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CARDIOVASCU7AR! PHAMCOLOGY and CHENIISTRY ~'Y' :~w,:: ~ -': Na•625 ..~ #~:,'' Nicotine effect upon cardiac membrane enzymes '*} ~ 4. Proposed Starting Date: October 1st, 1967 R . Anticipated Duration of this Specific Study: 2 years 6. Brief Descripton of Objectives or Specific Aims: ,_ . _ _ . . , .. . . . ~ • . . in experiunental am.mals. We propose here to extend this observation to a study of the primary :.~r....,., . _:.It has been reported that the administration of nicotine elevates serum cholesterol level cardiac tissues. :: and secondary effects of nicotine upon enzymes associated with subcellular membranes of A ~-' ;=:The specific aim of the proiect is to test the thesis that nicotine in chan--inQ• the •nature : muscle. ,... of those enzymes which. are bound; in the native state, to membrane portions of cardiac ~ of the lipid pool, from which cellular membranes are synthesized, may alter the function :.. ._ Give a brief statement of your working hypothesis. It is known that nicotine intervenes in, the biosynthesis of lipids and this: in turn may tration affects the ion transport system and so is an event preliminary to cardiomyopathy. ; systems which are part of the membrane structural network. It is primarily the ion transport, membrane adenosine triphosphatase and the hormonally -controlled adenyl cyclase systems which we propose to study. The thesis of membrane change will• be further examined by a consideration of serum enzymes whose levels in serum, are relateable to cellular changes. We hypothesize that the reported change in lipid composition which follows nicotine adminis- lipid composition of the membrane system will result in a changed function of those enzyme ., sarcoplasmic reticular and plasma membranes. From, this, we may predict that the changed change the nature of the lipid pool available for and ultimately incorporated into heart muscle

T-- 1:.'IILP....-:.,s ' , , . . . . .- __ . - • . ~ . --'8 DetailsofExperimento(DesignandProceduresr(AttachSeparatePages) Rodents and rabbits will be used as experimentab animals. Continued ..F{~i~ . .. . ., . ~ . ~irV A. Biological matenal ...t Injection of nicotine following a dose rate based upon, Kershbaum, et al. (Am, Hearo Jour. B. Observation of serum lipids. • - :: During the course of administration of nicotine, the level of serum lipids will assayed by thin-layer chromatography using silicic acid. Additional experimentaL pro- : differenbes observed in consequence of nicotine administration may be intensified in another =_;' cedures will be designed,upon the basis of the changed pattern of serum lipids so that the .,:Aw~... , ' . [ . ...•. • . . 206, 1965) will be undertaken. `" the prime'experimental group. 4• group of experimental animals by the inlection of hpids in ratios relative to those foundiin s ea o re e It h be ur a t find th t th d trati f 20 25 dia h 1 t I s e m o a zac o es ero in As In natural myopathic states, erythrocytes and muscle tissue are altered . ;.^L'_ _._ l•rls+'in1•s•. p:4 None C 11. @iographical sketches of alliprindpal andprofessional personnel'(append). t• cells and muscle membranes. . to experimental animals greatly affects-the activity pattern of transport A'TPase of red blood a a , n g a uus on (continued page 2A) 9 Physical Facilties Available (Where Other than Administering Organization indicale Geographical Lbcation) 10. AdditionalRequirementse 12. List obpuhlications: (Five most recent as pertinent) (append) . (see page 2A) (see page 2B) (see page 2C)

