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1 , Renewed: , ..- 9/1/66 .THE COUNCIL FOR TO:;ACCO Rip.,SizARCII - U.S.A. Renewed: ". 9/i/67_ N3.W YORS. 11t. Y. 10017 633 THIItD APErP[SE ` ' PEE COMVII;T • -Chm:- _~. Dr. Birig,-• -Dr. •Cattell Dr. Jacobson Application For Research Grant Dr. Lynch 1. Name of InvesiigatoE(s): (include Title and Degrees) 2. Institution & Address: . .. CARDIOVASCUTAR; PHARI~ACOLOGY and CI~ISTRY N0. 467-R2-SUB,'~ - Activated: 9/1/65 Thomas C. Westfall, A.B., M.S., Ph.D. Department of Pharmacology University of Virginia,School of Medicine Charlottesville, Virginia 22901 3. ShortTitleofProject: Supplemental Grant Request " Funds to aid in the purchase of a Scintillation Spectrometer (to supplement grant entitled, "Action of Nicotine on Subcellular Distribution of Catecholarnines in Brain and-Heart" ) 4. Proposed Starting Date: . October 1, 1967 5. Anticipated Duration of this SpecifirStudy: on separate sheet •6. Brief Descripton of Objectives or Specific Aims: on separate sheet 7. Give a Brief Statement of your Working Hypothesis: on separate sheet v

C.1. Eiographical sketches of all prinGpal and professional personnel (append). on separate sheet 9. Physical Facilties Available (Where Other than.Administering Organization Indicate Geographical Location) `_ see append'ed curriculum,vitae l 12. List of publications: (Five most recent as pertinent) (append) . - see appended curriculum vitae

To aid in the purchase of a Packard Automatic Tri Carb Scintillation Spectrometer Sub-Total 7,500 Total 7,500 Estimated Future Requirements: Salaries Consumable Suppl. Other Expenses PermanenbEquip. Overhead - Total Year 3 his understood Ihat,the appficant and institutionaf officers in applying for a grant have read and found acceptablh the Council's "Statement,of Policy Containing Conditions ond Terms Under Which Projed Grants Are Madm" Signature -0 Dir.r.tarol Pcoi.d y~/ Telephone Signature a72Y W un.u 0117- of th. luNluKaw Telep.hane ~ Q qw=

List flnandol support for research from oil sourca, insludln9 own inslitution, }o+thfs ond/or nlat.d m.orch pro/se1s. • i Catecholamine Metabolism Following Cigarette Smoking and Nicotine Administration The American Medical: i Association Education and - Research Foundation Committee foT Research on Tobacco and Health

Duration of Specific Study " ~.~•~~. 4; This equipment would be used to tielp•complete the project . listed above which is currently being sponsored.by The Council. .'In addition, it is anticipated that this instrument will be used in many future projects under contemplation by the principle investigator. `Imgortance of a Scintillation Spectrometer to the Specific Aims and Obiectives of the Principle Investigator. The purpose of this supplemental grant application is to request funds which will aid in the purchase of a scintillation .spectrometer. This instrument is greatly needed in achieving the following overall specific aims. 1) To ascertain.the importance of certain biogenic amines in helping to explain the central and;peripheral actions of nicotine. 2) To specifically determine if •there is an adrenergic mechanism involved in the central action of nicotine. 3) To specifically determine whether or not nicotine is exerting; any of its peripheral action by the release of norepinephrine directly from,adrenergic nerve termina:ls. CENTRAL ADRENERGIC ACTIONS OF NICOTINE Attempts at determining whether or not there is an adrenergic mechanism involved in the central action of nicotine are currently being:carried out in this laboratory by studying the effect of nicotine on the subcellular distribution of certain amines,in specific-brain regions where these amines are present in large }y concentrations. Studies have been made on brain areas containing O large concentrations in an attempt to prevent a masking of a O possible effect that might be taking place due to tissue which co has very little or no amine.content. Our results, to date, ~ indicate that there are certain changes in the level of these amines in various regionz of the central nervous system ~ (Westfall, et al., 1967). These studies are limited., however, j~ to measurement of changes in the• static storage levels of ~ catecholamines and other amines in the brain,. In addition,• they have not included areas such as the cerebellum which contains very little endogen.ous norepinephrine but may be very important because of the active metabolism of amines recently observed to be taking place there. Although these above mentioned studies can produce important results and can give us some ideas of.a release of amines by nicotine, they tell us nothing about the dynamic aspects of metabolism of amines in the brain. _

