Bull. Environm. Contam. Toxicol. 20, 31-34 (1978)

Nicotine Decreases Guanylate Cyclase Activity David L. Vesely and Gerald S. Levey 1 Division of Endocrinology and Metabolism, Department of Medicfne, University of Miami, School of Medicine, Miami, Fla. 33152

INTRODUCTION The cyclic nucleotide, guanosine 3',5'-monophosphate (cyclic GMP) appears to serve a critical role in normal cell growth, DNA synthesis, and carcinogenesis (GOLDBERG et al. 1977). Tobacco smoke and hydrazine, a carcinogen which occurs in tobacco and tobacco smoke, have been shown to stimulate guanylate cyclase [E.C. 4.6.1.2], the enzyme that catalyzes the conversion of guanosine triphosphate to guanosine 3',5'-monophosphate (cyclic GMP), (ARNOLD et al. 1977, VESELY and LEVEY 1977a,b). Nicotine, the major alkaloid in tobacco, has been reported to be absorbed by all tissues and sixty-seven percent of nicotine in cigarette-smoke is retained in the lungs of smokers (LARSON et al. 1961). Since nicotine has some tumor promoting activity (BOCK and TSO 1975) we examined the effect of nicotine on guanylate cyclase activity in a variety of tissues. METHODS Tissues used in these experiments were obtained from male Sprague-Dawley rats, weighing 150-200 grams that had been maintained ad libitum on Purina Laboratory chow. Alumina oxide, neutral activity I for column chromatography, was obtained from E. Merck, (Darmstadt, Germany). The [32p] GTP was from International Chemical and Nuclear Corporation, (Irvine, Calif.). Nicotine was obtained from Aldrich Chemical Company, Inc., (Milwaukee, Wis.) and dissolved in distilled water to prepare the concentration specified in the text. Guanylate cyclase activity was measured as previously described (VESELY and LEVEY 1977a,b,c). The various tissues were homogenized in cold 0.03 M Tris HCI, pH 7.6, and centrifuged at 37,000 g in a Sorval refrigerated centrifuge at 4 ~ for 15 minutes. The nicotine was added to the supernatant enzyme prior to addition to the reaction mixture. The final concentrations of nicotine noted in the text were calculated by their concentration in the final incubation mixture. The supernatants plus nicotine were assayed at 37 ~ for i0 minutes for guanylate cyclase activity, using a reaction mixture consisting of 20mM Tris HCI, pH 7.6; 5 mM MnCI2; 2.67 mM cyclic GMP (5 mM creatine phosphate, 11.25 U creatine phosphokinase); I00 ~g bovine serum albumin; 20 mM caffeine; [~-32P]-GTP, approximately 5 x 105 cpm; and the enzyme Please send all correspondence to: Gerald S. Levy, M.D., Professor of Medicine, Division of Endocrinology and Metabolism, Department of Medicine, P.O. Box 520875, Miami, Florida 33152

0007-4861/78/0020-0031 $01.00 9 1978 Springer-Verlag New York, Inc.

