b,arnal of Clinical Pharmacology 8:42-53, 1968 Seven drugs used as antihistamines were ad- ministered to 65 nonpatient volunteers in a partial t rnssover study design. On the basis of drug group -!,ita. it appeared that diphenhydramine and pro- methazine produce locv-energysedation (decrease in i!"(; aniplitude with increase in its time course •.,iriabilitv). Chlorpheniramine, diphenhydramine, ,)hrnindamine. tripelennamine, and a necver agent tr itadine. bring about increases in both EEG am- ,>litude and time course variability (high-energy ,wdation). Placebo does not produce any detectable ,fft!ct. Individual scoring of the EEG states of the Subjects revealed a very high variability in response both within and between drugs. No associations between predrug status and drug effect could be ti emonstrated. The state referred to as hiQh-ener~~• sedation was shown. on the basis of frequency analc-ses. to be due to increase in the electrical activity at both low- and high-frequenc~- bands. with minimal c:han~f, in the alpha range. Other support: U. S. Public Health Service. 12. Effects of high congener intake by human subjects on the EEG Henry B. Vlurphree, Robert E. Schultz. and Allan G. Jusko Department of Psvchiatry. Rutgers Medical School. and the Center of Alcohol Studies. Rutgers C'niversiti•. .\'en- Brunsccick. \'e~r ferseY Quan:erh- fournal of Studies on Alcohol. Supplement 5:50-61. 1970 Previously reported studies of the effects of alcoholic beverages on the electroencephalogram (EEG) are briefly reviewed. Some preliminary and tentative findings resulting from a new technique of ' time-serial frequency analysis of the EEG are also reported. Baseline EEG recordings from 3 scalp areas of both hemispheres were made in 10 healthy, light to moderate drinkers (2. women) aged 21 to -t0, after which they drank randomly either bourbon or vodka in amounts providing 0.8 g of alcohol per kg of body weight, superbourbon providing 0.1 or 0.2 g of alcohol per kg and 4 or 8 times as much congeners in the same proportions as in bourbon, or orange juice placebo. Recordings were repeated at hourly in- tervals for 6 hours. In some subjects who had large amplitude alpha before alcohol ingestion, bourbon produced a fast (beta) activity in the frontal areas (between 16 and 28 cycles with peaks at 23 per second). This activity was not seen in the parietal or the occipital areas. No reciprocity occurred between alpha-band frequen- cies and "fast" activity in the frontal areas. A quantitatively unique but not uniform effect was noted after superbourbon. After the lower dose of superbourbon (yielding a blood alcohol level of about 15 mg per 100 ml) the spectra'were initially almost indistinguishable from baseline recordings: occipitals showed moderate alpha, parietals re- flected this in muted form and the frontals presented no characteristic peak frequency activity. During succeeding recordings the alpha amplitude dimin- ished and prominant activity appeared in the par- ietals, peaking at 8.5 per sec. The increased variabil- T](mN 0115929 T200420 185

Nervous Sc'stem ters of the rat. but not when the drug was introduced at other sites in the hypothalamus. Systemic in- jection of atropine prPv~ented the hy-pothermic re- sponse to subsequently injected acetvIcholine. These results support the concept that central con- trol of body temperature involves cholinergic re- ceptors in the thermoregulatory centers. Other support: U. S. Public Health Service. 79. The effect of nicotine on thermosensitive units in the rostral hypothalamus G. V. Knox. and P. Lomax Department of Pharmacology. School of Medicine and Brain Research Institute. University of California. Los Angeles. California Proceedings of the Western Pharmacology Society 15:179-183. 1972 Nicotine has been shown to cause hypothermia following systemic injection in the rat and this effect has been reproduced by intraventricular administra- tion of the drug. The present study in rats was undertaken to determine the site and mechanism of action of nicotine on thermoregulatory function. The data obtained indicate that the cholinergic receptors in the thermoregulatory centers respond to nicotine and this leads to a fall in body temperature. The temperature changes are mediated, at least in part, by increased heat loss by vasodilation. There is a rapid development of tolerance to this response and a second injection of the drug is ineffective 2 hr to 13 davs later. A rise in core temperature fre- quently occurs in tolerant animals receiving a sub- sequent dose of nicotine; this response is similar to that seen after injection of cholinergic blocking agents into the thermoregulatory centers. The onset of peripheral vasodilation appears to be a more significant and reliable test of the drug effect than the accompanying small fall in core temperature. The latter is a function of both the intensity and duration of the response and tends to be small when the duration is short. The increase found in firing rate of hypotha- lamic units in response to thermal stimulation of the skin (tail) may represent the neural correlate of a lowering of the set point which would be expected to occur when the animal is exposed to a high environmental temperature. Infusion of nicotine into the rostral hypothalamus also increased the fre- quencc• of these same units. W'ith continued infusion of nicotine the firing rate of the units gradually declined to a level belo«• that of thecontrol period. This phenomenon may be analogous to the blockade which follows the initial stimulation when nicotine is applied to peripheral autonomic ganglia. This blockade could not be overcome by subsequent thermal stimulation of the skin. These data indicate that central cholinergic receptors are sensitive to nicotine and that in the case of the thermoregulatory centers the drug causes changes in the hypothalamic set point. Other support: U. S. Public Health Service. 80. The effects of acetylcholine and nicotine on unit activity in the hypothalamic thermoregulatory centers of the rat G. V. Knox, C. Campbell, and Peter Lomax Department of Pharmacology, School of Xtedicine and the Brain Research Institute, University of California. Los Angeles. California Brain Research 51:215-223, 1973 Multiple and single unit activity were recorded in the rostral hypothalamus of rats anesthetized with urethane. Units which increased their firing fre- quency in response to radiant heating of the animal's tail also exhibited an increase when acetvlcholine or nicotine were injected locally or when nicotine was administered systemically. In the case of nicotine the increased frequency was transient and was followed by a prolonged period in which the firing rate was below that of the control period. It is suggested that the frequency change in these neu- rons may represent the neuronal correlate of the downward setting of the hypothalamic thermostat that occurs when the animal is exposed to elevated environmental temperatures. This resetting appears to involve a cholinergic receptor that can be ac- tivated by exogenous cholinomimetics and such a lowering of the set point could explain the hypother- mic effect of these drugs. The response of, this receptor to nicotine resembles that of cholinergic receptors in peripheral autonomic ganglia in that initial stimulation, followed by blockade, occurs. Other support: U. S. Public Health Service. TIMN 0115950 206 T200441

\ert'rtu5 5\'Stetri rariuus ;ubst<tn(t'-; 3.5)1. Nic:otine causes an_ initial t.(epularizatitln ,tnd a ~ubsequent repolariza- tion of the endplate membrane. The actions _ of lobeline more closely resemble those.of tubocurarine rather than nicotine (132). There is further_ support for the notion that lobeline has an antinicotine action (130). C. .\'irntine Derivatives Studies of nicotine derivatives are of importance for both practical and theoretical reasons. A number of necc quaternary products of nicotine have been s}'nthesized (136. 137). Pharmacological investiga- tions of these compounds reveal a wide spectrum of alterations of nicotine actiritc• at the neuromuscular junction as well as in whole animal studies (138). D. ['iral Cheniotherapeutic Encephalomyoc.arditis virus infection in mice and tissue cultures is reduced by a water soluble and Pthanol-insoluble fraction of tobacco leaf, of ciga- rettes and tobacco smoke (139. 140). None of these extracts acted to induce serum interferon although the ci¢arette smoke agent did protect mice from encephalomyclcarditis by pretreatment. E. F D.rmrtw. \[. D.. Professor of Pharmacology. The Cnicersitr of Mic.hiqan \ledir.al School. prepared the summarc of this Section on The \r•nuus tik,tenu. Dr. Domino. a iong-time colleague of Dr. Maurice H. Sew rr,. ,rr reptPd this task following death of Dr. Seevers. A. Central Nervous System 1. Cigarettes and the college freshman Dorothy F. Dunn DPpnrtment of Health Science. Student Health Center. t'nirersitr of Illinois. Urbana. Illinois Journal of the American Medical Association 199:19-22. 1967 In January 1965 at the University of Illinois, Urbana, from information selected from the student data system and questionnaires, 102 variables were obtained on selected demographic, socioeconomic. attitudinal, and academic characteristics and the smoking habits of 3.567 freshmen enrolled in Rhet- oric 101. The difference between smokers and non- 182 smokers was significant at the 0.001 level of probability for the following variables: freshmen approval of teenage smoking. academic achieve. ment. organization membership and use of leisure time. belief that lung cancer was more likelv to develop in smokers than in nonsmokers, smoking habits of parent. place of residence when in high school. smoking habits of campus roommates. and belief that teenage smoking is a public health problem. Sixty percent of the freshmen did not smoke. and 43% of those who smoked wished to discontinue. 2. Smoking-Academically speaking! Dorothy F. Dunn Department of Health Science. Student Health Center. trniversitr of Illinois. Urbana. Illinois Journal of the American College Health Association 15:162-167. 1966 The population studied was the same as described in the preceding article (D. F. Dunn, JAMA 199:19. 1967) and findings presented are essentially the same. The number of the smokers in the group whose grade-point average was below middle C was above the average for the group as a whole. The same was also true in other areas of academic recogni- tion. 3. Cigarettes and the college senior Dorothy F. Dunn School of Public Health, University of Illinois at the Medical Center, Chicago. Illinois Journal of the American College Health Association 21:224-226, 1973 Information for Phase II of the author's study was obtained through questionnaires mailed to the 1,831 students surveyed as freshman in Phase I, who were still on the Urbana campus four years later. From the data obtained, the smoking profile of the 1968 senior respondent who smoked is summarized as follows: Over 50 percent had been smokink a pack or more of cigarettes a day. Approximately the same number had been smoking this amount for over a year. In Phase I, 27.7 percent had smoked a pack or more a day. However, fewer smokers in Phase II inhaled. TIMN 0115926 T200417 Stt <t t) t,t Ph S[r Pfi ,[tl nlt c i_ ill tin tht -t. t p ri Dot .5't •1 Wo lou 22: isti l' n tiilc ill !3o tinl t he t)r,t hat dnc inj[ mil fro Co[ the ing a re of tho (x) r not

