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Effects of ethanol on the cyclic AMP system of the dog gastric mucosa
275
European Journal of Pharmacology, 38 (1976) 275--279 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
EFFECTS OF ETHANOL ON THE CYCLIC AMP SYSTEM OF THE DOG GASTRIC MUCOSA JUHANI PUURUNEN, HEIKKI KARPPANEN, MATTI KAIRALUOMA and TEUVO LARMI
Departments of Pharmacology and Surgery, University of Oulu, SF-90 220 Oulu 22, Finland Received 9 Septemper 1975, revised MS received 27 January 1976, accepted 3 April 1976
J. PUURUNEN, H. KARPPANEN, M. KAIRALUOMA and T. LARMI, Effects of ethanol on cyclic AMP system of the dog gastric mucosa, European J. Pharmacol. 38 (1976) 275--279. The effect of ethanol on the cyclic AMP system of the dog fundic mucosa was studied in vitro. The gastric mucosal content of cyclic AMP was increased by 2.5% ethanol, whereas 10 and 20% ethanol decreased the mucosal content of cyclic AMP. The activity of adenylate cyclase was increased by 2.5 and 5% ethanol, whereas 10% ethanol did not significantly affect it. The activity of cyclic AMP phosphodiesterase was inhibited by ethanol in a competitive manner. The increase in the gastric mucosal content of cyclic AMP, induced by low concentrations of ethanol, is apparently due to the stimulation of adenylate cyclase and inhibition of phosphodiesterase. Changes in the phosphodiesterase or adenylate cyclase activities do not explain the decrease of the mucosal content of cyclic AMP by higher concentrations of ethanol. The mechanism of the decrease is discussed. Gastric acid secretion Adenylate cyclase
Gastric mucosa
Cyclic AMP
1. Introduction Low concentrations of ethanol in the gastric lumen increase the secretion of gastric acid in various species, including man and dog (Beazell and Ivy, 1940; Goodman and Gilman, 1970). However, in anaesthetized rat, orally administered ethanol inhibits the secretion of gastric acid at concentrations down to 1% (Puurunen and Karppanen, 1975). Also in the dog, higher ethanol concentrations decrease the content of hydrogen ions in the gastric juice {Beazell and Ivy, 1940; Davenport, 1967; Weisbrodt et al., 1973; Sernka et al., 1974). In the dog, the stimulatory effect of ethanol on the output of acid may be partly due to the release of gastrin from the antral mucosa (Woodward et al., 1955; Woodward et al., 1957}. However, ethanol also increases the secretion of acid when applied directly into the Heidenhain pouch {Davenport, 1967; Weisbrodt et al., 1973). These findings suggest
Phosphodiesterase
Ethanol
that ethanol also stimulates the parietal cells locally. Cyclic AMP may be an intracellular mediator of the hormone-stimulated gastric acid secretion in various species (Harris et al., 1965; Domschke et al., 1972; Bieck et al., 1973; Karppanen and Westermann, 1973; Ruoff and Sewing, 1973; Karppanen et al., 1974). Ethanol affects the cyclic AMP system in many tissues (Gorman and Bitensky, 1970; Greene et al., 1971; Volicer and Gold, 1973). Ethanol inhibits the activity of adenylate cyclase in the rat gastric mucosa (Tague and Shanbour, 1974; Puurunen and Karppanen, 1975) and decreases the content of cyclic AMP both in vivo and in vitro (Puurunen and Karppanen, 1975). Low ethanol concentrations stimulate the adenylate cyclase and increase the content of cyclic AMP in the human gastric mucosa in vitro (Karppanen et al., in press). The findings in man and rat suggest that ethanol might influence the secretion of gastric acid by chang-
276
ing the mucosal content of cyclic AMP (Puurunen and Karppanen, 1975; Karppanen et al., in press). The involvement of cyclic AMP in the canine gastric acid secretion has been challenged (Mao et al., 1972). However, other studies suggest that cyclic AMP may also regulate the secretion of gastric acid in the dog (Robertson et al., 1950; Bieck et al., 1973). In the present work we have studied the effects of ethanol on the cyclic AMP content of the isolated canine gastric mucosa. The effects of ethanol on adenylate cyclase and phosphodiesterase, the enzymes which control the intracellular level of the nucleotide, were also studied.
J. P U U R U N E N E T AL.
2. Materials and methods
riod, 2 ml of ice cold 10% trichloroacetic acid was added. Thereafter, the samples were homogenized for 40 sec with an ultrasonic homogenizer (Fischer Ultrasonic Probe ®, manufactured by Blackstone Ultrasonics Inc.). The total cyclic AMP in the tissue and the incubation medium was thus measured. After homogenization the samples were centrifuged for 2 min at 8,000 X g. Aliquots (0.2 ml) of the supernatant were washed 3 times with 1 ml of ether in the presence of 20 pl of 1 N HC1, in order to remove excess trichloroacetic acid. After lyophilization, cyclic AMP was determined by the protein binding assay of Gilman (1970). At the concentrations used, alcohol does not interfere with the assay procedure Puurunen and Karppanen, 1975). Protein was determined by the biuret method (Layne, 1954).