p r e p a r a t i o n having 0.2 to 0.6 m g protein. N i c o t i n e did not alter the pH of the r e a c t i o n m i x t u r e (pH 7.6). The r e a c t i o n was terminated by the a d d i t i o n of I0 ~i of 0.i M EDTA, p H 7.6, c o n t a i n i n g about 30,000 cpm of [3H]-cyclic GMP (to estimate r e c o v e r y in the s u b s e q u e n t steps) and b o i l i n g for three minutes. A f t e r c o o l i n g in an ice bath, the [32p] -cyclic GMP formed is i s o l a t e d b y sequential c h r o m a t o g r a p h y on D o w e x - 5 0 - H + and a l u m i n a using the m o d i f i c a t i o n of K R I S H N A and K R I S H N A N (1975). The r e a c t i o n m i x t u r e s w e r e d i l u t e d with 0.5 ml of d i s t i l l e d w a t e r and t r a n s f e r r e d to a D o w e x 50-H + column (i0 x 75mM). The columns were then e l u t e d w i t h a n o t h e r 0.5 ml d i s t i l l e d H20 and the e l u a t e s (i ml) were discarded. The second 1 ml of w a t e r fraction e l u t e d from the D o w e x - 5 0 - H + column was a l l o w e d to d i r e c t l y pass t h r o u g h a column of dry neutral a l u m i n a (i0 x 75 mM). The alumina columns were then e l u t e d w i t h 2 ml of 0.03 M T r i s - H C l buffer, pH 7.6. The above three mls of elutant from the a l u m i n a coluntn were c o l l e c t e d d i r e c t l y into s c i n t i l l a t i o n vials c o n t a i n i n g 15 mls of BRAY'S s o l u t i o n (1960). The eluates were then c o u n t e d in a P a c k a r d T r i - C a r b L i q u i d Scintillation spectrometer. The o v e r a l l r e c o v e r y of cyclic GMP after the two-stage c h r o m a t o g r a p h i c p r o c e d u r e was 90 to 95%. AIl of the [32p]-containing m a t e r i a l was i d e n t i f i a b l e as cyclic GMP as d e t e r m i n e d by thin layer c h r o m a t o g r a p h y on c e l l u l o s e PEI (Brinkman) u s i n g 1 M LiCI as s o l v e n t and C h r o m a r sheets (Mallinckrodt, St. Louis, Mo.) d e v e l o p e d w i t h absolute a l c o h o l and c o n c e n t r a t e d N H 4 O H (5:2, v/v). P r o t e i n was d e t e r m i n e d b y the m e t h o d of L O W R Y et al. 1951). RESULTS N i c o t i n e m a r k e d l y d e c r e a s e d g u a n y l a t e cyclase a c t i v i t y in all tissues tested (Table I). The r e s u l t a n t d e c r e a s e s in cyclic GMP a c c u m u l a t i o n s e c o n d a r y to guanylate cyclase i n h i b i t i o n w e r e h i g h l y significant. In terms of m a x i m a l i n h i b i t i o n w i t h n i c o t i n e in v i t r o stomach > lung > p a n c r e a s > liver > h e a r t > colon > spleen. The dose response r e l a t i o n s h i p for n i c o t i n e is shown in T a b l e 2. M a x i m a l inhibition w i t h n i c o t i n e was found at a c o n c e n t r a t i o n at w h i c h h y d r a z i n e bas a m a x i m a l s t i m u l a t o r y a c t i v i t y (VESELY and LEVEY 1977a). DISCUSSION The role of g u a n y l a t e cyclase and cyclic GMP in cell b i o l o g y has been d i f f i c u l t to define. As a r e s u l t of a decade of r e s e a r c h a t t e n t i o n has been focused on the role of cyclic GMP in normal and abnormal cell g r o w t h (KRAM and T O M K I N S 1973, K I M U R A and M U R A D 1975, V E S E L Y et al. 1976, D E R U B E R T I S et al. 1976a, G O L D B E R G et al. 1977). R e c e n t l y g r e a t interest has b e e n e v o k e d by the findings of several l a b o r a t o r i e s that chemical c a r c i n o g e n s influence the a c t i v i t y of g u a n y l a t e cyclase b y e i t h e r i n c r e a s i n g or d e c r e a s i n g its activity. Hydrazine (VESELY and LEVEY 1977a,b), n i t r o s a m i n e s and n i t r o s amides (DERUBERTIS and C R A V E N 1976b, V E S E L Y et al. 1977c,d), tobacco smoke and n i t r o u s oxide (ARNOLD et al. 1977), and b u t a diene d i e p o x i d e (VESELY and LEVEY 1977e) increase g u a n y l a t e

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TABLE 1 Effect of Nicotine on Guanylate Cyclase Activity

in Various Tissues

Cyclic GMP (pmoles accumulated/mg protein/10 minute incubation) a Tissue

Control

Lung Liver Pancreas Stomach Colon Spleen Heart

1375 275 201 390 605 831 163

Nicotine

• 12 • 6 • 6 • 6 • 8 i 12 • 6

117 32 17 15 80 112 18

• • i • • i •

(i00 mM) 4b 4 6 4 6 8 4

aEach value is the mean • S.E.M. of triplicate determinations each of three separate experiments.

in

bp < 0.001 for all tissues with nicotine compared to control by Student's t test for unpaired values. TABLE 2 Dose Response Relationship

of Nicotine on Hepatic Guanylate Cyclase Cyclic GMP (pmoldes accumulated/mg protein/10 minute incubation) a

Concentration(mM) 0 100 50 20 i0 1

Nicotine 285 34 230 261 284 286

• • • • • •

6 4b 6b 6 NS 6 NS 6 NS

Hydrazine 285 • 6 9000 • 20 2000 • 12 -250 • 6 --

aEach value is the mean • S.E.M. of triplicate determinations each of three separate experiments.

in

bp < 0.001 with nicotine as compared to control by Student's test. NS

Not significant.

cyclase activity.