slices from rat brain striatum. h~Ipothalamus. cortex and cerebellum were studied. The uptake of 1}C- nicotine is not dependent upon Na- present in the incubation fluid because a K--substituted (0-Na-) solution increased the "C-nicotine tissue space, a tris-substituted (0-Na-) solution decreased the "C- nicotine tissue space and a sucrose-substituted l0-Na-) solution did not change the amount of "C- nicotine taken up when compared to the'}C-nicotine tissue space obtained in a normal incubation so- lution. However, all three Na-free solutions elicited a sustained decrease in 14C-nicotine efflux. The increase in "C-nicotine space produced in a K-- substituted (0-Na-) solution was present in the slocti•er component of a two-component washout, whereas the decrease produced in a tris-substituted (0-Na-) solution produced an equal percentage de- crease in the size of both components. N1ost of the observed effects could be attributed to a linear relationship between the logarithm of the intracellu- lar K- concentration and the 14C-nicotine tissue space. In conclusion, it appears that there is an intracellular binding site for nicotine and that the degree of binding is dependent upon the concentra- tion of K-. 68. Effects of pharmacological agents on [14C]- nicotine distribution and movements in slices from different rat brain areas %t. T. Alderdice, and G. B. Weiss Department of Pharmacology, University of Texas Southwestern Nfedical School, Dallas, Texas Neuropharrnacol ogy 14:811-817, 1975 The effects of various pharmacological agents on [t;C]-nicotine accumulation, distribution and movements in rat brain cortex, striatum, hypothala- mus and cerebellum slices were studied. When present during incubation, mecamylamine (2.0 mM) decreased the ["C]-nicotine tissue space and both mecamylamine and nonradioactive nicotine (0.62 mM) acted primarily to decrease the amount of ['aC]-nicotine in the slower component of a two- component washout of [14C]-nicotine. Procaine (3.67 mM) also decreased [14C]-nicotine uptake; this decrease occurred almost exclusively within the slower component. Lobeline (0.62 mM), physostig- mine (1.0 mM) and, to a lesser extent, metanephrine (1.0 mM) also decreased the [14C]-nicotine tissue space in a competitive manner. No direct interaction with various cholinergic agents was demonstrated. f When added during washout of ["C]-nicotine, both mecamvlamine and procaine increased ['}C)-nico- tine efflux. It appears that the agents employed compete with ["C]-nicdtine for cellular binding sites or. in the case of procaine, might possibly also inhibit access of ['aC]-nicotine to the intracellular sites. 69. On 14C-nicotine distribution and movements in slices from monkey cerebral cortex M. T. Alderdice, and G. B. Weiss Department of Pharmacology, Universitv of Texas Southwestern Medical School, Dallas, Texas Archives Internationales de Pharmacodvnamie et de Therapie 209:162-171, 1974 Evidence obtained in this study indicates that the uptake and distribution of "C-nicotine in ca- puchin monkey brain cortex slices is both qualita- tively and quantitatively similar to "C-nicotine dis- tribution tribution and uptake values previously reported for rat brain area slices. Tissue spaces for 14C-nicotine increased as the extracellular pH or the temperature was raised; this additional accumulation could not be explained solely by a passive distribution in accord with either the membrane pH gradient or a diffusion-limited process. Alternatively, accumula- tion of a portion of the 14C-nicotine taken up in monkey cerebral cortex slices may be due to an energy-dependent process similar to that present in rat brain area slices. 70. Characterization of [1'C]-nicotine accumulation and movements in slices from different rat brain areas G. B. Weiss, and M. T. Alderdice Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas iv'europharmacology 14:265-273, 1975 The effects of pH and temperature on the uptake and cellular distribution of inethyl-[14C]-labelled nicotine were examined in slices prepared from rat brain cortex, striatum, hypothalamus and cerebel- lum. At incubation intervals of 30-60 min, ['4C]- nicotine uptake values at pH 7.20 were similar at 37°C and 0°C, whereas at pH 8.20 and 37°C the ['4C]- nicotine tissue space was 2.4-fold higher than that TIMN 0115947 T200438 203

Nert•ous System Extracellular space values were determined with either :4C-suc:rnse or ',C-inulin at different_ incubation time intervals (30 and 60 min) in rat brain slices prepared from the cerebellum, cortex, striatum and hypothalamus. At both time intervals and in all brain area slices. "C-sucrose spaces were larger than "C-inulin spaces. Efflux of "C-sucrose but not of inulin could be resolved into two linear components with halftimes of 1.6 and 15.0 min. Efflux of '=:V`a from each of the different brain areas also could be separated into two distinct components, the slower of which was increased bv ouabain. Similarities between 22Na and '{C-sucrose movements indicated that there is a cellular accumulation of sucrose but not of inulin. An accurate estimate of functional extracellular space in brain slices could be derived from either the "C-inulin space or the 1iC-sucrose space corrected for cellular uptake. Values obtained range from approximately 12 ml/100 g (cortex) to 20 ml/100 g (cerebellum). 65. Drug-induced alterations in respiration of rat brain cortex and striatum slices in a carbon dioxide-bicarbonate-buffered medium George B. Weiss, Leif Hertz, and Frank R. Goodman Department of Pharmacologc•. University_of Texas Southwestern Medical School. Dallas. Texas Biochemical Pharmacology 21:625-634. 1972 Rates of oxvgen utilization were measured, with an oxygen electrode in rat brain cortex and striatum slices. Values obtained for respiratory rates in cortex (approximately 85 µ moles/hr/g wet weight) and striatum (about 43 N. moles/hr/g) were similar in both bicarbonate and tris-buffered solutions. Addition of potassium chloride (to 70 mM) elicited an increase in oxvgen utilization in both preparations. Exposure to procaine (10 mM) reduced the respiratory rate to levels substantially below control values and pre- vented the K--induced increase in respiratory rate. Pentobarbital sodium (1 mM) and, perhaps to a lesser extent, caffeine (5 mM) also decreased the control respiration rates and inhibited the K'- induced stimulation of respiration rates. Nicotine (0.67 mM) had no effect on control or K*-stimulated respiration rates in cortex or striatum slices. Effects of procaine appeared to be additive with those of caffeine and pentobarbital, and are probably attained by different cellular mechanisms. Observation of similar respiratory rates in bicarbonate and tris solutions also suggest that correlations mav be attempted between brain tissue respiration studies in CO,-free media and measurements of ionic pa- rameters in the presence of bicarbonate-buffered solutions. 66. Dependence of "C-nicotine accumulation on intracellular K+ in rat brain area slices Marc T. Alderdice, and George B. Weiss Department of Pharmacology. University of Texas Southwestern Medical School, Dallas, Texas Federation Proceedings 34(3):346, 1975 The effects of variations in Na- and K- con- centrations on '4C-nicotine distribution were ex- amined in order to obtain information about the mechanism by which intracellular accumulation of "C-nicotine occurs. Substitution of portions of ex- tracellular Na` with equimolar concentrations of tris buffer or addition of 60 mM tris decreased 1-0C- nicotine uptake, whereas a K'-substituted (O-Na) solution increased 14C-nicotine uptake and a su- crose-substituted solution was without effect. Ob- served changes in'}C-nicotine distribution could not be explained by variations in intracellular pH and in the membrane pH gradient. The increased "C- nicotine uptake elicited with high K' was blocked by mecamylamine or procaine; both inhibitory agents prevent intracellular accumulation of "C- nicotine. The intracellular K' concentration, (K-]i, was increased in a K* substituted (O-Na) solution and decreased in a tris-substituted (O-Na) solution. The uptake of'{C-nicotine can be linearly correlated with the log [K]j. Thus, intracellular accumulation of 14C-nicotine appears to depend upon [K`]i and may result from K+-induced alterations in the binding capacity of a subcellular depot. 67. Ionic basis for intracellular 14C-nicotine accumulation in slices from different rat brain areas M. T. Alderdice, and G. B. Weiss Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas ' Archives Internationales de Pharrnacodynamie et de Therapie 218:252-267, 1975 The effects of ionic alterations on the accumula- tion, distribution and movements of 14C-nicotine in ' T1MN 0115946 T200437 202

f r 1 1 , t t ; f t 16. Abstinence effects in smokers H. B. Murphree, ana R. E. tit hultz \..,, JersNc Bureau o.t Research in Neurolo4e-and ;>;„ niatrY. Princeton. Vecr Ierset' r:,.,lrnnhon Prnceedings 27, part 1:220. 1968 \ possible reason ~chv smoking is c-erv difficult tc, ;i~~e up is that abstinence produces anxiet~~ as an ,spet~.t of a subtle but nonetheless compelling c~.•ith- ,Ir,iwal syndrome. To test this possibility 11 moder- at,• to heavc smokers took part in 37 trials. Each trial hr;an at 8:30 .~.~t. Subjects had a light. low fat hroatkfast without caffeine one and one half hours e,irlier. They were allowed to smoke ad lib. until the beginning of the experiment, then were deprived for h hours. The following were recorded hourly: 10- minute quantitative EEG. supine blood pressures ,ind pulse rates after lving down 10 minutes for the E:E:G. and orthostatic blood pressures. Placebo or mc'probamate. 100 mg, or no medication was given immediately after initial baseline recordings. The IPAT 8-Parallel-Form Anxiet_v Battery was also ad- ministered hourlv. After the 6 hour recording, sub- ivi ts smoked a cigarette in 10 minutes; inhaling tI,-oplv. Then another set of recordings was made to ,Iotermine the effects of resumption of smoking after tI,-privation. No relationship was found between ,1,-privation and anxiety as measured by the IPAT. AI,;tinence caused reduction in blood pressures and ,Iu("' rates which returned to or overshot baseline t.,luws on resumption of smoking. Changes in EEGs %%'vre most prominent when the_ subjects resumed 'nu,king. Medication did not antidote the depriva- ',-n or resumption effects. 17. EEG study of caffeine and amphetamine as possible surrogates for smoking in humans f1. B. Murphree. R. E. Schultz. and A. G. Jusko : i. t,,,rtment of Psrchiatrv. Rutgers ?ldedical School and Center of .~Icohol Studies. Vew Brunswick, N.J. %'l:urmncolvaist 12:207. 1970. The present work was planned to assay two .timulants as surrogates during abstinence. Eight snu,kers were deprived for 6 hours. After a baseline t0-minute recording, they were given decaffeinated , ~)ffee plus lactose tablets, or 250 mg of caffeine Na h-nzoate was added to the coffee, or the tablets t.nntained 5 mg d-amphetamine, according to latin- square double-blind procedure. Then recordings were made hourly. Caffeine was ineffective and amphetamine doubtfullv effective in reversing the EEG depression. 18. Effect of acute deprivation of smoking on aggression and hostility Martin D. Schechter and Michael J. Rand Department of Pharmacology. University of Melbourne. Parkville. Victoria. .-t ustralia Psychopharmacologia 35:19-28. 1974 Smokers (12) and non-smokers (1-1) were tested on the Buss aggression-machine to determine their aggression scores and visual reaction times during two sessions. The mean aggression scores of the non- smokers did not differ significantly between ses- sions whereas there was a significant increase in aggression scores for the chronic smokers in their non-smoking (deprivation) session compared with their smoking session. This increase in aggression in deprived smokers is discussed as a factor in the continuance of the smoking habit and is positively correlated to rated hostility scores on the Buss- Durkee Hostility Inventory. 19. Effects of selection on mortality Carl C. Seltzer, and Sec•mour fablon Harvard School of Public Health. Boston, Nifassachusetts, and Ivtedical Follow-up Agency. National Academy of Sciences .Vational Research Council. bVashington. D.C. American Journal of Epidemiology 100:367-372, 1974 The effect of the bias of medical selection on subsequent mortality by cause, was examined in a series of 85,491 white male World War II U.S. Army veterans who have been followed for a period of 23 years, 1947-1969. The results indicate that the selection process for military service exerted a profound effect on mortality of Army veterans after separation from service. Generally, the mortality rates are well below those of the general population within the first few years of discharge, but thereafter gradually approach those of the parent population. The effect of the bias of selection on, subsequent mortality rates may persist even after 23 years from Army discharge. The effect of the bias varies con- siderably according to the nature of the cause of death. TIIVIN 0115931 T200422 187