by the administration of diazacholesterol. This phenomenon we attribute to a change in r;=r'the lipid components of the membrane s to which the protein enzyme is bound. The changed ,.r= ; ~~__... .{. 'k°~~~ ~: ctivity pattern• ( response to metals and to the cardiac steroid membrane ) represents an ; extremely sensitive indicator of membrane subcellular organization. : : . ,_.. .:... _. . .. .., _ - . A'Sw..4~. . . . ' . - - . .. . . Enzyrne assay . -_,' systems chosen for primary study are known to be associated with Yriembrane structures of the cell. The transport ATPase is probably the best documented ,.~.. . model for transport phenomena and its vectorial role in cell function closely relates to -,~~; u membrane structure The adenyl cyclase system appears to be part of a structure-related . _ complex of catalysts acting upon ATP as substrate. The pivotal position of adenyl cyclase, -:: whose catalysis is facilitated by epinephrine and whose product in turn controls the phos- .. .,t, s cataolism and its ycolysis may be a key to interpretation of ener.getic .~ `> phorolysi step of gi ., ~.. ' biological control. These two membrane related enzymes will serve as indicators of drug nieotine ) induced changes. . ~', . . _ Aldolase and creatine phosphokinase elevation in serum are widely used indicators of tissue degeneration. These will be assayed and correlated with the hypothesize .cardiomyopathy. - . • . : Blood will be collected in a haemolyzing solution. The blbod haemolyzate will be centrifuged at 20, 000 x g for 20 minutes. The supernatant discarded and the pellet washed and recentrifuged in the Tris buffer. The final pellet obtained will, be collected and ;; stored as the erythrocyte ghost preparation, and used as the source of ATPase activity. ,~; rt{ Muscles will be macerated and, sarcoplasmic reticulttm isolated by a differential centrifugation: ~ procedure in sucrose. Heart muscle membrane preparations prepared in, much the same ;~ way will be assayed for adenyl cyclase activity by ultra violet analysis following ion exchange" ~ F... chromatography. These methods and those for the assay of aldolase and creatine phospho- kinase activity in serum are presently in use in our laboratory. The ATPase will be assayed ;` by measurement of inorganic phosphate; aldolase by the Sibley-Leihninger method and creatin.e ~ phosphokinase by measurement of phosphoryl group transfer. -• '`- D. Analysis of data, r + Findings will be interpreted in terms of the effect of nicotine administration upon changes in membrane enzyme and in serum enzyme levels (presumably related to linkage from cells ). This deviation will be correlated with induced changes in the metabolic lipid pool and all factors considered as a function of possible cardiomyopathy. Facilities available. -Three laboratories- are in use by the principal'investigator. Many major analytic tools are available. Specific holdings directly available to this project are: Beckman, B flame spectrophotometer, International HR i refrigerated centrifuge, Beckman L ultra-centrifuge, RSCo fraction collector, ISCO fraction collector, deionizing column, water baths, Zeromatic pH meter, Cahn electro-balance, Roller-Smith balance, Beckman DB recording spectrophotometer, refrigerators, freezers, necessary minor laboratory devices and ware. Held by a colleague in an adjoining laboratory, and available to the project are F and M gas chromatographs, Cary 14 spectrophotometer, and P-E infrared spectrophotometer. 1003547000

R: REDACTED MATERIAL ~ ~ Additional common facilities held by the Department in another building, available to this project, include a 2000 square. foot laboratory, animal rooms, refrigeration equip= ment, Mettler balance, free electrophoresis unit r analytic ultracentrifuge and recording .. , . . • spectrophotometer. Also the resources of the Institution include electron microscope <. ;._. r, . . •.. facilities, infrared spectrophotometer, Beckman amino acid analyzer, scintillation '_counter, and•generally, most of the instrumentation and facflities•appurtaining to the -laboratory investigation. A unique dual differential microcalorimeter is in use in this 1a13o.ratozy and appropriate to aspects of the proposed study: . . Biographical sketches of all principal and professiona•1 personnel. Harry Darrow Brown m., 3 c. Ph.D. A.M. B.S. 1964- t 1964- 1963 -1964' ' 1961-1963 1957-1961 1954-1956 Occasional Anil B. Patel M.S * M.Sc. B .Sc . 1966- 1963 -1965 ` 1962-1963 91 1957 Columbia. University. Faculty of Pure Science 1952 same 1950 Long Island University - Assistant Professor, Department of Biochemistry and Nutrition, University of Texas Medical Branch; Galveston, Texas. Collaborator, United States Dept. of Agriculture. Chemist ( Biochemist ), USDA, New Orlea.ns. Associate Professor, Southern Illinois University, Edwardsville. Assistant Professor, Loyola University, New Orleans. Lecturer, Hunter College, New York. Editor: Macmillan, Acad. Press, Schol. Lib. University of Cincinnati 1961 University of Bombay 1953 University of Bombay Research Associate, Department of Biochemistry, The University of Texas Medical Branch; Galveston, Texas. Research assistant in orgaze.i.c chemistry, University of Cincinnati. Teaching assistant in chemistry, University of Cincinnati. * Residence requirements complete; thesis in preparation.