We are becoming.more and more aware that the storage-levels of norepinephrine, dopamine and serotonin are not static but"r'•eflect dynamic.equilibria between the rates df formation and the rates of utilization of the amines (see reviews by Glowinski and . Baldessarini, 1966; Hornykiewicz, 1966).. In other words, there is a constant amount of. norepinephrine in nerve endings because,_._ a continuous synthesis or input is balanced by a continuous • efflux onto receptors or metabolizing enzymes. The level of norepinephrine will be altered, therefore, if one of these rates h bhd N iti hhid tht asseen cange.umerousnvesga+orsave empaszea ..syn'Chesls, storage release anCt metabolism are not unrelatec! phenorn.ena and should.be considered together in interpreting ~drug action (Neff; et al., 1965; Costa,.et al., 1966). Therefore, it may be thats changes in the turnover rate of these amines are .a better measur•e of drug action than mere changes in the concentrations of the amines which could remain constant or even ' decline despite an increased rate of syntHe-sis. ., •' Recent studies by Iversen and Glowinski (1966), and Glowinski and Iversen (1966a!,b.) have shown that different turnover rates of norepinephrine occur in several diff erent regions, of the rat brain. These investigators observed that the brain regions seemed.to fall into three classes. The cerebellum appears•to have the fastest turnover of norepinephrin;e, with a half-life of about 2 hours. Although this brain region has such a fast turnover, it is quite interesting that this area has a! very low endogenous norepinephrin!e content.. This would seem to indicate that the small stores of norepinephrine are in an unusually active metabolic state. This.rapid turnover may even be correlated in some way with its ability to form adenosine 3',5'-phosphate. Along with the low endogenous norepinephrinScon~tent, the cerebellum seems to `- accumulate very little H-norepinephrins. On the other hand, the bellu r a e s to ine hrine c c l t b lit f H t m pp ar e a cumu a norep p e a es o e more -me o when compared to brain regions with slower turnover times. Following the cerebellum, the brain regions with the next fastest turnover rates seem-to be the cortex and hippocampus with ~ half-lives of about 3 hours. The regions with the slowest rates W- : of turnover of norepinephrine are the hypothalamus and the medulla j~ oblong,ata with half-lives of 4 hours. Interestingly, these same rA regions have the highest concentration of endoger~ous norepinephrine ~ and seem'to accumulate thg greatest amounts of H-norepinephrine ~ and the least amount of H-metabolites. These experiments point out the important fact that an inverse relationship between•turnover and, N endogenous concentrations of norepinephrine in various parts of the brain seem to exist. .This proba•bly indicates that areas with small endogenous levels actually h,ave a faster active metabolism of catecholamines than regions containing higher levels of norepinephrine. These observations emphasize, therefore, the importance of studying turnover rates rather than only measuring steady state levels of catecholamines in various brain regions following drug administration. . At the present time there appears to be three available methods for measuring the rate of turnover of norepinephrine in the brain. Two of these methods involve the use of labelled techniques. The first method measures catecholamine turnover by introducing

0 removal of an,equivalent amount of labelled and unlabelled amine. -:.turning over of the endogenous norepinephrine stores at a -con stant rate, by the continual synthesis of norepinephrine and •..decline of H3-norepinephrine that occurs is interpreted as'the labelled norepinephrine into the lateral ventricle and follows the changes in specific radioactivity with time. The'exponential `:-as tyrosine, dopa or dopamine and measurina-the amount of formed ~,~norepinephrine. The second is by using;labelled precursors such turnover rate and;therefore, the rate of synthesis of endogenous ~,The slope of the exponential decline is a reflection of the , .':; amine stores. :: central stores.gives a measure of the rate of utilization of i y disappearance of endogenous catecholamines from~peripheral and ;inhibiting catecholamine biosynthesis with such,drugs as aC-methyl-p--tyrosine When s nthesi s is blocked~ the rate of .norepinephrine from these precursors. The final method is by . Of these three methods of measuring catecholamine turnover in ''• the brain, the use of exogenous tritiated n,orepinephrine appears to have distinct advantages. The use of A-methyl-p-tyrosine, for •°-';instance, is very limited by the difficuity of measurina very low other precursors is an improvement over the previous method'but may ,r : q es ai a e, e use o - opamine or ' concentrations of endogenous norepine.phrine in discre~e brain areas ~ ~' with the chemical techni u av ' 1 bl Th f H d complicated by the possibility that a prolonged synthesis of ,-to suggest that this fact does not occur to any important extent ":`with H3-d`opamine (Iversen and Glowinski, 1966)1. :.-of the labelled precursor. The available evidence, however, seems H3-norepinephrine may occur in the presence of persistant amounts -It can be concluded, therefore, that equipment necessary to ''which has been demonstrated to have a rapid;metabolism but which has 'This seems particularly important for regions such as the cerebellum -;would give a direct measurement of the influence of nicotine on the r;dynamic metabolism of catecholamines in the central nervous system. ;of catecholamines in the brain in response to nicotine.• These studies measure labelled substances is required to adequately measure turnover : t b d t di f _;s;;~.'...•_. ;.,no een s even u e or changes in static catecholamine levels .because ot the technical problems in measuring the low levels 1003545793 •.(see IMestfall, et al., 1967) present there. PERIPHERAIL.ADRENERGIC ACTIONS OF NICOTINE Another important use of this instrument will be its application in studies aimed;at further evaluating the role of nicotine on peripheral amine stores in adrenergic nerve terminals. As in the central nervous system, there is likewise a dynamic equilibria between the rates of formation and rates of utilization of catecholamines in peripheral sympathetic nervous structures. Therefore, it is equally • important to study the influence of nicotine on the turnover of norepinephrine in adrenergically innervated:organs such,as the heart and spleen. Similar to different brain, regions,'there are differences in the turnover rate of norepinephrine in various organs as well ' .(Burack and Draskoczy, 1964).