Nen'ous Svstem 53. Effects of nicotine on the exploratory behavior of female rats John A. Rosecrans Department of Pharmacology. Medical Collese of Virginia. Richmond. Virginia Pharmacologist 11(2):246, 1969 The effects of various doses (200-800 µg/kg; s.c.) of nicotine (N) on exploratory behavior were studied in CD rats of both sexes. Naive CD rats were initially depressed (0-15 min), while a secondary stimulation of activity was observed in females (15-45 min). ale rats remained depressed throughout. The ini- M tial depression induced by N in female CD rats was reduced or reversed if rats were presented a single training session prior to a dose of nicotine. Similar effects were observed in female rats selected for High (H) or Low (L) Activity (A) with the latter group more responsive to N's excitatory effects. 400 µg/kg (N) was the most effective stimulatory dose, and was also most effective in facilitating avoidance behav- ior. This dose induced a maximal stimulation of exploratory behavior following three daily injec- tions. This latter effect was observed in four different experiments, and apparent tolerance appeared by the 5th dose: H. A. female rats also appeared to be more responsive. The results obtained indicated that N allowed rats to more easily habituate to a novel environment, and was somewhat effective in reg- ulating exploratory behavior. 54. Brain area nicotine levels in male and female rats of two strains J. A. Rosecrans, and M. D. Schechter Department of Pharmacology, Medical College of Virginia. Richmond, Virginia Archives Internationales de Pharrnacodynamie et de Therapie 196:46-54, 1972 The relationship between brain nicotine levels and behavioral effect in different rat populations has been investigated. Male and female CD (Sprague- Dawley) and CDF (Fisher) rats were given several doses of nicotine (400 µg/kg, subcutaneously) and brain area drug levels were determined following behavioral (activity) study. Nicotine reached maxi- mum levels in each brain area within 20 minutes of a single dose in both sexes and strains. These levels were maintained over a 40 minute period at which time they had declined by 1/3 of the peak con- centration. Females of both strains accumulated more nicotine than similarlv treated males, and were also more sensitive to its behavioral (stimulatory) effects. Inter-strain variability was minimal, but CDF rats appeared more sensitive to the behavioral effects of nicotine, while drug levels were generally higher in the CD strain. Variability between sex and strains was somewhat dependent upon the brain area studied and the rank order of nicotine accumulation from highest to lowest appeared as follows: Dien- cephalon > Telencephalon > Brainstem (minus cerebellum). It is suggested that these differences in brain area nicotine distribution could be related to such pharmacological processes as drug metabo- lism, absorption and/or cerebrovascular circulation. Furthermore, sex hormones and pituitary-adrenal function are suggested as possibly playing predomi- nant roles in affecting these processes. Few significant relationships were obtained br,- tween drug level and behavior, and it is suggested that better behavioral methodology must be devised before such correlations could be obtained. 55. Brain area nicotine levels in male and female rats with different levels of spontaneous activity J. A. Rosecrans Department of Pharmacology, ,1,fedical College of Virginia, Richmond, Virginia Neuropharmacology 11:863-870, 1972 The present investigation was designed to study brain area nicotine distribution in an attempt to determine why, in previous research, rats of differ- ent activity levels were differentially affected by this drug, at least insofar as its effects on behavior and 5- hydroxytryptamine turnover were concerned. Brain area nicotine levels were evaluated in male and female CD strain rats grouped according to different levels of spontaneous activity; drug levels were studied 15 and 30 minutes following the subcuta- neous administration of 400 µg/kg of nicotine. The results obtained indicated that there were few sig- nificant differences between experimental groups, but whether high or low active rats were analyied, brain area nicotine levels were higher in the female rat. However, these differences also appeared to be related to other factors such as whether each rat received a single dose or was dosed on five suc- f 198 TIMN 0115942 T200433

N'ern•ous Svstem aromatic monoamines on the same tissue section is described. The use of aqueous solutions is com- pletelr avoided. thus eliminating artifacts due to diffusion and dislocation of the labeled water-solu- ble drug and endogenous amines. The combination of these two techniques provides a means for obtain- ing data hitherto unavailable to correlate the site of binding and/or storage of water-soluble drugs with monoaminergic nerve cells and terminals. Other support: U. S. Public Health Service. and institutional support (V.A.). 85. Brain amine changes in stressed and normal rats pretreated with various drugs J. A. Rosecrans Department of Psychiatry. Yale University. Vecc Haven. Connecticut. and the Department of Pharmacology. Medical College of Virginia. Richmond. Vi 6inia .-lrchires lnternationales de Pharmacodynamie et de Therapie 180:460-470. 1969 Rats pretreated with various drugs (p-chloro- phenvlalamine. pargyline, a-methyl-p-tyrosine, pro- benecid) plus saline controls, were subjected to an acute horizontal oscillation stress for one hour. Following stress. the animals were sacrificed and 5- hvdroxvtryptamine (5-HT) metabolism and norepi- nephrine (NE) concentrations were determined in two brain areas, the forebrain and midbrain + hindbrain. The results of these experiments indicate that during this stress NE neurons were activated; while 5-HT sr_ stems were concurrently depressed. 5- HT srstems. however, were activated if this amine was depleted prior to stress, indicating the presence of a 3-HT negative feedback system. Lastly, there were differences observed in brain area responsive- ness to stress. indicating the existence of different neuronal populations in the areas studied. 86. Effects of acute stress on forebrain 5-hydroxytryptamine metabolism and pituitary adrenal function John A. Rosecrans Department of Pharmacology. Medical College of Virginia, Richmond. Virginia- European Journal of Pharmacology 9:170-174, 1970 %fale rats were subjected to either of two types of stress, one hour horizontal oscillation or passive avoidance. The former stress appeared to inhibit 5- hydroxytryptamine (5-HT) turnover, but the severity ~.~-........,_...._.._..._...~. of this stress may have prevented a true analysis of these results. To understand better the dvnamics of the relationships between 5-HT systems and stress, male rats were paired according to equivalent spon- taneous activity (curiosity), and were subjected to a more subtle stress of passive avoidance (one trial learning). This procedure consisted of determining how much time a rat would spend in a cage in which it had been shocked 24 hr previously. Pituitart•- adrenal function was stimulated in rats who had learned this response, while non-learners exhibited no such change. In contrast, forebrain 5-HT me- tabolism was reduced in learners and stimulated in nonlearners. No conclusive explanations can at pres- ent be offered, but it does appear that this latter procedure may be useful in studying behavioral and chemical correlations of fear in rats. These studies also present further evidence of the complexity of the indoleamine response to acute stress. 87. Differences in brain area 5-hydroxtryptamine turnover and rearing behavior in rats and mice of both sexes John A. Rosecrans Department of Pharmacology. Nfedical College of Virginia. Richmond, Virginia European Journal of Pharmacology 9:379-382, 1970 Rats and mice of both sexes were compared as to rearing behavior and 5-hydroxytryptamine (5-HT) turnover in forebrain and areas caudal to this. Turnover rates were calculated from changes in 5- HT metabolism in animals pretreated with the monoamine oxidase inhibitor, pargyline (75 mg/kg i.p.). The results indicated that the female of each species has both a more functional 5-HT system and a higher rate of rearing. These data were interpreted on the basis that 5-HT systems normally depress the ability of an animal to habituate to a novel en- vironment. 88. Forebrain biogenic amine function in high and low active female rats John A. Rosecrans I Department of Pharmacology, Medical College of ' Virginia, Richmond, Virginia Physiology and Behavior 5:453-458, 1970 Female rats selected for High (H) or Low (L) Activity (A) were administered the amine synthesis r TIMN 0115952 T200443 in tnu t h, di, nu Tl all pt it\ fe .1C ~.• hf 8i bi Sf Jo D, [' F.i c t 208