List of five recent publiLations pertinent to the proposed research. .. .. . . .. . _.., . .. . . . ..:,, ,~ . ..,.: . ......, . ~ .,. ... . .., • ~ . ~. .: . ... - _ .. . . mahty ui human myopathy. Ms, submitted. `Brown, H.D,, Chattopadhyay,S.K., andPatel, A.B., (1967) Erythrocyteabnor- •..Glycoside effect upon membrane enzymes of erythrocytes: and muscle in duck : Brown, H. D., Chattopadhyay, S. K., Patel, A., and Rigdon, R, H., (1967) - 'myopatny. Experientia, in press. '~ ' Brown, H. D., Chattopadhyay, S. K., and Patel, A. (1966) SarcoDlasmic- reucuium pi-trase upon a solid support. Bioch. Bfoph. Res;. Comm. 25 (3): : Brown, H. D., (1966 ) A characterization of the ouabain sensitivity of heart micro- Pharmacol., 15: 2007-2011. Brown, H. D., (1966) Azasteroids. and heart adenosine triphosphatase. Biochem . somal ATPase. Bioch. Bioph. Acta 120: 162-165.

R: REDACTED MATERIAL -; " -11. Salaries (Personnel by names) :, Professionai Principal investig-4tor ' Research associate,, Anili Patel Technical . Secretarial services TPrhniral aSSistant o&Sr, wct ~ I!. Consumable Supplies (fistiby categories) •. Chetnicals 380~ :•- t-:nart paper, stanonery aW. Hardware, small tools 75. Glassware 160'. Silica curvettes (41 80: C. Other Expenses (itemize) Craftsman's services 70. Communications, xerox 80. T`tavel (to consult with-colleagues and to present results ) 450. Publication costs 70.. Animalsand animal care 800. D. Permanent Equipmentfi(itemize)' •.Tanks and- accessories for thin-Iayer chromatography 350. E. Overhead (7 5% oF A+ B+ C) aao-wrar Sub-Tota1 I 1591. 15911. Total Estimoted Future.Requirements: 12548, Salaries Consumable Suppl. Other Expenses Permanent Equip. Overhead Total Yean2 ~ 745. 1470. none I654•. 12681. i l Year 3 It is understood that the applicant and instituiional officers In applying for a,grani have read and found acceptable the Council's "Statement of Policy Containing Conditions and Terms Under WHith Project Grants Are Mbde." " Signature~ ~ _ Dir.amraf Fr~.e 713'SO 5 - 4="K `_

List finoncial support for reswrch from oll sourc.s, lncluding own Institution, for this and/or ralated research projeds. No support for this p Source ~oject is available from other sources. ~%d Amount Duration ~