Previou,s publications from this laboratory have pointed out the inconsistencies and controversies that exist in the literature concerning the ability of nicotine to release norepinephrine from storage sites in adrenergically innervated organs (Westfall, 1965a,b;'Westfall, et al., 1967). One of the main reasons f or these discrepancies is probably a result of the difficulty in measuring and detecting,the'small changes in endogenous amine -that are taking place following the admin-istration of nicotine.. A more convenient method for studying cardiac nor:epine hrine in the rat has been used by Herttin and: co-workers (1961~ and by Daly, et al., in the mouse (19663. In both cases they have shown that following tracer doses of labelled norepinephrine the amines are taken up into the heart, equilibrate with,endogernous amine and most important are affected in a similar manner by agents that cause or inhibit norepinephrine release. This method;has been described as being rapid, simple, economical and quite reliable. Other methods have been described:using a perfused heart preparation (Nash, et al., 1967). It is anticipated that similar methods would be very valuabl!e and useful in studying; the influence of nicotine on norepinephrine stores. These methods appear even, more valuable when it is realized that d,rugs can-act by releasing norepinephrine from,nerve endings without arn appreciable change in the endogenous level as measured by the currently available biochemical methods. Because of the dynamic equilibria that exists between formation and utilization, any release of amine may be quickly replaced by synthesis and/or reuptake into nerve terminals. With the use of labelled amines described above, such a release can be much more easily detected. - Another potential use of the scintillation spectrometer would be to study the influence of nicotine on the uptake of norepinephrine o into adrenergic nerves. Such a ossible effect is suggested by the data.of Nedergaard, et al. (1966). These investigators have reported that nicotine can potentiate the response of vascular smooth muscle to nerve stimulation by up to 60%. The same doses of nicotine occasionally produced small increases in resting tone. Similar types of potentiation of sympathetic effects exist for a large number of drugs such as cocaine or imipramine which are well known to block uptake or reuptake of norepinephrine into adrenergic nerve termin,als (Carlsson, 1966; Trend;elenburg, 1966). Therefore, it is q:uite possible that nicotine also possesses some ability to antagonize the uptake of norepinephrine into adrenergic nerve terminals. Such a possibility certainly deserves investigation. 7.. Workinq Hypothe si s. The synthesis, storage, release and metabolism of biogenic amines are not unrelated phenomenon and should be considered:tog,ether in interpreting the action of nicotine in the central and peripheral nervous system. Changes im the turnover rate of amines such as norepinephrine will give a much clearer picture of the effect of nicotine on the dynamic metabolism,of amines in brain and,peripheral organs.

. Details of'Experiments Utilizing a Scintillation Spectrometer. Ky" . . • " . ... •S TURNOVER OF CATrCHOLANEINES IN TNE CENTRAL NERVOUS SYSTEM BEFORE AND AFTER NICOTINE ADMINISTRATION— in specific activity of exogenously administered tritiated .:.norepinephrine. • offer the most advantages is the one which follows the chang,es As mentioned above, there are three available methods for x=•-studying the turnover of catecholarnines in then brain at the `, present time. Of these three methods, the one which seems to doses will be administered;prior to or following the intraventricular The labelled,amine will be injected into the lateral ventricle of rats anesthetized lightly with-pentobarbital, using the recently described method of Nobel, et al. (1967). Nicotine in various be dissected:out on an ice cooled glass plate including the following. -and brains quickly removed, blotted and chilled. Seven regions will decapitation at various tumes--after the injecti.on of norepinephrine : injection ot DL-H-3-norepin-ephrine. T11e rats will be Kilied by cerebellum, medulla oblongata hypothalamus d) midbrain e) striatum f) hippocampus g) cortex . Glowinski and Iversen (1966). In addition, the plates in,"Craigie's Complete details of these dissections are given in the report by :'Neuroanatomy of the Rat" (Zeman and Innes, 1963) will be closely ~i;''-.f ol lowed . ... . ... ~.- . The tissues will be quickly weighed and homogenized in 10 to 15 ml of cold 0.4 N perchloric acid in a Ultra-turrax homogenizer. The homogenates will then be centrifuged at 10,000 x g for 10 min. After the addition of 0:1 ml of 1% disodium ethylenediamine- ~,& tetraacetate (EDTA) and 0!.1 ml of a freshly prepared 1% solution 0 of ascorbic acid, the pH will be adjusted to 8.3 - 8.5, with N 0 normal RaOH. The sample will then be passed through a column GJ containing aluminum oxide-to remove the free catecholamines while Q1 the combined effluent and:washing! will be used for the estimation ~ of the o-methylated metabolite, n-ormetanephrine. Elution of the ~ catecholamines fromf the alumina column, will be carried out• by the addition of 0.2 N HC1. An• aliquot of this eluate will be used ~ to determine the radioactivity of the free norepinephrine, while the tritiated d!eaminated metabolites will be extracted from the alumina-eluates according to the method of Kopin, et al. (1961). Briefly, this involves taking an.aliquot, acidifying it with 6 N HC1 and saturating the solution with NaCI. The nonamine catechols are then extracted into ethylacetate. After centrifugation, an aliquot of the organic phase will be evaporated in a current of air