\er-,•ous System pared from rat brain striatum and hypothalamus. Nicotine inhibited the uptake of both "C-labelled norepinephrine and dopamine only in hvpothala- mus slices. Under similar incubation c_.onditions. washout experiments « •ith striatum and hypothala- mus slices indicated that nicotine decreased the amount of '{C-\E and 14C-DA present in the slow washout component of the hypothalamus but did not alter "C-labelled contents in the striatum. Ad- dition of 0.62 m.1vl nicotine produced a sustained increase in ''C-NE efflux from brain slices: the presence of desipramine or the absence of either sodium or calcium ions in the washout solution did not alter this nicotine-induced increase. These find- ings support the hypothesis that the nicotine-in- duced release of norepinephrine in the hypothala- mus occurs by a specific and direct action at cellular amine binding sites or stores. 96. Effects of different potassium ion concentrations and of procaine and pentobarbital on ['4C] glutamate fluxes in rat brain-cortex slices George B. Weiss. and Leif Hertz Department of Biochemistn• A. University of Copenhagen. Denmark Biochemical Society Transactions 2(2):274-277. 1974 The effects of elevated K- concentrations as well as of procaine. pentobarbital and nicotine on the fluxes of "C-glutamate in rat brain cortex slices were investigated. As the [K-] is increased (from 5 mM to 35. 55,85 and 125 mM), the 60-90 min'}C-glutamate tissue space is decreased. Rate of "C-glutamate uptake is not altered by high K', but efflux is markedlv increased. Procaine decreases "C-gluta- mate efflux in both control and high K' solutions by equivalent amounts whereas pentobarbitaJ specifi- callv inhibits the high K--induced increase in '4C- glutamate efflux. The effects of high K- can be attributed to a decreased binding or retention of '-'C- glutamate within the cells. Lack of effect of high \'a-. 15 m.%l Cs- or up to 6.7 mM nicotine on '*C- glutamate fluxes as well as lack of specificity of procaine on effect of high K- indicate a lack of correlation of glutamate fluxes with effects on de- polarization, 0, uptake or swelling. 97. Effect of central catecholamine depletions by 6-hvdroxvdopamine on morphine antinociception in rats Nfar%• A. Elchisak. and John A. Rosecrans Department of Pharmacolo5r. Medical College of ['irginia. t"irginia Commomcealth C'niversitr. Richmond. t"irQinia Research Communications in Chemical Patholo,-t• and Pharmacology 6:349-352. 1973 Rats treated intracisternallv at 2 weeks of age with 6-hvdroxvdopamine, which depleted both brain norepinephrine and dopamine, showed an- tagonism of morphine antinociception 6 weeks later when measured by the hot-plate and tail-flick tests. Antagonism was most marked in the hot-plate test. Rats which were treated with desmethylimipramine plus 6-hydroxydopamine showed preferential and enhanced depletion of brain dopamine, greater an- tagonism of morphine antinociception in the tail- flick test, and complete antagonism in the hot-plate test. These results suggest that a critical level of brain dopamine is necessary for the antinociceptive effect of morphine. 98. Morphine and the electrophysiology of the cingulum: A preliminary study S. Jacobs, and P. Lomax Department of Pharmacology and the Brain Research Institute, University of California, Los Angeles, California Proceedings of the Western Pharmacology Society 15:12-15. 1972 Preliminary studies are reported on the effects of intravenous administration of morphine, heroin and naloxone on the normal firing patterns of the cin- gulate gyrus (area 24) in rats. The results obtained demonstrate that morphine can modify the firing patterns of units in the cingulate gyrus. That this is a specific effect on receptor sites for the drug is indicated by the reversion to the control firing rate when the narcotic antagonist naloxone is adminis- tered. A correlation of these electrical events in relation to the phenomenon of addictive drug- seeking behavior and the withdrawal syndrome is needed. Such studies are of particular value in establishing a neurophysiological model for deter- mining the most appropriate site for anterior cin- TIlbIN 0115956 212 T200447

\ert•nus SA•stem ytimulation tieemin,ly depends less on enhance- ment nf epinephrine ;a>c retion and more on aug- mentation of a hypnthalamopituitarv mechanism. possibly increased ACTH secretion. Other support: U. S. Public Health Serv•ice, the U. S. Atomic Energy Commission. and LTCI-CC%I Research and Educational Foundation. 102. Immunoassay of plasma growth hormone in cats following fasting and administration of insulin, arginine. 2-deoxyglucose and _ hypothalamic extract \orio Kokka. Joseph F. Garcia. \,lerrilc %forgan. and Robert George Department of Pharmacology. University of California Colle;e ot' .%tedicine. Irrine. Latrrence Radiation Laboratorr. C'nic•ersitr of California. Berkeley. and Department of Pharmacoloac•. The Center for the Health Sciences and Brain Research Institute. University of California. Los Angeles. California Endocrinology 88:359-366. 1971 Plasma GH levels were measured in unanesthe- tized cats following various procedures known to stimulate GH secretion in man. The radioimmunoas- sav scstem used in the present study could measure plasma GH in concentrations between 4 and 100 ng/ml. Antiserum to cat GH cross-reacted with purified rat and rabbit GH but did not bind GH or ACTH from man or LH and TSH from sheep. Similarly. pituitary extracts from cat, rat, rabbit and dog inhibited binding of labeled cat GH to its antibodv. whereas human and monkey pituitary extracts had little effect. Immunoreactive plasma GH levels in cats were not changed by insulin hy- poglycemia. fasting. intravenous glucose, arginine infusion or by injection of sheep hypothalamic extract. Plasma GH rose significantly (p < 0.02) only after 2-deox-,•glucose infusion. The present results indicate that GH does not play an active role in the short-term homeostatic adjustments to blood glucose changes in cats. Other support: U. S. Public Health Service, and the [.`. S. Atomic Energy Commission. 103. The effects of intravenous nicotine on blood glucose. plasma corticosterone and growth hormone levels in rats and rabbits Robert George Department of Pharmacologt• and Brain Research Institute. School of ~fedicine. Universih• of California. Los .~1 ngel es. Cal i f orn ia Unpublished Report to the A.%tA-ERF Intravenous nicotine (free base) at a dose of 0.25 mg/kg failed to alter blood glucose and corticoste- rone levels in both rats and rabbits one hour after its administration. A dose of 0.5 mg/kg of nicotine increased significantly the blood glucose levels in rabbits 30 min after injection and they remained elevated for 3 hours; corticosterone levels were significantly elevated but only at 30 min after which time they returned to normal levels. In rats 0.5 mg/kg of nicotine significantly ele- vated blood glucose and plasma corticosterone lev- els, one hour after administration. In an adrenal demedullated group of rats the blood glucose and plasma corticosterone levels rose significantly one hour after administration of 0.5 mg/kg of nicotine while plasma growth hormone levels were unal- tered. These data indicate that nicotine in high doses may elevate blood glucose and plasma corticoste- rone levels and these effects are not mediated via the sympathoadrenal system. Also, the doses of nicotine required to produce hyperglycemia and increased corticosterone secretion were usually convulsant in their action. 104. Effect of intracerebral injection of nicotine and carbachol on corticosterone secretion in the rat Robert George Department of Pharmacology and Brain Research Institute, School of Medicine, University of California. Los Angeles, California Unpublished Report to the AMA-ERF These studies were done to localize the central sites of action of nicotine on corticosterone secre- tion. The injection of 1 µl of a 3 µg doSe into various regions of the limbic-midbrain circuit was examined since this circuit has been implicated in the control of ACTH secretion. It was found that focal injection of nicotine into the ventromedial and posterior hypothalamic areas as well as the amygdala and TIMN 0115958 T200449 214

r,.,rtit'd the rat',; .rbility to discriminate between .-thanl)l and saline. 22. C.N.S. effect of nicotine as the discriminative stimulus for the rat in a T-maze Martin D. Schechter. and John A. Rosecrans ;).•prrrtmvnt of PharmacoloaY. Medical Colle;e of t ir,urrn. Richmond. Virginia t rr. Srienr;es 10. Part L:821-832. 1971. Rats were trained to make a specific behavioral rrspunse for food reward in a T-maze apparatus un(liticinal upon whether they were injected sub- utLtnec,uslc with 400 µg/kg of nicotine or saline. l'his differential response was dose- and time-re- I,rtrci. Pretreatment with 750 µg/kg hexamethonium ;,, I,eripheral ganclionic blocking agent) had no offrc t on the rats' ability to discriminate the cueing ,ffe< t of nicotine. tvhereas. pretreatment with 500 mecamylamine (with peripheral and central !,1r„ i.in,,Y rffec:tsl significantly inhibited this effect of iriccrtine. It appears that whatever the cues were to which the rats differentially responded the effect is m0diated b}• nicotine's action on the central nervous .~ stem. 23. Effect of inecamvlamine on discrimination between nicotine- and arecoline-produced cues `.lartin D. SLhechter. and John A. Rosecrans i),•prrtrnent of Pharmacology. Medical College of t':r,_,inia. Richmond. Virginia la:r•,pttnn lournal of Pharmacology 17:179-182. 1972 Rats were trained to respond differentially in a three compartment shock-escape apparatus condi- tional upon whether they were injected subcuta- nonusly with nicotine. 0.4 mg/kg, or arecoline, 0.5 :nsz: k;. The specific cueing effects of these two hrrlinergic agonists suggests the presence of dis- tiru.t C.\.S. receptor sites of action for muscarinic- ,rnd nicotinic-cholinergic drugs. Pretreatment with nic,c.amvlamine. 0.5 mg/kg, inhibited the nicotine- i nduced cue but had no effect on the arecoline rosponse. 24. Nicotine as a discriminative stimulus in rats depleted of norepinephrine or 5-hydroxvtryptamine Martin D. Schechter. and John A. Rosecrans Department of Pharniacolo;Y. Medirnl College uf ['irginia. Richmond. Virginia Pst-chopharmacolo;ia 24:417-429. 1973 Rats it•ere trained to make a specific behavioral response for food recvard in a T-maze apparatus conditional upon whether they were injected sub- cutaneouslv «•ith 0.4 mg/kg of nicotine or saline. Depletion of brain levels of 5-hvdroxytr~•ptamine bv orallv administered para-chlorophenc•lalanine had no significant effect on the rat's ability to dis- criminate nicotine. However, both insoluble alpha- methyl-p-tyrosine and its ester (which deplete brain norepinephrine) at intraperitoneal doses of 90 and 135 mg/kg, respectively, significantly decreased dis- crimination of nicotine at 180 and 270 minutes post- administration. At these doses and times. saline discrimination was not altered. The experimental evidence indicates that nicotine's CNS cueing effect is mediated by norepinephrine, and this is discussed in the light of the Burn and Rand hypothesis. It is proposed that nicotine may act on a specific nico- tine-sensitive cholinergic receptor in the C\S. which causes release of norepinephrine «•hich. in turn, produces the interoceptive cueing effect that enables the rats to make the appropriate behavioral response. 25. Nicotine as a discriminative cue in rats: Inability of related drugs to produce nicotine-like cueing effect Martin D. Schechter. and John A. Rosecrans Department of Pharmacology. Medical College of Virginia. Richmond. Vi oinia PsYchopharmacologia 27:379-387. 1972 Rats were trained to enter one arm of a T-maze for food reward after the subcutaneous administra- tion of 0.4 mg/kg of nicotine and to enter the opposite arm following the injection of an equal volume of saline. Administration of 0.4 mg/kg tlico- tine isomethonium iodide hydroiodide, 0.4-10.0 mg/ kg lobeline sulfate, 2.0 and 4.0 mg/kg d-amphet- TIMN ®115933 T200424 189