2.1. Isolation o f gastric mucosa
2.3. Assay of adenylate cyclase activity
Fundic gastric mucosa of three mongrel dogs was utilized for the in vitro studies. Dogs were fasted for 20 h and anaesthetized with pentobarbital (Nembutal®), 30 mg/kg i.v. at 9.00 a.m. The abdomen was opened with a longitudinal midline incision and the stomach was removed. A piece of the fundic mucosa was dissected from the other layers of the stomach and placed immediately in a carbogen-gassed, ice cold Tyrode solution. Assays were performed within an hour of the resection.
Pieces of fundic mucosa were homogenized manually in 40 mM Tris-HC1 buffer, pH 7.4, by means of a teflon homogenizer. The homogenate was centrifuged for 2 min at 1,000 X g in order to remove mucus from the sample. Then the supernatant was centrifuged for 20 min at 10,000 X g at 4°C. The supernatant was discarded and the pellet was resuspended in 40 mM Tris-HC1 buffer, pH 7.4. The incubation mixture for adenylate cyclase contained 40 mM Tris-HC1 buffer at pH 7.4, 15 mM MgSO4, 1 mM ATP, 2 mM theophylline, 20 mM phosphoenolpyruvate, 6.5 pg pyruvate kinase and tissue homogenate (0.2-0.3 mg protein/sample) in a total volume of 100 pl. The reaction was linear for at least 10 min. The samples were incubated for 10 min at 37°C. The reaction was stopped by placing the tubes in a boiling water bath for 3 min. The samples were then diluted with 400 #l of redistilled water. After centrifuging for 2 min at 8,000 X g duplicate 50 pl aliquots of the supernatant were used for the determination of cyclic AMP by the protein binding assay of Gilman (1970). The calibration standards, blanks and recoveries were done as described
2. 2. Determination o f cyclic AMP The in vitro effects of ethanol on the mucosal content of cyclic AMP were studied by the method previously described in detail by us (Karppanen et al., 1974). In the present study, the fundic mucosal cells were scraped off the muscle layer of mucosa and incubated in a volume of 2 ml. The protein content of the samples was 3--5 mg. The samples were further handled as described in the original method. Immediately after the 5 min incubation pe-
E T H A N O L A N D G A S T R I C M U C O S A L CYCLIC AMP
in detail by Schwabe et al. (1974), but the standards, like the samples, contained 1 mM ATP in order to correct for possible interference with the protein binding assay of cyclic AMP.
277 6
.s
2.4. Assay o f phosphodiesterase activity Pieces of fundic mucosa was homogenized in 40 mM Tris-HC1 buffer, pH 7.4, by means of a teflon homogenizer at 600 rpm for 1 min. The homogenate was centrifuged for 5 min at 1,000 × g. The activity of the cyclic AMP phosphodiesterase of the supernatant was assayed by the method of Thompson and Appleman (1971) with slight modifications as described by Klotz et al. (1972). 2. 5. Agents used Pent®barbital sodium (Nembutal ®, Abbot Laboratories Ltd., Queenborough, 3H(8)-labeled cyclic adenosine 3',5'-monophosphate, specific activity 30 Ci/mmole (The Radiochemical Centre, Amersham); cyclic adenosine-3',5'-monophosphate (cyclic A M P ) a n d adenosine-5'-triphosphate (ATP) (Boehringer, Mannheim); anion-exchange resin Dowex @ 1 ×2, 200--400 mesh, p.a. quality (Serva, Heidelberg); phosphoenolpyruvate, pyruvate kinase, cyclic AMP dependent protein kinase, protein kinase inhibitor and crotalus atrox (Sigma Chemical Co., St. Louis); theophylline (Merck, Darmstadt) and ethanol, 94% (Oy Alko Ab, Helsinki). All other chemicals were of reagent grade or of the best commercially available grade.
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Fig. 1. E f f e c t of e t h a n o l o n t h e c o n t e n t o f cyclic AMP of t h e dog gastric mucosa. E a c h p o i n t represents t h e m e a n ± S.E. o f 1 7 - - 1 9 e x p e r i m e n t s . * p < 0.05 a n d * * p < 0.001 i n d i c a t e t h e statistical significance o f t h e changes w i t h r e s p e c t t o t h e c o n t r o l value.
AMP by about 50% (p < 0.001). With 20% ethanol the decrease of the cyclic AMP content was still more pronounced, approximately 70% (p < 0.001).