::':in a glass vial and the radioactivity determined-. The o-methylated metabalite,=normetanephrine will be--:assayed by the method described by Iversen, et al. (1966). Briefljr;., the effluent and. washing:s from the alumina column used to extract -' the catecholamines will be adjiusted. to pH 6.5•and passed through _ a column (6 x 20 mm) of Dowex 50W-X4 in the sodium form. The ;.:resin will then be washed with glass distilled water, and the ._ normetanephrine eluted with a mixture of equal quarts of 6 N hydrochloric acid and ethanol. Aliquots of this eluate will then':44: -.The free catecholamines, normetanephrine and the deaminated metabolites will be assayed in a scintillation spectrometer after the addition of 4 ml ethanol and 10 quantities of 0.4% 2,5-diphenyloxa:zole and 0.005% 1,4-di(2-5-phenyloxazole)benzene in toluene. Tritiated o-methylated, deaminated metabolites can be estimated by the difference be~ween total radioactivity of tissue extracts and the sum of (H ) norepinephrine and other ~ :~;-;'~; metabolites. A flow chart, enciosed in this report, gives a quick overall picture of ths-extraction procedures. In some experiments, various subcellular constituents of the various brain regions will be assayed for their f ree catecholamine and metabolite content. In these experiments, the tissues will be initially homog:enized in 0-.3 M sucrose. On,e-fourth of the sample will be taken for assay of total radioactivity and the remalning;extract spun at a low speed (10,000 x g;- 10 min) to,remove the coarse fraction. The low speed supernatant will then be spun at 100,000 x g for 40 min, yielding a• supernatant and particulate fraction. The low speed sediment,high speed supern-atant, high speed particulate and.totai tissue samples will then be extracted in 0.4 N perchloric acid and extracted according:to the procedure described above for the differentiation of free, deaminated, o-methylated and deaminated ~ ~"o-methylated amines. . .~ _., ~- . . ., . - . yY STUDY OF THE RELEASE OF NOREPINEPHRINE FROM ADRENERGIC NERVES BY NICOTINE As ha;s been mentioned in the previous section, controversies still exist as to whether or not nicotine releases appreciable quantities of norepinephrine from adrenergic nerves. Most of this controversy exists as a result of discrepancies in the literature because of the difficulty of available chemical methods for determining the small amounts of norepinephrine that might be released. _ In the present studies, the influence of nicotine on the . release of norepinephrine will be studied by measuring the amount of labelled morepineph,rin,e remaining in the heart at a g;iven period of time, following;the prelabeiling of the norepinephrine stores. 4 p Y • ~~' be eva orated to dr ness in vials -~-~, .;.,

These studies can be carried out in both,mice and rats. assayed according to the method of von Euler and Lisha•jko (1961). centrifuged (10,000 x g for 10 min) and the supernatants taken for assay in a:scintiilation spectrometer, similar to the method described above. Endogen-ous norepinephrine will be simultaneously •turrax apparatus. Following homogenization, the extract will be after 3 hours by a blow on the heads and the hearts quickly removed. The hearts will be immediately exsanguinated:and , homogenized in 10 vol of 0.4 N perchloric acid:in an Ultra- norepinephrine will be injected into the tail vein of male white mice. Nicotine in various doses will then be administered subcutaneously one hour later. The mice will then be sacrificed 50 mg of heparin/1) of 1.0i'mµ mole (5µ curies) of tritiated For mice, a 0.1 ml solution (isoton-ic sodium chloride,'con.taining {.• except that about 10,µC of tritiated norepinephrine will be administered by a tail vein_ and the animals sacrificed by decapitation. In the studies on rats a similar procedure will be followed distribution of the labelled norepinephrine will be studied. In In some experiments the influence of nicotine on the subcellular • ultracentrifuge. All fraction-s resulting from the differential centrifugation will then be taken for assay in a scintillation . and extracted•in 10% trichloroacetic acid for assay of total heart. The remaining homogenate will be spun in a refrigerated centrifuge at a slow,speed (10,000 x g, for-10 min). The slow speed supernatant will then be spun at 100,000 x g for 45 min in a these experiments, the tissues will be homogenized in ice cold potassium phosphate buffer, pH 7.5. An aliquot will be taken counter or adsorbed on- alumina, for endogenous norepinepttrine determination following,extractions in 10% trichloroacetic acid. INFLUENCE OF NICOTINE ON THE UPTAKE OF NOREPIN'EPHRINE The influence of nicotine on the uptake of norepinephrirne can be conveniently studied by administering the nicotine prior to the intravenou.s injection of tritiated norepinephrine as described above. Similar experiments can be carried out on the perfused guinea pig heart where, in addition.to net uptake, the arterial-venous difference can also be studied. This.can be accomplirshed-by measuring,the perfusate content as well as the tissue content of N norepinephrine. ~ CW ~ . ~ . ~

EXTRACTION, SEPARATION AN D ANALYSIS OF CATECHOLAMINES AND METABOLITES Animals injected, sacrificed (decapitation), tissues dissected out and weighed. - W " Homogenization, (0.4 N perchloric acid, if Ul'tra-turrax apparatus) Centrifugation (10,000-x g - 10 mm) W Ascorbic acid, EDTA added pH adjusted to 8.3-8.5 (NaOH!) W Alumina Columns washed with water y Effluent + washings .(o-methylated metabolites) I Dowex column (50W-X4 ), washed with water eluted with 6 N H.C1+Ethan,ol if added Ethylacetate ~ Contrifugati tY Assayed" for Assayed in Assayed in Normetanephine in Scintillation Counter Scintillation Coun Scintillation Counter Ad j usted to-Ph• 6. 5 Aliquot (taken for total radioactivity determination) Catecholamintes e.luted with 0.2 N' MC1 A1:iquot (assay Aliquot(dearninate of norepinephrine) metabolites), I - 6 N HC1, NaC 1003546798 Ira