nhihitors p-chlorophenVlalanine (p-CPA) or a- ;;wth% l-p-tyrosine (a-N1PTj. and were subjected to ei- 'her ht'havioral or chemical studies. The data in- ,!ii.itt'd that 5-hcdroxytryptamine systems were ;:u,re functional in the forebrains of H.A. rats, while :,-t.PA ~vas less active behaviorally in these animals. : i•hv reverse situation was evident in L.A. rats in •.,hich forebrain norepinephrine systems appeared m„rt> functional. and a-IMPT was similarly behavior- •i{lk lrss active in these latter animals. These ex- ,wrimt,nts indicate a good correlation between acti.t•- i tv it>cNl and forebrain biogenic amine function in fvmale rats. However. whether these amine systems ,ino directl}- involved in the control of activity, or t%hwther these changes are the results of these lxehaOoral differences has not been clarified. 89. Effects of nicotine on behavioral arousal and brain 5-hydroxytryptamine function in female rats selected for differences in activity lohn A. Rosecrans !)Npartment of Pharmacology, Medical College of t'ir,inia. Richmond. Virginia (•:uropean Journal of Pharmacology 14:29-37, 1971 The effect of various doses of nicotine (200, 400 ,ind 800 N.g/kg, subcutaneously) on exploratory be- !iaoor and forebrain 5-hvdroxytryptamine (5-HT) metabolism were studied in female rats selected for tlifferences in spontaneous activity. The results in- dicated that nicotine affected each activity group differentlv: high activity rats (H.A.) appeared more ,usceptible to the chemical effects of nicotine (200 .ind 400 µg/kg) as 5-HT accumulation in pargyline • inunoamine oxidase inhibitor) treated rats was sig- nific:antlv reduced. while low activity (L.A.) rats •1ppeared more susceptible to behavioral stimulation due to nicotine (200 and 800 µg/kg). The 400 µg/kg .i-ise. which induced both a maximal facilitation of .+rousal levels and a significant reduction of 5-HT Ai.umulation was studied in greater detail, and was libseered to initiate similar effects following re- peated doses. However, in acute situations nicotine appeared to modulate behavior as evidenced by a stimulation of behavior in L.A. rats and a depression in the H.A. animal. Further chemical correlation studies indicated that nicotine (400 µg/kg) reduced 5-HT turnover (as measured using the monoamine oxidase inhibitor, pargyline) to a greater extent in the forebrains of H.A. rats, while caudal (brainstern) areas were inhibited more in L.A. rats. This' in- % vestigation presented evidence which indicated that the nicotine-induced elevation of arousal levels was correlated (at the 400 µg/kg dose) to an inhibition of 5-HT systems. While these data were essentially preliminary in nature, this study does give en- couragement to the concept that nicotine's variable effects on the behavior of animals of different temperaments could be related to its effects on the forebrain 5-HT projection system. Furthermore, this study supports previous research in this laboratory which suggests that there may be a significant relationship between animal temperament and fore- brain biogenic amine function. 90. Effects of nicotine on brain area 5-hydroxytryptamine function in male and female rats separated for differences of activity John A. Rosecrans Department of Pharmacology, Nfedical College of Virginia, Richmond, Virginia European Journal of Pharmacology 16:123-127, 1971 Male and female rats separated according to differences in activity were administered nicotine in daily dose of 400 µg/kg, subcutaneously, for five days, during the first four of which effects on spontaneous activity were recorded in comparison to controls receiving distilled water. Fifteen minutes following the fifth dosings, the rats were adminis- tered the monoamine oxidase inhibitor, pargyline (75 mg/kg, intraperitoneally), and were sacrificed 10 or 60 minutes thereafter for estimation of 5- hydroxytryptamine (5-HT) function (accumulation) in diencephalon and brainstem areas. It was ob- served that 5-HT accumulation was significantly lower in nicotine treated high activity (H.A.) rats of both sexes. These effects were most evident in the di- encephalon of the female, while brainstem areas were similarly affected in the male rat. 5-HT func- tion appeared to be unaffected by nicotine in low activity (L.A.) rats, but there was some evidence that this system was stimulated, especially in the dien- cephalon of the female rat (+ 31%). Correlations between the stimulatory effects of nicotine on activ- ity and 5-HT accumulation in the H.A. rat supported previous research which indicated that nicotine can inhibit this chemical system. Pooled data (H.A. + L.A.) reduced the significance of these findings providing further support for using such preselec- TIMN 0115953 T200444 209

Nervous System obtained at 0°C. Efflux experiments with striatum and hc.pothalamus slices placed the additional amount of [''C]-nicotine present at pH 8.20 and 37~C within the slower washout component. The dis- tribution of ["Cl-nicotine could not be attributed solelv to diffusion and membrane pH gradient at 37°C and pH 8.20: a linear increase in ["Cl-nicotine uptake at 37°C resulted as extracellular pH was increased from 7.20 to 8.20. The ["C]-nicotine up- take was decreased by addition of nonradioactive nicotine (0.62-6.2miv1) and by iodoacetic acid (3mM). Thus there appears to be a component of ['aC]-nicotine uptake which is present in lesser amounts at physiological pH levels and increases as the extracellular pH is raised. Possibly this ["C]- nicotine, bound or stored at intracellular sites, may alter other cellular processes. b. Sensori' SYsterns 71. Effect of nicotine on cochlear function and noise-induced hair cell loss Richard P. Bobbin, and Maria 1. Gondra Kresge Hearing Research Laboratory of the South. Department of Otorhinolar7.-ngology, Louisiana State L'niversitv Medical Center. `'ew Orleans. Louisiana Annals of Otology. Rhinology and Laryngology 85:2-17-254. 1976 Nicotine administered in high doses intra- venously (1 mg/kg) and intracochlearly (10 mM) exhibited very little effect on guinea pig cochlear potentials. ~t'hen administered chronically (1-20 mg/ko. i.p. for 20 days), nicotine demonstrated no effect on cochlear potentials or the number of observed hair cell scars monitored 21 days after the treatment was terminated. Neither chronic nicotine treatment (1-20 mg/kg, i.p. for 20 days) nor acute reserpine pretreatment (5 mg/kg, i.p. 24 hours be- fore) altered the hair cell damaging effects of an intense pure tone (4 kHz, 126 dB SPL, 30 min). No nicotine ototoxicity was detected by the methods used in this studv. Other support: The Kresge Foundation, and the National Science Foundation. 72. Spontaneous vertical eve movements on closing the lids Martin Spector Department of Otolo;t•. C'niversitt• o ' I Pennsv/rania Medical School. Philadelphia. Pennsi•lvania EYe. Ear. Nose and Throat Monthlc• 50:396-;398. 1971 One hundred normal individuals were studied bv vertical nystagmus recordings with the eres closed. Fifty-seven percent exhibited spontaneous nystagmus or "spikes." The individuals were sub- jected to positional tests to evaluate the effects of positioning. In most cases, an identical pattern of response was maintained by the subject with the head in the various test positions: but in three percent, a difference was recorded. Repeat tests on different davs showed a marked labilitv of thF. findings. Consistent recordings were obtained in only forty-five percent. On these grounds, the prac- tice of recording vertical nystagmus behind closed lids is deprecated. 73. Horizontal nystagmus in routine subjects by electronystagmography Martin Spector Department of Otology. Universitv of Pennsylvania Nfedical School. Philadelphia, Pennsylvania Tournal of Laryngology and Otology 85:1039-1045, 1971 One hundred routine subjects. ostensibly nor- mal, were studied for horizontal nystagmus in four positions with eyes closed. In about 8 per cent of the test positions, easily read nystagmus was found. In about 23 per cent of the test positions, an irregular nystagmus was present. In 37 out of 58, mrstagmus was not duplicated on another day. Figures are given on the effects of alerting. 74. Acute effects of smoking on the labyrinth Martin Spector Department of Otolaryngology. Kensington Hospital. Philadelphia, Pennsylvania Unpublished Report to the AMA-ERF , The acute effects of smoking a cigarette on auditory and vestibular function were studied in 100 subjects. There was no depression of hearing and there were no consistent and definite changes in horizontal nystagmus, detectable with electronys- tagmography. 'I'ITVIN 0115948 T200439 , r 1 < f 204

Elevating the extracellular magnesium concen- tratiun from 4 m\1 to 20 m\l blocked spontaneous t,.p.s,p.'s. i.p.s.p.'s and prevented orthodromic stim- ulation. In elevated magnesium. nicotine caused a depolarization-repolarization sequence similar to that in normal magnesium. It differed in that the ,implitude of the depolarization was decreased and the duration increased. The results indicate that the cholinergic re- ceptors of snail neurons closely resemble the cholin- ergic receptors of frob sartorius muscle. A preliminary account of this study appeared in Federation Proceedings 32(3):806. 1973. 61. The separation of two cholinergic systems in the brain stem C. D. Barnes, A. H. Hassen. and E. J. Grey Department of Life Sciences. Indiana State University, Terre Haute. Indiana Prnceedings of the [t'estern Pharmacology Societv 16:184-187. 1973 In experiments performed on decerebrate cats, the spino-bulbo-spinal inhibitory system is shown to have a muscarinic component. The weight of evi- dence is in favor of the cholinergic system involved %.•ith REM (rapid eye movement) sleep being nico- tinic in nature. The possibility of some muscarinic offects on REM cannot be eliminated at the present, however. 62. Behavioral evidence for two types of cholinergic receptors in the C.N.S. Martin D. Schechter, and John A. Rosecrans Department of Pharmacology. .lledical College of Virginia. Richmond. Virginia (':uropPan lournal of Pharmacology 15:375-378, 1971 Rats were trained to make a specific behavioral rt,sponse for food reward in a T-maze apparatus t.0nditional upon whether they were injected sub- : utaneously with 0.4 mg/kg of nicotine or saline. Pretreatment with 0.25 mg/kg of atropine sulrate had no effect on the rats' ability to discriminate the central cueing effect of nicotine. The administration of 0.25 and 0.5 mg/kg of arecoline hydrobromide produced effects similar to saline. The results pro- vide behavioral evidence for the possible existence of specific muscarinic- and nicotinic-cholinergic receptors in the central nervous system. 63. Estimation of ionic concentrations and intracellular pH in slices from different areas of rat brain L. Hertz. A. Schousboe, and George B. Weiss Department o,f Pharmacologr. Medical Collelge of Virginia. Virginia Commoncrealth C'nirersih•. Richmond. L'irg inia Acta Physiologica Scandinavica 79:506-515. 1970 The present study represents an attempt to obtain comparative values for tissue compartment sizes and for cationic contents and distribution at different incubation time intervals in slices prepared from different areas of the rat brain. Tissue spaces occupied by "C-sucrose and 'H- inulin were measured at different incubation time intervals (15-120 min) in rat brain slices prepared from cortex, cerebellum, caudate, diencephalon and midbrain. Total water content, total Na- and K- contents and "C-DMO (5,5-dimethvl-2.-l-oxazoli- dinedione) spaces were obtained from the same tissues. At all time intervals "C-sucrose spaces were larger than corresponding 3H-inulin spaces. After 60 min incubation, the mean values for "C-sucrose spaces in preparations from different brain areas ranged from 31.5 to 38.1 ml/100 g, whereas the 'H- inulin space mean values were 19.3-26.1 ml/100 g. Average intracellular pH was calculated from "C- DMO spaces in the presence and absence of 3.67 mM procaine. The pH; values obtained using inulin- derived extracellular spaces were 6.91-6.99, and those calculated from sucrose spaces were 6.77-6.89. The contents of K- and Na- showed no distinct regional differences, but potassium uptake with time could only be observed in preparations from cerebral and cerebellar cortex. 64. On the measurement of extracellular space in slices prepared from different rat brain areas F. R. Goodman, G. B. Weiss, and M. T. Alderdice Department of Pharmacology. University of Texas Southwestern Medical School, Dallas, Texas Neuropharmacology 12:867-873, 1973 The present study was undertaken to obtain an accurate measure of the functional extracellular space in slices prepared from different rat brain areas. • . TIMN 0115945 T200436 201