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A concentration of 1.25% (v/v) ethanol did not significantly change the mucosal content of cyclic AMP, whereas 2.5% ethanol increased it {p < 0.05). At a concentration of 10%, ethanol decreased the level of cyclic
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"1, E T H A N O L
3. Results 3.1. Effects of ethanol on gastri c mucosal cyclic AMP (fig. 1.)
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1.25
5
5
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1.25 2.5 5 10 "/. ETHANOL (V/v) /
Fig. 2. E f f e c t o f e t h a n o l o n t h e a d e n y l a t e cyclase activity o f t h e dog gastric mucosa. On t h e left, t h e effect o f e t h a n o l o n t h e basal activity o f a d e n y l a t e cyclase is s h o w n . O n t h e right, t h e e f f e c t of e t h a n o l on N a F - s t i m u l a t e d (10 m M N a F ) a d e n y l a t e cyclase is s h o w n . * p < 0.05 a n d ** p < 0.01 i n d i c a t e t h e statistical significance o f t h e increase in the activity o f a d e n y l a t e cyclase w i t h r e s p e c t t o the c o n t r o l value.
278
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Fig. 3. I n h i b i t i o n b y e t h a n o l of the cyclic AMP p h o s p h o d i e s t e r a s e activity o f the dog gastric mucosa. T h e e n z y m e activity o f t h e 1,000 x g s u p e r n a t a n t of t h e m u c o s a l h o m o g e n a t e was t e s t e d in t h e p r e s e n c e o f 5 x 10-7.to 2 x 10 -6 M cyclic AMP as substrate. V is expressed as n m o l e s of cyclic AMP h y d r o l y z e d p e r m g protein per •in. Symbols: control • ~, 5% ethanol x - - x , 9.4% e t h a n o l • m. Each value is the m e a n o f 4 e x p e r i m e n t s , p e r f o r m e d in duplicate.
3.2. Effect of ethanol on gastric mucosal adenylate cyclase (fig. 2.) At concentrations of 2.5 and 5% (v/v), ethanol increased the basal activity of adenylate cyclase by 36 and 22% respectively. The NaF-stimulated activity of adenylate cyclase was increased approximately 42% by 2.5% ethanol and 27% by 5% ethanol. However, the latter increase was not statistically significant. 10% ethanol had no effect on the activity of adenylate cyclase.
3.3. Effect of ethanol on gastric mucosal phosphodiesterase (fig. 3.) In the presence of low concentrations (0.5--2 #M) of cyclic AMP, ethanol inhibited the phosphodiesterase activity of the 1,000 X g supernatant in a competitive manner. The Ki-value was 9 X 10 .2 M, which would correspond to an ethanol concentration of 5%
AMP. This effect of ethanol is explained by the stimulation of adenylate cyclase and the inhibition of phosphodiesterase activity. However, at higher concentrations, ethanol decreased the content of the nucleotide in the incubate. The present results do not explain this effect since the activity of adenylate cyclase was not inhibited. Furthermore, the higher ethanol concentrations should be more p o t e n t inhibitors of the breakdown of the nucleotide than the lower concentrations. Our recent findings in the rat suggest that prostaglandins may be involved in the ethanol-induced inhibition of gastric acid secretion (Karppanen and Puurunen, in press). Prostaglandins affect the formation of cyclic AMP in various tissues (Kahn and Lands, 1973). Therefore, the ethanol-induced decrease of cyclic AMP might be secondary to changes in the formation of prostaglandins. Another possibility is that the portion of cyclic AMP which is released into the incubation medium (Karppanen et al., 1973) is further increased by higher concentrations of ethanol, which increase the permeability of the mucosal cells (Davenport, 1967; Kuo et al., 1974). This pool of cyclic AMP would be rapidly destroyed by the high phosphodiesterase activity which is also released from the minced mucosa (Karppanen et al., 1973). We have previously suggested that ethanol may influence the secretion of gastric acid by changing the gastric mucosal content of cyclic AMP (Puurunen and Karppanen 1975, Karppanen et al., in press), or possibly, by increasing the formation of prostaglandins Karppanen and Puurunen, in press). Further studies are needed to show whether the dual effect of local ethanol on the gastric acid secretion in the dog is due to the changes in cyclic AMP.
(v/v). Acknowledgements 4. Discussion In the isolated gastric mucosa of the dog, 2.5% ethanol increased the content of cyclic
T h e skilful technical assistance o f Mrs. Sirpa Rut a n e n and Miss Tiina H e i k k i n e n is gratefully a c k n o w l edged.
