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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 ~ . .. .. - ..