„A ~, '•i+ rt',;ult from (.hanges in passive :urml)r,u1V pt'rmvabi(it}•. rt. The tlnlt'-CUUrtio i)t host-iarbachol htperpo- iari/+t(c~ci acc.orded with a\a extrusion process rate was directly proportional to [Na ~, with a c:c,nstant of 0.38 = 0.02 min' at 23-3: 'C. y. t\"ith increasing concentrations of carbachol. il}t. amplitude of the hyperpolarization increased in t,r„luI rtion to the preceding depolarization. but the r,tt,, l<,nstant of the hyperpolarization was un- h<ut,E'.ci. 10. The after-hyperpolarization was reduced in t,rop{,rtion to the depolarization b~• hexamethonium. I) ut was not affected by atropine. hvoscine or tetro- (if t i. A hyperpolarization also followed depolar- irition by acet}•Icholine or by EDTA in Ca-free ,nlution. 1•. Nicotine-depolarization was not followed by ,t 11yperpolarization unless hexamethonium was ,ided to the washout fluid. This was attributed to prolonged receptor-stimulation by nicotine. 13. It was concluded that the after-hyperpolar- irttion was due to the electrogenic extrusion of Na- ,te;cumulated in the gangiion cells during the preced- i tlj depolarization, evith no involvement of specific niuscarinic receptors. The relationship of this pro- t f'ss to post-tetanic hyperpolarization and to other forms of druo induced hyperpolarization is dis- t ussed. Some of the observations described in this paper hiwe been briefly reported by the authors elsewhere t British Journal of Pharmacology 37:51 1P, 1969, and 40:559-561P. 1970). 110. Nicotine washout rates from isolated rat a,anglia in relation to recovery from nicotine depolarization D. A. Brown. and C. N. Scholfield UNpartment of Pharmacology. St. Bartholomew's t-fospital Medical College, Charterhouse Square. London. f•:ngland - British lournal o/ Pharmacology 45:29-36, 1972 1. Isolated rat superior cervical ganglia recov- ered more slowly from the depolarizing action of nicotine than from that of carbachol or acetylcho- line. This was due to sustained high nicotine con- centrations in the c-icinit%- of the receptors. since recovery was hastened b%- adding hexamethonium to the washout fluid. 2. Ganglia incubated for 4 min in 80 µ,%l 'H- nicotine accumulated nicotine to a level exceeding the extracellular space. as judged from the uptake of 'H-mannitol. 3. The subsequent efflux of 'H-nicotine into non-radioactive solution could be largel~• resolved into two exponential components. with rate con- stants of 0.55 = 0.04 and 0.094 ± 0.007 min-'. The former was similar to that for total mannitol efflux. and so might be largelv ascribed to clearance of extracellular nicotine. The slower efflux might be due to clearance from intracellular compartments. Nicotine efflux rates were not affected by hexame- thonium indicating that receptor-activation did not modify the slow efflux. 4. Efflux of choline compounds ('H-acetvJcho- line, 'H-choline and 'H-carbachol) showed an ad- ditional. very slow component (rate constant 0.001 to 0.002 min-'). 5. It was suggested that slow efflux of intracellu- lar nicotine might sustain depolarization on wash- ing by maintaining high perineuronal concentra- tions of nicotine. With choline compounds the efflux rate from such sources may be too slow to affect perineuronal concentrations. 111. Intracellular pH in rat isolated superior cervical ganglia in relation to nicotine- depolarization and nicotine-uptake D. A. Brown, and J. V. Halliwell Department of Pharmacology. St. Bartholomew's Hospital I'Vedical College. Charterhouse Square. London. England British Journal of Pharmacology 45:349-359. 1972 1. The intracellular pH (pH,) of rat isolated superior cervical ganglia incubated in normal Krebs solution (pHo = 7.37) was estimated to be 7.33 from the uptake of a weak acid, '{C-5,5-dimethyloxazoli- dine-2,4-dione (DMO). Addition of 30 µM nicotine for 30 min reduced the DMO-estimated pH, by 0.15 units to 7.18. This effect was prevented,by hexame- thonium (2.5 mM) or by depolarizing the ganglion with K` (124 mM). 2. 'H-nicotine (30 µM) was concentrated within the ganglia to an intracellular/extracellular concen- TIMN 0115961 T200452 217

Nervous System to be located t'\I.IuSiVell' on the postsynaptic mem- brane. However. the su¢aestion has been made that the presynaptic nerve terminals are also sensitive to acetvlcholine. and that. during normal transmission. the acet}•(choline initiall}• released by the pregan- glionic nerve impulse might depolarize the ter- minals and release a further quantity of acetrlcho- line. so amplifying the transmitter release process. In the present study. the neural acetvlcholine stores of the superior cervical ganglion of the cat were labelled by perfusion of the ganglion with 'H- choline added to the perfusion fluid. Subsequent stimulation of the preganglionic nerve trunk at 10 Hz while perfusing with unlabelled choline regu- larlv increased the amount of tritium in the effluent perfusate. Separate measurement of acetvJcholine and choline in the perfusate showed that this in- creased radioactivity arose entirelv from an in- creased level of 'H-acetvlcholine. However, injection of acetvlcholine into the perfusion stream in doses that produced contractions of the nictitating mem- brane which were frequently as great as, and some- times greater than. those elicited by preganglionic nerve stimulation. did not release appreciable amounts of 'H-acetvlcholine. Carbachol. nicotine, tetraethvlammonium. and methacholine, were no more effective than acetvlcholine in releasing 'H- acetylcholine. These findings are considered to pro- vide strong evidence that a presynaptic action of acetv)choline does not play any part in the transmis- sion process. This study was the subject of a preliminarv report appearing in the British Journal of Pharma- cology 38:446P-448P. 1970. Other support: Grants from the governors of St. Bartholomecv's Hospital. 108. A note on the effect of dithiothreitol (DTT) on the depolarization of isolated sympathetic ganglia by carbachol and bromo-acetylcholine D. A. Brown, and D. Kwiatkowski Department of Pharmacology, The School of Pharmacy. University of London. London British Journal of Pharmacology 56:128-130, 1976 The S-S reducing agent, dithiothreitol (DTT) altered the properties of nicotinic receptors in rat superior cerc•ic. Qanglia such that (i) c:arhachol became less act..e as a depolarizing agent and (ii bromo-acety(choline produced an irreversible de- polarization. The latter was temporarily annulled by hexamethonium (which retained antagonistic pro p. erties). but returned when hexamethonium was removed. It is concluded that ganglionic nicotinic receptors might be quite similar to those for mono. quaternary agonists in.leech dorsal muscle. 109. Origin of the after-hyperpolarization that follows removal of depolarizing agents from the isolated superior cervical ganglion of the rat D. A. Brown, M. J. Brownstein. and C. \. Scholfield Department of Pharmacology. St. Banholomelr's Hospital Nfedical College. Charterhouse Square. Lonrfon, England British Journal of Pharmacology 44:651-671. 1972 1. Potential changes in isolated rat superior cervical ganglia following addition and removal of depolarizing agents were recorded using a movino- fluid extracellular electrode svstem. 2. Ganglionic negativity produced by carbachol was followed by a pronounced ganglionic positivity on washing. This after-positivity was attributed to hyperpolarization of the ganglion cells since it was unaffected by crushing the postganglionic trunk. 3. The after- hyperpo larizat ion was selectively depressed by (a) cooling (Q10 2.3), (b) metabolic inhibitors (cyanide, azide, 2.4-dinitrophenol). (c) reducing [K-]o or substituting Cs- for K-, (d) oubain. and (e) substituting Li- for Na-. This suggested a close dependence on active Na- transport. 4. When K' was restored to K--free solution. or the preparation was warmed rapidly. or when meta- bolic inhibitors were washed away, the hyperpolari- zation was rapidly regenerated. The effect of restor- ing K- indicated that the hyperpolarization was generated directly by the Na- pump. 5. The hyperpolarization was not altered by replacing C1- with isethionate, indicating that the voltage change produced by the Na- current was not modified by passive Cl- movements. , 6. Hexamethonium added to the washout fluid augmented the after-hyperpolarization, suggesting that there was a high (cationic) leak current due to continued receptor-activation on washing with nor- mal Krebs solution. 7. The hyperpolarization was reduced by omis- sion of Ca*- and restored by addition of Mg". This 216 T~M1~ 0115960 T200451