Burack, W. R. and P. R. Draskoczy. The turnover of endogenously labelled catecholamines in several regions of the sympathetic nervous system. J. Pharmacol. exp. Ther., 144: 66-, 1964. •Carlsson~ A. Pharmacological depletion of catecholamine stores. : Pharmacol. Rev., 18: 541, 1966.. Costa, E., Bouilin,, D. J., Hammer, W., Vogel, W. and B'. B'. Brodie. Interactions of drugs with adrenergic neurons. Pharmacol. Rev., 18: 577, 1966. f°' Daly, J. W., Greveling, C. R. and. B. Witkop. The chemorelease o noreninephrine from mouse hearts. Structure-activity relationships. I. Sympathomimetic amines and relatediamines. J. Med. Chem., 9: 273, 1966. ~ Daly, J. W.,_Creveling, C. R. and B. Witkop. The chemorelease of norepinephrine from mouse hearts. Structure-activity relationships.' II. Drugs affecting!the sympathetic and central nervous system. J. Med. Chem,., 9: 280, 1966. • Glowinski, J. and R. J. Balidessar•ini. Metabolism of n-orepinephrine in the central nervous system. Pharmacol. Rev., 18: 1201, 1966. Glowinski, J. and L. L. Iversen. Regional studies of catecholamines in the rat brain. I. The disposition of (H~) norepinephrine, ~:....(H3) dopamine and (H3) dopa in various regions of the brain. J. Neurochem., 13': 655, 1966. .Glowinski, J. and L. L. Iversen. Regional studies of catecholamin-es ...in the rat brain. III. Subcellular distribution of endogenous and exog;enous,catecholamines in various brain regions. Biochem. : Pharmacol., 15: 977, 1966. Hertting•, G., Axelrod, J. and R. W. Pa~trick Actions of cocaine and ~ -norepinephrine in the N tyramine on the uptake and release of H .. heart. Biochem. Pharmacol., 8: 246, 1961. CO Hornykiewicz, 0,. Dopamine (3-hydroxy tryptamin,e) and brain functions. Pharmacol. Rev., 18: 925; 1966. Iversen, L. L. and J. GLowinski, 0,. Regional studies of catecholamines in the rat brain- II. Rate of turnover of catecholamin.es in various brain region,s. J. Neurochem., 13: 671, 1966. Kopin,, I. J., Axelrod J. and E. Gordon:. The metabolic fate of H3- epinephrine and Ci4-metanephrine irn the rat. J. Biol. Chem., 236: 2109, 1961. Montanari, R., Costa, E., Beaven, M. A. and;B. B. Brodie. Turnover rates of norepinephrine in hearts of intact mice, rats and guinea•pig-s. using tritiated noTepinephrine. Life Sci., 2: 232, 1963'.

• Nash, C. W.,,Wolff, S. A. and B. A. Ferguson. The action of from isolated perfused: rat hearts. Fed. Proc., 26: 570, 1967. svnpathomimetic amines on the release of noradrenaline ~• , •. 7 Neff, N. H., Tozer, T. N., Hammer, W. and B. B. Brodie. -Kinetics of release of norepinephrine by tyramine. Life Sci.,.4: 1869, 1965. . • . Noble, E. P., Wurtmann, R. J. and J. Axelrod. A simple and rapid method for injecting H3-n,orepinephrine into the lateral ventricle of the rat brain. Life Sci., 6: 281, 1967. Irend'elenburg, U. Mechanisms of supersensitivity and subsensitivity ..to sympathomimetic amines. Pharmacol. Rev., 18: 629, 1966. Westfall, T. C. Effect of nicotine and nicotine analogues on tissue •• and urinary catecholamines in the rat. Acta physiol. Scand., 63: 77, 1965. .Westfall, T. C. Tobacco alkaloidis and the release of catecholamines, in tobacco. alkaloids and related compounds. Ed. by U. S. von Euler, Pergamon Press, 4: 179, 1965. • Westfall, T. C.,.Fleming, R. M., Fudger, U. K. and W. G. Clark. Effect of nicotine and related substances on amine levels in the brain. Ann. N. Y. Acad. Sci., 14 2: 83, 1967. Zeman, W. and J. R. M.. Innes. Craigie's Neuroanatomy of the Rat. Academic Press, New York, 1963.