In contrast, azo dyes, polycyclic

aromatic

hydrocarbons, aromatic amines, aflatoxins (VESELY and LEVEY 1977f) dimethylhydrazine, and hydrazine sulfate (VESELY et al. 1977b) decrease guanylate cyclase activity. Nicotine, wh• has been reported to be absorbed in all tissues (LARSON et al. 1961), inhibited guanylate cyclase activity in every tissue tested including liver, lung, pancreas, stomach, colon, spleen, and heart~ The relationship of the guanylate cyclase inhibition to the deleterious effects of nicotine such as

33

its reported tumor-promoting activity (BOCK and TSO 1975) is unclear. However, the connection may reside in the area of DNA synthesis. Cyclic GMP increases DNA synthesis (KRAM and TOMKINS 1973 , RUDLAND et al. 1974, W E I N S T E I N et al. 1974) and this may account, at least in part, for the effects of cyclic GMP on normal and abnormal cell growth. Hydrazine increases DNA synthesis (BANKS et al. 1967) and increases guanylate cyclase activity. Conversely, nicotine has been shown to decrease DNA synthesis (VALADON and MUMMERY 1974, ROSZMAN et al. 1975). Thus, the observation that n i c o t i n e - i n d u c e d a decrease in guanylate cyclase activity and cyclic GMP generation is consistent with, and may be the m e c h a n i s m by which, nicotine decreased DNA synthesis. ACKNOWLEDGEMENTS Dr. Levey is an Investigator of the Howard Hughes Medical Institute. This work supported by NIH grant HL 13715-07. REFERENCES ARNOLD, W.P., ALDRED, R. and F. MURAD: Science 198, 934 (1977). BANKS, W.L., CLARK, D.A. and E.R. STEIN: Proc. Soc. Exp. Biol. Med. 124, 595 (1967). BOCK, F.G. and T.C. TSO: Proc. 3rd World Conference Smoking Health. (U.S. Dept. of Health, Education and Welfare) Vol. i, 161 (1975). BRAY, G.S.: Anal. Biochem. ~, 279 (1960). DERUBERTIS, F.R., CHAYOTH, R. and J.B. FIELD: J. Clin. Invest. 57, 641 (1976a). DERUBERTIS, F.R. and P.A. CRAVEN: Science 193, (1976b). GOLDBERG, N.D. and M.K. HADDOX: Ann. Rev. Biochem. 4_66, 823 (1977). KIMURA, H. and F. MURAD: Proc. Natl. Acad. Sci. USA 72, 1965 (1975). KRAM, R. and G.M. TOMKINS: Proc. Natl. Acad. Sci. U S A 70, 1659 (1973). KRISHNA, G. and N.A. KRISHNAN: J. Cyclic Nucl. Res. i, 293 (1975). LARSON, P.S., HAAG, H.G. and H. SILVETTE: Tobacco, Experimental and Clinical Studies. Williams and Wilkins Co., Baltimore, Md. 1961. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and R.J. RANDALL: J. Biol. Chem. 193, 256 (1951). ROSZMAN, T.L., ELLIOTT, L.H., and A.S. ROGERS: Am. Review of Resp. Disease !ll, 453 (1975). RUDLAND, P.S., G O S P O D A R W I C Z , D. and W. SEIFERT: Nature 250, 741 (1974). VALADON, L.R.G. and R.S. MUMMERY: Microbios. 10A, 97 (1974). VESELY, D.L., CHOWN, J. and G.S. LEVEY: J. Mol. and Cell Cardiology 8, 909 (1976). VESELY, D.L. and G.S. LEVEY: Biochem. Biophys. Res. Commun. 74(2), 780 (1977a). VESELY, D.L., ROVERE, L.E. and G.S. LEVEY: Enzyme, in press. (1977b). VESELY, D.L. and G.S. LEVEY: Proc. Soc. Exp. Biol. and Med. 155(3), 301 (1977c). VESELY, D.L., ROVERE, L.E. and G.S. LEVEY: Cancer Res. 37, 28 (1977d). VESELY, D.L. and G.S. LEVEY: Enzyme, in press, (1977e). VESELY, D.L. and G.S. LEVEY: Clin. Res. 25(3), 502A (1977f). WEINSTEIN, Y., CHAMBERS, D.A., BOURNE, H.R. and K.L. MELMON: Nature 251, 352 (1974). 34