Nerc•ous Sc•stem tion techniques when attempting to study the phar- macolog~- of a subtle psychoactive drug such as nicotine. Female rats appeared to be. chemically and behaviorallv. more sensitive to nicotine which ap- peared to be somewhat related to the fact that female 5-HT accumulation was faster than in similarlv treated male rats. 91. Brain 5-hydroxvtryptamine correlates of behavior in rats: Strain and sex variability John A. Rosecrans, and Martin D. Schechter Department of Pharmacology. Medical College of Virginia. Virginia Commoncrealth University. Richmond. ['iro in ia Phtsiology and Behavior 8:503-510. 1972 Male and female rats of two strains. CD(Sprague- Dawlev) and CDF(Fischer) were compared behavior- al)v and chemica)lv in order to learn more about the behaviora) importance of 5-hydroxytrvptamine (5- HT) svstems. These studies yielded the following results: (1) female rats of both strains appeared generally more active, and the order of rearing behavior was as follows: CD ~= CD d> CDF 4> CDFd: (2) in vitro telencephalic 5-HT metabolisms rates appeared to correlate positively to rearing behavior between, and within each strain: and (3) in vivo and in vitro estimates of 5-HT function was significantly higher in the females of both strains. The oc•erall conclusion of this research indicated that 5-HT svstems mav be linked to some aspects of behavioral arousal and suggested that 5-HT function was positively correlated to activity. However, in forming such correlations are aspects other than chemical turnover at specific nerve endings must be considered. Other support: U. S. Public Health Service. 92. Effects of chronic nicotine administration and age on various neurotransmitters and associated enzymes in male Fischer-344 rats Jeremy H. Thompson, Che Su, Jean C. Shih, Dorothea Aures. Leslie Choi. Sherrel Butcher. William S. Loskota. Marcia Simon. and Douglas Silva Departments of Pharmacology and Experimental Therapeutics. Psychiatry. and Oral Diagnosis, UCLA Schools of Wedicine and Dentistry, Los Angeles, California, and Psychopharmacology Research. Veterans Administration Hospital, Sepulveda. California Toxicology and Applied Pharmacology 27:41-59. 1974 The acute, subacute and chronic effects of nicotine have been studied in male Fischer-344 rats. 210 Tcvo-month-old animals were injected s.c. with ei. ther 1.0 ml of 0.850•o w/v NaCl/dav. or 1000 µb nicotine base/mllkg for 4 days or for 2 or 22 months; injectables were formulated in 60'o gelatin. Nicotine pretreatment did not significantly alter 5-hydroxv, tn•ptamine (5-HT) or norepinephrine (NE) concen- trations in a variety of tissues. nor whole brain choline, acetylcholine and acetylcholinesterase ac- tivity. y-Aminobutyric acid concentrations in 8 brain areas were all higher after chronic nicotine ad- ministration, but histamine (HM) concentrations were variablv affected in a variety of tissues. The uptake of 5-HT into the heart and gastrointestinal mucosa, and of NE into the heart and aorta. was not affected by nicotine treatment. However, uptake of NE into synaptosomes prepared from an homogenate of whole brain was depressed. Urinary 5-hydroxvin- doleacetic acid (5-HIAA) excretion was significantlv higher in nicotine-treated rats compared to control animals both during ad libitum feeding and during food deprivation. In addition, urinary 5-HIAA ex- cretion was significantly higher in nicotine-treated ' rats with food deprivation compared to ad libitum feeding. Histidine decarboxylase (HD) and dopa decarboxylase (DD) activities in a variety of tissues were variably affected by nicotine: however, both enzymes showed diminished activity in the gastric mucosa following the alkaloid. Cardiac, aortic and hepatic catechol-O-methyltransferase (COMT) activ- ity was not altered by nicotine administration. Hepatic, aortic and cerebral monoamine oxidase (MAO) activity was unchanged, but an increase in myocardial activity, and a decrease in hepatic activ- ity were observed depending upon the substrate. Several changes were observed between young (4-month-old) and old (24-month-old) rats. Bone marrow (femur) 5-HT and HM concentrations de- creased with age, whereas 5-HT levels in the gas- trointestinal area, and HM concentrations in general, increased with age. Uptake of NE and 5-HT into the heart was reduced with age, whereas HD and DD activity showed no consistent change in the tissues examined. COMT activity significantly increased in the liver and aorta, but not in the heart, with age. Conversely, MAO activity increase~ in the myo- cardium but not in the liver, brain or aorta with age. TIMN 0115954 T200445

, . ~Intor S}vstemt 75. Characteristics of sustained tremor induced by nicotine in pilocarpine-treated animals ,a. Tsujimoto. and T. Dohi Drpurtment of Pharmacology. Hiroshima L'nirersitY ti~ hnol of Dentistry I:asumi. Hiroshima. Japan veuropharmacoloQr 15:421-426. 1976 _ Sustained tremor (nicotine-pilocarpine tremor) cv<ns produced by the administration of nicotine to tiilocarpine pretreated mice and rats, and its charac- teristics were studied. Nicotine-pilocarpine tremor cc,is composed of tremor of the head and limbs and occasional sharp head movements up and back- c.-,trds. Pilocarpine caused hyperthermia in most of the rats kept in a high ambient temperature (30-320C) ,ind also induced hypothermia under normal con- ditin+is (2"_ = 2`C). Nicotine produced the sustained tremor onlv in rats which showed a rise in body temperature by pilocarpine pretreatment. In mice pretreated with pilocarpine, nicotine produced the sustained tremor under normal conditions as well. The time course of nicotine elimination from brain was dissociated from that of the intensity and duration of the sustained tremor. Atropine. nico- tinok•tic drugs. promethazine, phenobarbital and prnpranolol blocked the induction of nicotine- t,ilocarpine tremor. L-Dihydroxvphenvlalanine. wmethyldopa. reserpine and tolazoline were not othec tit'e. It is suggested that nicotine-pilocarpine tremor does not solely result from the inhibition of hepatic dru, metabolism by pilocarpine, and that the central t holinergic system might be involved in the in- durtion of nicotine-pilocarpine tremor. ~ rl. Temperature Regulation 76. Cholinergic and adrenergic interactions in the thermoregulatory centers of the rat Peter Lomax. R. S_ Foster, and W. E. Kirkpatrick Department of Pharmacology. School of Medicine and the Brain Research Institute. University of California. Los.-ingeles. California 13rrtin Research 15:431--138. 1969 S}•stemic administration of pilocarpine causes a fall in body temperature in the rat. This effect can be reproduced by direct injection of the drug into the rostral hypothalamic thermoregulatory centers. The fall in temperature following systemic administra- tion of pilocarpine can be attenuated or blocked by prior injection of norepinephrine into the rostral hypothalamus. Injection of a-blocking agents into the thermoregulatory centers. immediately preced- ing injection of norepinephrine at the same site, prevents the inhibitory effect of norepinephrine on the hypothermic response to systemic pilocarpine. It appears that temperature regulation involves a cho- linergic link in the rostral hypothalamus. Catechol- amines may also play a role in the control of body temperature by changing the degree of polarization of these cholinergic neurons and thus adjusting the sensitivity of the thermoregulatory centers. Such an adjustment of the membrane potentials by endoge- nous catecholamines could be a factor in the physi- ological regulation of the set point of the central thermostat. Other support: U. S. Public Health Service. 77. Drugs and body temperature Peter Lomax Department of Pharmacology. School of Medicine. and the Brain Research Institute. University of California. Los Angeles. California International Review of aVeurobiolooy 12:1-43. 1970 Current knowledge on the effects of drugs on thermoregulation is reviewed. Attention is directed mainly to agents for which there is evidence indicat- ing a direct action on the thermoregulatory centers in the central nervous system. Other support: U. S. Public Health Service. 78. Temperature changes following iontophoretic injection of acetylcholine into the rostral hypothalamus of the rat Lv. E. Kirkpatrick. and P. Lomax Department of Pharmacology, School of Medicine and the Brain Research Institute. University of California. Los Angeles. California Neuropharmacology 9:195-202, 1970 A method is described for iontophoretic in- jection of acetylcholine in the central nervous sys- tem of experimental animals. A fall in body tem- perature occured when acetylcholine was delivered into the rostral hypothalamic thermoregulatory cen- TIMN 0115949 T200440 205

Section IV Sidman's avoidance session held during the same fixed time each day. The dvoidance rate during the nicotine available period did not differ significantly from that of the saline control period. Although the monkev•s tended to receive more electrostimulation during the nicotine available period, an attempt was made to observe the influence of these electrostimu- lations on cigarette smoking behavior. The results. hocti•ever. were inconclusive because of erratic smok- ing response rate. 145. Army rank and subsequent mortality by cause: 23-year follow-up Carl C. Seltzer and Seymour Jablon American Journal of Epidermiology 105:559-566. 1977 Mortality among veterans has been studied in relation to military rank at separation in a series of 85.491 men discharged from the US Army in 1946 and traced through 1969. It was found that although the mortality of privates was very close to ex- pectation based on population rates, non-commis- sioned officers had a 23% advantage and com- missioned officers about a 40% advantage. The relative advantage of the veterans who had higher rank held not only for deaths from all causes but also for most of the specific causes examined and there was only a small tendency for the difference to diminish with the passage of time during the 23-year period of follow-up. TIMN 0115974 T200465 230

i l 114. Dissociation of depolarization and ganglionic blockade induced by nicotine Gerard L. Gebber t~,,partment of Pharmacolo~r. .~lichigan State t'nirersitr. East Lansing, Nfichigan )nurnal of Pharmacologr and Experimental Therapeutics 160: t _'-1-13-t• 1968 Transmission blockade and depolarization ,'Voked bv nicotine are considered generally to be related and dependent events. In the present study, however. it was found that the characteristics of the blockade of transmission evoked by nicotine in cats kcere not those usually associated with depolariza- tion-induced ganglionic jsuperior cervical] inexcit- abilitv. The following observations suggest that the blockade of transmission and the simultaneously occurring ganglionic depolarization were not caus- all%' related: (1) tetanic preganglionic stimulation antagonized the block occurring during depolariza- tion evoked by nicotine; (2) tubocurarine antago- nized depolarization evoked by nicotine, but en- hanced the simultaneously occurring blockade of transmission; (3) the intravenous infusion of nico- tine evoked a postganglionic discharge which was maintained when transmission was abolished; (4) discharges evoked by nonnicotinic ganglionic stim- ulating agents were enhanced during the blockade of transmission and depolarization evoked by nicotine. In many respects the blockade of transmission which L'oincided with depolarization was similar in charac- ter to the block which persisted beyond the period of depolarization evoked by nicotine. In addition, the data suggest that the site at which nicotine evokes ganglionic depolarization is distinct from that at ~vhich the compound blocks the actions of neu- rogenicallv released acetylcholine. A preliminary account of this work appeared in the Pharmacologist 9:226, 1967. 115. Observations on drug-induced activation of cholinoceptive sites in a sympathetic ganglion Gerard L Gebber, and David W. Snyder Department of Pharmacology, Michigan State University, East Lansing, Michigan Journal of Pharmacology and Experimental Therapeutics 163:64-74, 1968 In studies on cats, the mode of administration played an important role in determining the pharma- cologic characteristics of the ganglionic (superior cervical) responses evoked bt• tetramethylammo- nium (T"vfA) and acetvlcholine (ACh). The postQan- glionic discharge evoked bv a single injection of T%1A or of nicotine was abolished by hexametho- nium (G;) and was unaltered by atropine. In contrast. the discharge evoked during the infusion of TNIA was blocked by either C6 or atropine, whereas the discharge evoked during the infusion of nicotine was again blocked by C5 but not by atropine. The infusion rates of T%1A employed did not alter transmission. The data suggest that infusion of T%IA initiated an interaction between the C5 sensitive and atropine-sensitive excitatory ganglionic cholinocep- tive sites. It is proposed that the spread of de- polarization from the C6 sensitive site markedly enhanced a weak muscarinic stimulating action of TMA. Thus, the postganglionic discharge evoked during the infusion of TMA appeared to emanate mainly from the atropine-sensitive site. In contrast to the C6-sensitive firing evoked in an unconditioned ganglia by a single injection of ACh, constant rate infusion of the compound usually elicited a dis- charge which was blocked by atropine and not by C5. The role of the described facilitatory interaction in the normal integrative functions of sympathetic ganglia is yet to be determined. 116. Comparison of differential secretion of adrenal catecholamines by splanchnic nerve stimulation and cholinergic agents Larry R. Klevans, and Gerard L. Gebber Department of Pharmacology, Michigan State University, East Lansing, Michigan Journal of Pharmacology and Experimental Therapeutics 172:69-76. 1970 The venous blood of the in situ acutely denerva- ted cat adrenal gland was analyzed for epinephrine (E) and norepinephrine (NE) by the trihydroxyindole method. A comparison was made of the differential secretion of E and NE produced by frequencies of splanchnic nerve stimulation (SNS) and doses of acetylcholine, dimethylphenylpiperaZinium and nicotine which released equivalent amounts ofj total catecholamines. Total catecholamines secreted rose TIMN 0115963 T200454 219