FUNDS FOR THE PURCHASE OF A LIQUID SCINTILLATION SPECTROMETER Present Reques Additional Funds: Second year budget of a grant entitled, Action of Nicotine on Subcellular Distribution of Catecholacnines,in Brain and Heart, Council for Tobacco Research -'-U.S.A. . . . . . Research and Development Fund University of Virginia School of Medicine . . . . . Total. . $15,500.00 Cost of Instrument. . $15,500.00- 0

A we171 equipped Department of Pharmacology with adequate ;`differen.tial estimation of epinephrine and norepinephrine, the ' ~;•:.fluorometer with the necessary f ilter combinations for for this project includes: Farrand Model A photoelectric .laboratory and office space is available. Equipment available. room as well'as rooms for animals within the Department and located in the Department of Biochemistry on the floorahove is also available. Our Department has a well equipped shop and dark ~ultra-centrifuge. The use of a Spinco preparative ultracentrifuge -'analytical balances, Grass polygraphs, and'Type 40 rotor for :Medical Electronics fractionator, Beckman spectrophotometers, .-homog;enizer, International refrigerated centrifuge, Gilson .Beckman expandomatic pH meter, chromatographic,column•s for :`.alumina absorption, ultra-turrax high speed tissue floor below) an Aminco-Bowman spectrophotofluorometer, ~°=use of (on loan from Cancer Research Laboratory, located on . ' ~rrecc + fl.n C.-hnnl n~s Medicine. - :} ~- ---- _.._...-- -~------- ---... _.. _..---- -- Staff. ` 1. Thomas'C. Westfall, Ph.D. Principle Investigator 2. Hirofumi Osada, M.D. Research Fellow 3. Leslie Frank .Research Assistant ~ •-'4. William Moore Graduate Student

R: REDACTED MATERIAL CURRICULUM VITAE • N ame • Thomas C. Westfall Date of Birth: ~- Place of Birth: I:atrobe, Penn,sylVania Marital Status: ; R- .. Education : - - in Pharmacology. 1963-1964. Naticsnal Heart Institute Postdoctoral 1959 A.B., Biology and Chemistry;:West Virginia University 1961 M.S., Pharmacology., West Virginia University 1962 Ph.D., Pharmacology, Dr. Daniel T. Watts, supervisor, West Virginia-University Education,al Awards: ~ - 1959-1962. National. Institutes of Health Predoctoral Traineeship Academic Positions Held•: Award. 1962-1963'. Instructor in Pharmacology, West Virginia University Medical. Center. 1965- Assistant Professor of Pharmacolooy, University of Virginia School of ?J:edicin,e.- Sweden, Professor U. S. von Euler, supervisor. 1964-1965. Assistant Professor of Pharmacblogy, West Virginia : University Medical Center. 1963-1964. Research Fellow of National Heart In,stitute, Department of Physiology, Karolinska Irnstitute, Stockholm; Teaching Experience: I 1962 thru 1965. Gave approximately 20 lectures per year to medical, nursing and graduete students at West Virginia University Medical Center. This in,volved participation in the laboratory course of instruction to the same groups. 1966 to present. Gave about one-fourth of-he lectures'in Pharmacology to the sophomore medical class at- the University of Virginia. This also-involved active participation in all = laborator.y, exerci ses. • Active teaching at the grad-uate. 1eve1 and supervision of graduate students was also carried out. Committee Assionments. (University of Virginia. School of Medicine): 1965-1966. Research Society, School of Med_c"ne. 1966-1967. Search Committee for Pharmacology Chairman.. • YI"RO KERO' • • ~XERO~ yr~'O ~./...~ _-

R: REDACTED MATERIAL Karolinska Institute, Stockholm, Sweden. Philadelphia General Hospital, Cardiology Division, Philadelphia, Duke ;University School of Medicine, Durham., N. C. Medical College of Virginia, Richmond,'Va. SYmposia Participation (by invitation).: • 1.966-196.7. 1967- . 1967- . ; Search Committee for Psychiatry Chairman.. AD,HOC Curriculum Committee of Basic Sciendest,-,~.AD HOC Committee, Cell Biology course for first ~~._ .year medical students. ;Society Memberships: REDACTED Professional Lectures (by invitation): REDACTED University of Texas School of Medicine, Galve.stsdn, Texas. .Turku Uni,versity School of :lhedicine, Turku,--Finland. and the Release of Catecholamines. Fourth International Wenner-Gren Center Symposium.. Tobacco Alkaloid•s and Related Compounds.. Held in:Stockholm, Sweden, February, 1964. Paper presen-ted: Tobacco Alkaloids New York Academy of Sciences Symposium on The Effects of Nicotine ..•, and Smoking on the Central N'ervous System. Held, in New. York City, April 7-9, 1966. Paper pre-sented: Effect of N icotine and Related Substances on Amine Levels.in the Brain. Central and peripheral autonomic effects of nicotine. Influence of drugs on uptake,storage, release and inactivation of biocenic amines. ;XEnp: ' X:aq ., gQa. . , ~ r. . American Medical Association Workshop on•Tobacco and Health. Held' at Broadmoor Hotel, Colorado Spring.s., Colorado, November 1-3, .1966. 'Paper presented: Influence of Nicotine on Catecholamine Metabolism. N[ajor Fields of Investigation : _ XCnJ XCno •ci-•r

