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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 -~-~, .;.,