81. Temperature chanaes following microinjection of histamine into the thermoregulatory centers of the rat ll. E. Brezenoff. and P. Lomax Depurtment of Pharmacologc. School of Medicine and the Brain Research Institute. C'nirersitr of California. t,,,; .-1n,eles. California ' F'.xpNrien tia 26:51-52. 1970 %ticroinjection of histamine (1 µg) into the rostral hypothalamus of the rat produced an im- mt'diate fall in body temperature which continued for about 20 min. At the highest dose emploved (5 µg i the niean fall is core temperature was 2.0 '- 0.3°C I5.E.%1.) in 4 animals. Threshold effects were ob- :orved with as little as 0.5 µg. Histamine had no effec.t in animals pretreated with the antihistamine, c hlorcyclazine (5 mg/kg, i.p.). 1 h prior to the intracerebral injection of either 2.5 or 5 µg of histamine. It is tentativelv suggested that histamine should be considered among the candidates for a role as a neurotransmitter in the thermoregulatory centers of the hypothalamus. Other support: U. S. Public Health Service. 82. Temperature changes induced by chlorpromazine and N-methyl chlorpromazine in the rat lt". E. Kirkpatrick. and P. Lomax Department of Pharmacology. School of Medicine and the Brain Research Institute. University of California at t.vs lnoeles. California \'europharmacology 10:61-66. 1971 Chlorpromazine and its quaternary analogue, N- meth}•l chlorpromazine, both cause a fall in body tf~mperature when administered systemically to rats. Injection of either drug directly into the rostral hypothalamic thermoregulatory centers led to a rise in temperature. The tertiary amine was more potent in producing these responses and had a more prolonged effect. These results indicate that the hypothermia after systemic administration of chlor- promazine was mediated at sites outside the nervous srstem. The hyperthermic action of the drugs could have been due to blockade of cholinergic receptors in the thermoregulatorv centers. Alternativelv. sta- bilization of neuronal membrane potentials by the drugs could have led to an upward setting of the hypothalamic thermostat. Other support: LT. S. Public Health Service. 83. The sites and mechanisms of action of drugs affecting thermoregulation P. Lomax, and G. V. Knox Department of Pharmacologc•. School of Medicine and the Brain Research Institute. L'nirersitr of California. Los Angeles. California The Pharmacology of Thermoregulation. S}'mposium. San Francisco 1972, pp. 146-154 (Karger. Basel 1973) Pharmacologists are interested in determining the site and mechanism of action of drugs. In the case of agents which cause a change in bodv temperature following systemic administration this can be extremely difficult. The problem may be compounded due to the drug affecting more than one component of the temperature regulating sys- tem, possibly in different directions in terms of heat loss or heat conservation. Although rigid criteria for defining the above parameters have not yet been established, techniques for investigation now avail- able should allow such localization with a rea- sonable degree of certainty. This presentation is directed towards the methods available for the investigation of this gen- eral problem, including contributions from the au- thors' laboratory. Other support: U. S. Public Health Service. e. Neurotransmitter Interaction 84. A combined procedure for the histochemical fluorescence demonstration of monoamines and microautoradiography of water-soluble drugs David Masuoka. and Gian-Franco Placidi (Robert W. Earle) Psychopharmacology Research Laboratories. Veterans Administration Hospital. Sepulveda. California. and Department of Pharmacology. University of California. Irvine-California College of Medicine. Los Angeles. California I Journal of Histochemistry and Cytochemistry 16:659-662. 1968 A procedure for combining microautoradiogra- phy and the histochemical fluorescence method for ` TIMN 0115951 T200442 207

Nervous Sc•stem There was no evidrnt.e nf drug-induced firinQ dur- ing blockade of conduction by nicotine-like drugs. Injection of a second dose of D`IPP or other nicotinic drugs 1 to 3 minutes after recorery of conduction from first dose of nicotinic drug caused a signifi- cantl}• smaller degree of blockade. Bv contrast, con- duction blockade by potassium chloride (0.5-1.0 mg) or procaine (100-5300 µg) was unaffected by prior injection of nicotine-like drugs. Similarlv. prior injection of potassium chloride or procaine did not affect response to nicotine-like drugs. The ability of the nicotine-like drugs to antagonize the response to a second injection of a nicotine-like drug did not occur in nodose ganglia perfused with a saline solution. Thus. the actions of nicotinic substances on peripheral nerve resembles those seen at junc- tional sites. This study was the subject of a preliminary report appearing in The Pharmacologist 10:217. 1968. Skel etal Neurom uscular 128. Comparison of the effects of procaine, chlorpromazine and their quaternary derivatives on nerve action potentials M E. Kirkpatrick. and Peter Lomax Department of Pharmacology. School of Medicine and the Brain Research Institute. University of California at Los Angeles. California Research Communications in Chemical Pathology and Pharmacology 1:1-19-155. 1970 The isolated frog sciatic nerve preparation was used to compare the ability of procaine, chlorproma- zine and the quaternary derivatives of these drugs to block neural conduction. Procaine (6mM) caused 50°o reduction in the height of the action potential in 28 min; chlorpromazine (0.6mM) achieved this in 15 min. N-methvl procaine (12mM) was without effect on the propagated action potentials whereas N-methyl chlorpromazine (0.6mM) proved to be slightly more active than its tertiary analogue. There was no recovery of conduction after prolonged washing of nerves treated with the phenothiazine derivatives. These results suggest that the blockade produced by chlorpromazine is due to an action on the outside of the nerve membrane and that the mechanism bv which conduction is abolished is fundamentallc• different than that of the clinicallv useful local anesthetics. Other support: U. S. Public Health Service. 129. Nicotine-induced depolarization and stimulation of potassium efflux in striated muscle Edward G. Henderson, and John C. Hancock Department of Pharmacology. Schools of Medicine and Dental Medicine. University of Connecticut. McCook Hospital. Hartford. Connecticut Journal of Pharmacology and Experimental Therapeutics 177:377-388. 1971 The influence of nicotine on potassium (;=h i exchange in denervated frog sartorius and rat exten- sor digitorum longus muscles was examined. Nim- tine (0.005-0.2 mhl) did not alter the uptake of'ZK iii any of the muscles tested. Efflux was significantly increased during the first one to five minutes of nicotine exposure and returned to normal monoton- ically within an hour in both types of denervated muscle as well as in the innervated frog sartorius muscle. After removal of nicotine, there was a long period (> 1.0 hour) during which 42K efflux was decreased 20 to 50% below control values. The time course of ;2K efflux stimulation paralleled the time course of muscle cell membrane depolarization. Both the nicotine-induced depolarization and stimu- lation of 4ZK efflux were blocked by prior administra- tion of d-tubocurarine and, therefore, were probably initiated by an interaction of nicotine with che- mosensitive sites on the muscle cells. The decreased rate of 42K efflux after nicotine removal was not blocked when d-tubocurarine was added immedi- ately after exposure to nicotine. Conduction in sartorius muscle cells was not blocked by low concentrations of nicotine (< 0.06 mM) and action potentials recorded during the peak of depolariza- tion were followed by a "late positive afterpotential" of long duration (> 150 msec). The magnitude of the late positive afterpotential was increased, by reduc- ing the potassium concentration of the bathing medium. This study was the subject in part of a pre- liminary report appearing in Federation Proceedings 29(2):548, 1970. TIMN 0115968 T200459 lot c:k de rot 0 f tie cI•t iz<i de- int c h( a< in po lot hat in rar t hE du (1- mi pai del "K to stil ni( ula Ieb me inc bel 224

.\:errous System trodes using standard techniques. Quantal content (m) was calculated from the coefficient of variation of 110 repetitively evoked EPPs at 1 Hz. Both d-tubocurarine (10-5%1) and lobeline (10-'M and 2 x 10-'M) decreased the amplitude of the evoked EPP without causing significant change in the resting potential of the end-plate membrane. Nicotine (10-5M and 3 X 10-'M) causes initial depolarization and subsequent repolarization of the end-plate membrane. During both these phases, the EPP amplitude progressively declines. The decrease in EPP amplitude caused by d- tubocurarine and lobeline is due primarily to an interference with the postjunctional action of the transmitter as evidenced by a decrease in quantal size (q) without significant alteration in m.. In contrast, the decrease in EPP amplitude caused by nicotine during its phase of maximum depolariza- tion is accounted for by a decrease in both q and m. The depression of the EPP amplitude when the end- plate membrane has repolarized under the influence of nicotine is due to receptor desensitization. These results indicate that at the frog neu- romuscular junction the actions of lobeline more closely resemble those of d-tuhocurarine rather than nicotine. The depression of m during nicotine- induced depolarization of the end-plate membrane may be related to a similar depolarization of the motor nerve ending. This study was the subject of a preliminary report appearing in The Pharmacologist 13:264, 1971. 133. Receptor desensitization by lobeline and nicotine Robert L. Volle and Linda Reynolds Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut .\'aunrn-Schmiedeberg s Archives of Pharmacology c_ology 276:-k9-5-k. 1973 In frog sartorius muscles, lobeline had no effect on resting membrane potentials but prevented de- polarization of the fibers by nicotine. The blockade caused by lobeline was not antagonized by elevated concentrations of nicotine. Procedures preventing depolarization of the muscle by nicotine had no effect on receptor-desensitization (anti-nicotinic ef- fects) caused by nicotine. From these two lines of evidence. it was concluded that receptor desensitiza. tion in skeletal muscle does not require depolariza- tion of the fibers. 134. Blockade by lobeline of potassium exchange in skeletal muscle: Relationship to receptor desensitization at the endplate Robert L. Volle Department of Pharmacology, University of Connecticut, Health Center. Farm(ngton. Connecticut Vaunyn-Schmiedebero's Archives of Pharmacology 282:335-347. 1974 Lobeline (0.05 to 2.5mM) caused depolarization of muscle fibers in frog sartorius muscle bathed in chloride-free solution. When the extent of depolar- ization was plotted against the log of the concentra- tion af lobeline, the relationship was described by a straight line with a slope of 57mV for a 10-fold change in the concentration of lobeline (0.2 to 2.0mM). Like lobeline, b