BIBI:IOGP.APxY ~ of . Thoma s C. Westf all: Malor Publication•s • Westfall, T. C. and Watts, D. T. The on t?-.c cardiovascular respon-se to h siol., 17: 471, 1962'. . Westfall, T. C. and Watts, D. T. The effect of•cigarette Exp. Biol. Med;., 112: 843, 1963. :_ smoke on epinephrine secretion in the dog. _roc. Soc. 3.- Westfall, T. C. and Watts, D. T. Catecholamine bxcretion in smokers and nonsmokers. J. Aool. Physiol., 19: 40, 1964. , . • • +i+L... M. . Westfall, T. C. and Watts, D. T. The effect of nicotine on brain and urinary amines in the rat. J. Neurochem., 11: 397, 1964. - - 5. Westfall,' T. C. Tobacco alkaloid-s and. the release of catecholamines ir, Tobacco Alkaloid:s and.Related• Compounds, Ed. by U. S. von,Euler, Pergarnon Press, 4: 179, 1965. 6. Westfall, T. C. Effect of nicotine and nicotine analogues •.• on tissue and urinary'catecholamin,es in the rat. Acta: physiol. Scand., 63: 77, 1965. 7. Westfall, T. C. Uptake and exchange of catecholamines in rat tissues after d and 1-adrena~line. Acta ohysiol. Scand., 63: 336, 1965. ... • .. . . .:~ . . 8. Westfall, T. C. and Peach, M. J. Action of angiotensin on myocardial and renal catecholamines in the rabbit. Biochem. Pharmacol., 14: 1916, 1965. - 9. Westfall, T. C., Cippoloni, B. and Edtnundowicz, A. Influence of propranolol on the hemodynamic changes and pla•sma catecholamine levels following cigarette , smoking;and nicotine. Proc. Soc. Exo. Biol. Med, 123: 174, 1966. 10. Westfall; T. C., Fleming, R. M., Fudg;er,. M. K. and. Clark, W. G. Effect of nico•tine and related substances on amine levels in the brain. Ann. N. Y. Acad. Sci., 142: 83, 1967. ;xr_rro' . • ixF.PO• .xr.wp ~...

• • -.-- Westfall, T. C. Accumulation of norepinephrine in rat ' _ tissue following treatment- with three bet&adr'energic • 67. antagonists. Arch. int. Pharmacodvn., 167:`69,:1.q . .' . .12. Westfall, :. C. and Anderson, G. P. Influence of nicotine on catecholamine metabolism in the~rat. Arch. int. Pharmacodyn., (in press) 1967. Westfall, T. C. Effect of beta adrenergic blockers on the-.:;; -.noradrenaline content of rat heart and spleen before : and after noradrenaline infusion. B•rit. J. Pharmac. Chomoi;ner., (in press) 1967. storage in the perfused guinea pig;heart. Influence of Beta adrenergic antagonists on norepinephrine Action of nicotine-on subcellular amine levels in brain • and.heart. stores following depletion with various pharmacological ~agents. . 7 Influence of adrenahectomy on repletion of catecholamine 20: 89, 1961. Westfall, T. C.* and V`latts, D. T. .hhe effect of reserpine on the cardiovascular response to smoking. Fed. Proc., 21: 193, 1962. Westfall, T. C.* and Watts, D. T. The effect of cigarette smoke on epinephrine secretion in the dog. Fed. Proc:, Westfall, T. C.* and Watts, D. T. Catecholamine excretion in' smokers and nonsmokers. Fed. Proc., 22: 1509, 1963'. . Peach, M. J. and Westfall, T. C. Action of angiotensin on 3. myocardial catecholamines in the rabbit. Fed. Proc., 24: 488, 1965. 5. Westfiall, T. C.* Influence of pron.ethalol, propran,olol and iproveratril on u^Lake and storage of norepinephrine. Fed. Proc., 25 : 260-, 1-966.. 6. Westfall, T. C.*, Fleming, R. P4-., Fudger, M. K. and Clark, W. G.. Effect of nicotin-e and related substances on aminee levels in the brain. Svmoo.sium abstract. Syrnao.sium on The Effects of Nicotine and Smoking on the Central.Nervo.us Sys;:em. N. Y. Acad. Sci., April, 1966. 7. Westfall, T. C.* Uptake and. storage of norepinephrine following the administi-ation, of three beta adrenergic an tag;omi st s. Va. J. Sci., 17 : 354, 1966•. XF_RO 4f•Y. . r. . .. . ~.:- ,. .'.itt:..a ~ . .. .. - ..

Westfall, T. C.* Influence of nicotine on catecholamine . metabolism. Symposium a: .tract_. Tobacco and Health. °, Amer. Med. Assn., November, 1966. ,--,- - Westfall, T. C.* Influence of beta adrenergic antagoni•sts on the norepinephrine content in,rat heart before and after NE infusion. Fed. Proc., 26: 569, 1967. - Westfall, T. C.* Influence of beta adrenergic blockers on norepinephrine storage in the perfu-sed guinea pig heart. Va. J. Sci., 00: 000,, 1967. lyestfall, T. C.* The effect of beta adrenergic blocking, drugs on the norepinephrine level in the perfused guinea pig heart following•NE infusion. The Pharmacologist, 9: 249, 1967. III. Texts Contributor to: _ Medicinal Chemistry, AlfredlBurger, Editor, third edition, John Wiley and Sons, Inc., Interscience Publishers, New-York. " Introduction to Neuropharmacology (co-author, with E. D. Brand). is XEPO •XERO `:°RO • XI'R.l cunr, 'C^c~r-' ~C.,