R: REDACTED MATERIAL Chemist (Biochemist), USDA, New Orleans. Assistant Professor, Loyrola_University, New Orleans. 1954-1956 1950-1953 Occasional 1557 Columbia: University. Faculty of Pure Science • . 1952 same 1950 Long Island University Assistant Professor, Department of Biochemistry and Nutrition,•" . - University of 1exa•s Medica I Br<nclr Galveston Texa.s: , , ,_. b U i d S l a te Col orator, n tates Dept: of Agriculture. Associate Professor, Southern Illinois University, Edwardsville. Lecturer, Hunter College, New York. Teaching Assistant, Columbia University. Editor: Macmillan, Acad. Press, SchoT. Lib. Present ent areas of scholarship: The enzymes of vectorial subcellular structure tc, enzyme function, Membrane ATPase and muscle disease; enzyme studies in the . ( transport ) metabolism, relationship of ' microcalorimetry applied to biochemical J myopathic mouse; routine calorimetric medical analysis; a book 'Biochemical under my editorship, will be published by Academic Press. Microcalorimetry' being written together with a group of distinguished collaborators; ' ...~ Recent bibliograPh y: . Patel, A. B., Brown, • H. D. (1967 ) On the mechanism of trypsin e sterase activity. Ms. submitted. Brown, H. D., Rigdon, R. H., Chattopadhyay, S. K., and Patel, A. (1967) O Enzyme studies ir. the myopathic duck. 1`~Is, submitted. . C, in human myopathy'. Ms. submitted. norma ty Brown, H. hattopadhyay, and• Patel, A. B. (1967) Erythrocyte a ., b li C D CW ~ ~. .~ . O'' Cl1~ , i

; Brown, H. D-., Chattopadhyay, S. K., and. Patel, A. B, (1967) Erythrocyte ghost ATPase. Ms. submitted. . Patel, A. B., and Brown, H. D'. (1967) Selective deamination of.nucleosides by 2, 4 dinitrophcnyl hydrazine, Nature, 214: 402. Brown, H. D., Chattopadhyay,'S. K., and Patel, A. (1967) Properties of butanol- extracted and detergent-solubilized membrane ATPase. Arch. Biochem. Biophys. . 120: 222. Schuster, C. F., Shannon, G. R., and Brown, H. D. (1967) Temperature control of collected. fractions. Ms, subn;ittcd. ~:;; adh a S K and Patel A (1967 ) The a arent multi- Brown H D Chatto pp y ; p y .,,,••,,• >^r{ plicity, of membrane ATPa se activities. Biophys. J. Supp , VII, Abst. VYA8. y' Chattopadhyay, S, K., Patel, A., Rigdon, R. H., and Browns H: D. (1967 ) ~' _;'~~ s~ Transport enzyme abnormality in myopathy. , Proc. 7th Intl. Biochem. Congress (abst,) in press, ,- . Brown, H. D., Chattopadhyay, S. K., Patel, A., and Rigdon, R. H. (1967 ) ; ;- Glycoside effect upon membrane enzymes of erythrocytes and muscle in duck myopathy. • 'Experientia, In rress, =, Brown, Ii.D„ Chattopadhyay, S. K., and Patel, A. (1966) Sarcoplasmic-reticulum ATPase upon a solid support. Bioch. Bioph. Res. Comm. 25 (3 ): 304-308. Brown, H. D., Chattopadhyay, S. K., and Patel, A. (1966) Restructuring, by =tY' linkage to insoluble matrices, of enzymes solubilized from cell particles. J. Cell Biol., 31 (2): 17A (abst.). - Brown, H• D•, Chattopadhyay, S. K., and Patel, A, (1966 ) Characteristics of an ATPase in membrane particles, solubilized, and linked to a cellulose matrix. i E l i .,.. nzymo og a n press ,. Brown, H. D. (1966) Simultaneous 3-element microcalorimetry of experimental .' and control enzyme reactions. Proc. Second Intl, Biophysics Congress. #6 (abst. 92), Brown, H. D., Patel, A., and Chattopadhyay, S. K. (1967) Cellulose-matrix supported biological substances. Ms. submitted. ' Brown, H. D. (1966) Azasteroids and heart adenosine triphosphatase. Biochem.- Pharma.col., 15: 2007-2011. Brown, H. D., Jackson, R. T., and Wai.tzman, M. B. (1967) Ciliary process ATPase: azasteroid and erythrophleum alkaloid inhibition. . Life Sciences, in press.; ' Chattopadhyay, S. K., and Brown, H. D. (1966) A new detergent useable in enzyme solubilization. Texas Jour. Sci,, 18 (3): 324. Brown, H. D•, Patel, A., and Chattopadhyay, S. K. (1966) The properties of 1 : apyrase upon a solid support. Plant Physiol,, 41 (supp.) Txvi (abst.}, . • ~. ~.~. Hutfinan, Gary and Brown, H. D. (1966 ) Heart membrane ATPase-model system ~ for inotroPic activity Texas Reports of Biol and Med 24 ( 3 ): 512 (abst ) , , . • • Brown, H. D. (1966) Sampler for automated chemical• analysis. Chemist-Analyst. 55 (3): 88-89. Brown, H. D. (1966) A characterization of the ouabain sensitivity of heart micro- somalATPase, Bioch. Bioph. Acta 120: 162-165. Brown, H. D. (1966 ) Membrane preparation and steroid-azasteroid sensitivity of transport ATPase. In, Sneil, F., Membranes and transport phenomena. Biophy. Soc., St. Louis. Brown, H. D. (1966 ) Membrane. preparation and steroid azasteroid• sensitivity of transport ATPase. Biophys. Jour, VI Supp.: 135 (abst.). Brown, H. D•, Neucere, N. J,, Altschul, A. M., and Evans; W. J. (1965) Activity patterns of purified ATPase from Arachis. Life Sciences 4: 1439•1447. i ~ 100354'7006 • =