R: REDACTED MATERIAL CURRICULUM, VITAE Name: Hirofumi 0sad'a ~- .~:::~_~:~•--- . • P1'ace of Birth: Kawasaki, Japan Education: University of Tokyo, Doctorate of Medical Science (Internal Medicine), March 1964. University of Tokyo Hospital, Internship, April 1959 - Ma-rch 1960. Yokohoma Municipal Un-iversity, M.D., 'Ma.rch 1959.. Academic Positions: 1964-1966. Instructor in Internal Medicine, University of Tokyo. 1966-1967. Research Fellow, Division of -CardUology, Philadelphia General Hospital, Philadelphia, Pa. 1967- . Research Fellow, Depart:nent of Pharmacology, University of Virginia School of Medicine, • rrrrp ^t++.v Charlottesville, Val. Society Membershios ACTED AMED I ;xrwa •3w•. ,. .. ••..~.~t

Publications : _ . .... . ~ . . .:~'. • . , . _ • ... . .. . . .. • drug (Nialamide). In Japanese. J. Therap., 43: 1961, 1961. s2rotonin and catecholamine metabolism •of new anti-anginal - ' Shimizu, K. and Osada, H. Clinical experience and effects on : Kobayashi, T., Ito, Y., Kobayashi, M., Kajihara, T.; Takeuchi, M. 2. and Aoki, T. Acute cardiac death-of unknown etiology. In English. Jap. Heart J, 3: 4!42, 1962. ~.~;.;,` ~_ ti:i . . s In Japanese. Diaonosis and Therapy, 50: 925, 1962. , y- . . , . , f g > > > , , > > Makai N. Kono, R - Yo~shimura S Su ai M Komizo U -,; .{~... 3. Kobayashi, T., Ito, Y., Kajihara, T.', Kishii, T., Osada, H., f .:.: .:....... .4. KobaYalshi> T., Ito, Y.> Kajihara> T:> Shimizu> K.e Kishii,T. Osada, H. and Komizo, U. . Clinical experience and effects of (J,-methyl dopa). In Japanese. J1. Geriatrics, 7: 330, 1963. Ej,ima,'M., Osada, H. and Tkeda, T. Paragang:lioma. In Japanese. 5. Kobayashi, T., Ito, Y., Kajih,ara, T., Shimizu, K., Kishii, T., Osada, H. and;Komizo, U. A new anti-hypertensive drug : . ao. , , e J. Yoshimura, S., Kobayashi., T. and 0sada, R. Acute cardiac death. • d-methvl dopa on serotoni,n and catecholamine. In Japanese. '"'?Rrx`° 44 1962 1888 Th r Endocrinoloay and Metabol? sm, 4: 99, 1963. . Yoshimura, S., Kobayashi, T. and Osada, H. Acute cardiac death. .In Japanese. J. Jao. Med. Assn., 49: 1015, 1963. 8. Kobayashi, T., Osada, H. and Komizo, U. c{-methyi dopa as a,anti- hypertensive drug. In Japanese. Respiration and Circulation, 11: 507, 1963. 9. Osada, H., Komizo, U., Kondo, K., Kishii, T., Kajihera, T., KobaJyashi, T. and Okano, S. Experimental and clinica~l trial of H.-catecholamine, esp.ecialLy as tos:-methyl dopa. In Japanese. Saishin-loaku, 19: 198, 1964. 10. Kobayashi, T.,,Kajihara., T. and Osada, H. Some con•siderations on the causative diseases of acute cardiac death, In Japanese. J. Chest. Dis., 8: 465, 1964. 1]:. Yoshimura, S., Kobayashi, T., Kajihara;, T. and Osada, H. Acute cardiac death of un'•:no:;~n, etiology. In Japanese. J. -Chest.. _ Dis., 8: 529•, 1964. s...a'_..~... ~. . xcr~o~ XCpo 1003546809

, -.. _ . . i: .. '. 12. Osad_:a, H. The effects of reserpine, .(-methyl dopa, nialamide and dexamethazone on the uptake and-metabolism-of •H3-catecholamine:r' , In English. Jap. Heart J., 5: 224, 1964 Progress on research ='•s:rf: H and Osada Kajihara T 13 Koba ashi T , . , .: . y , ^.:.~.. ., t erapy o ~-~- n in the world in 1964. Etiology, diagnosls an x hypertension. In Japanese. Jan. Med. J., 2129': 3, 1965. . 14.- Kobayashi T. and Osada, H. Anti-hypertensive effects of reserpine. In Japanese. Jan. Med'. J., 2147: 139, i965• 15. Kobayashi, T. and Osada, H. Basa;l and'casual blood pressure. In Japanese. Jao. Med. J., 2140: 130-, 1965. 16•. Kobayashi, T., Osada, H., Kondo, K. and Tawara, I. Anti- anginal and anti-anrhythmic effects of propranolol. In Japanese. J. Therao., 48: 775, 1966.. 17. Kobayashi, T., Kajihara, T., Osada, H., Komizo, U. and Ishii, K. b lism t i t h l i H l • . a o am ne me -ca ec o ycer ne on The effect of nitfog In English. Jaot. Heart_J-., 7: 430, 1966: 18.__Kobayashi, T., Kajihara, T., Osada, H. and Okada, T. The effects of ethacrynic acid'. In Japanese. J. Therap., 1966 (under contribution). %. 19. Osada, H. Review and Practice. The rnethod's of ineasuiements of catecholamine•in urine, blood and tissues. In Japanese-. Librar for Chemical P.nal,sis, Vol. 6, Jap. Chemical Analystics Assn, in Tokyo under contribution). Y6fi0 eXYlt'. ~'COPY 'r,t.l'-:,