R: REDACTED MATERIAL = calorimetry: schema for the continuous observation of the movement of Brown, H. D., Evans, W..J., and Altschul,, A. M. (1965 ) Applications of glucose across a biological rrtembrane. Bioch,. Biophys. Acta 94 (1): 302-304. ': Altschul, A. M., Evans, W. J., Carney, W'. B., TVicCourtney, E. J., and Brown, H. D. :: (1964•) Some aspects of preparative electrophoresis on polyacrylamide gel: ' application to bovine serum albumin. Life Sciences 3: 611-615. . Brown, H. D., and Altschul, A. M. (1964) Glycoside-sensitive ATPase from -'fl,rachis hypogaea.--Bioch. Bioph. Res. Comm. 15: 479-483. N. J. (1964 ) Char- Altschul A. M., Evans and Neucere - Brown H. D. W. J. , ,• , , , , , acterizaUion of a purified ATPase from. Arachis hypogaea with special reference to the relationship-of'activity to conformation. Plant Physiol. 39 ( supp ) lxi-Jxi.i " (abst.). t3rown, H. U., tvans, W. J., antl Altsctlul, A. M. ( lyb4 ) Study oL A"1'Pase by differential calorimetry.. Fed. Proc. 23 (2): 175 ( abst .) . Brown, H. D., Jackson, R. T., and DuPuy, H. J. (1964) Transport of sugar i:n ' Allium: Effects of inhibitors and ethylene. Nature 202 ( 4933 ): 722-723. 0 calorimetry of ATPase activity in potato apyrase and red-blood-cell ghosts. - A Brown, H. D., Evans, W. J., and Altschul, A. M. (1964) Analysis by differential -`s- (1964) Properties of a preparative polyac .rytamide gel electropharesis column as •'Life Sciences 3: 1487-1492. ;: Altschul, A. M., McCourtney, C. J., Evans, W. J., Carney, W. B., and Brown, H "; they affect yield and column stability. ACS al?st. Chicago, Sept. 1964. Brown, H. D. (Ed.) (1963 ) Cell Interface Reactions. Schol. Lib., N~. Y. epidermis. J. Cell. and Comp. Physiol•. 61 (3): 215-222.. • Jackson; R. T., and Brown, H. IJ. (1963 )~ Cation and glucose transport in onion ~ Memberships: I