ETHANOL AND GASTRIC MUCOSAL CYCLIC AMP
References Beazell, J.M. and A.C. Ivy, 1940, The influence of alcohol on the digestive tract, Quart. J. Studies Alcohol 1, 45. Bieck, P.R., J.A. Oates, G.A. Robison and R.B. Adkins, 1973, Cyclic AMP in the regulation of gastric acid secretion in dogs and humans, Amer. J. Physiol. 224, 158. Davenport, H.W., 1967, Ethanol damage to canine oxyntic glandular mucosa, Proc. Soc. Exptl. Biol. Med. 126, 657. Domschke, W., S. Domschke, M. Classen and L. Demling, 1972, Histamin und die Magensekretion der Ratte: Adenosin-3',5'-monophosphat als 'second messenger', Naturwiss. 59,521. Gilman, A.G., 1970, A protein binding assay for adenosine 3',5'-cyclic monophosphate, Proc. Nat. Acad. Sci. 67,305. Goodman, L.S. and A. Gilman, 1970, The pharmacological basis of therapeutics, edn. 6 (The Macmilan Company, New York) p. 138. Gorman, R.E. and M.W. Bitensky, 1970, Selective activation by short chain alcohols of glucagon responsive adenyl cyclase in liver, Endocrinol. 87, 1075. Green, H.L., R.H. Herman and S. Kraemer, 1971, Stimulation of jejunal adenyl cyclase by ethanol, J. Lab. Clin. Med. 78,336. Harris, J.B. and D. Alonso, 1965, Stimulation of the gastric mucosa by adenosine 3',5'-monophosphate, Federation Proc. 24, 1368. Kahn, R.J. and W.E.M. Lands, 1973, Prostaglandins and Cyclic AMP. Biological and Clinical Applications (Academic Press, Inc., New York and London). Karppanen, H. and J. Puurunen, 1976, Ethanol, indomethacin and gastric acid secretion in the rat, European J. Pharmacol. 35,221. Karppanen, H.O. and E. Westermann, 1973, Increased production of cyclic AMP in gastric tissue by stimulation of histamine (H 2)-receptors, NaunynSchmiedeb. Arch. Pharmacol. 279, 83. Karppanen, H.O., P.J. Neuvonen and E. Westermann, 1973, Effect of some gastric stimulants on the production of cyclic AMP in the minced gastric mucosa, Naunyn-Schmiedeb. Arch. Pharmacol. 277, R35. Karppanen, H.O., P.J. Neuvonen, P.R. Bieck and E. Westermann, 1974, Effect of histamine, pentagastrin and theophylline on the production of cyclic AMP in isolated gastric tissue fo the guinea pig, Naunyn-Schmiedeb. Arch. Pharmacol. 284, 15. Karppanen, H., J. Puurunen, M. Kairaluoma and T. Larmi, 1976, Effects of ethyl alcohol on the adenosine 3',5'-monophosphate system of the human
279 gastric mucosa, Scand. J. Gastroenterol. (in press). Klotz, U., S. Berndt and K. Stock, 1972, Characterization of multiple cyclic nucleotide phosphodiesterase activities of rat adipose tissue, Life Sci. 11, 7. Kuo, Y.-J., L.L. Shanbour and T.J. Sernka, 1974, Effect of ethanol on permeability and ion transport in the isolated dog stomach, Dig. Dis. 19,818. Layne, E., 1954, Spectrophotometric and turbidimetric methods for measuring proteins, in: Methods in Enzymology, eds. S.E.P. Colowick and N.O. Kaplan (Academic Press, New York) p. 447. Mao, C.C., L.L. Shanbour, D.S. Hodkins and E.D. Jacobson, 1972, Adenosine 3',5'-monophosphate (cyclic AMP) and secretion in the canine stomach, Gastroenterology 63,427. Puurunen, J. and H. Karppanen, 1975, Effects of ethanol on gastric acid secretion and gastric mucosal cyclic AMP in the rat, Life Sci. 16, 1513. Robertson, C.R., C.E. Rosiere, D. Blickenstaff and M.I. Grossman, 1950, The potentiating effect of certain xanthine derivates on gastric acid secretory responses in the dog, J. Pharmacol. Exptl. Therap. 99, 362. Ruoff, H.-J. and K.-Fr. Sewing, 1973, Cyclic 3',5'adenosine monophosphate in the rat gastric mucosa after starvation, feeding and pentagastrin, Scand. J. Gastroenterol. 8,241. Schwabe, U., R. Ebert and P.S. SchSnhSfer, 1974, Sensitive determination for adenylate cyclase activity by cyclic adenosine 3',5'-monophosphate protein binding assay, Naunyn-Schmiedeb. Arch. Pharmacol. 286, 83. Sernka, T.J., C.W. Gilleland and L.L. Shanbour, 1974, Effects of ethanol on active transport in the dog stovhach, Amer. J. Physiol. 226,397. Tague, L.L. and L.L. Shanbour, 1974, Effects of ethanol on gastric mucosal adenosine 3',5'-monophosphate (cAMP), Life Sci. 14, 1065. Thompson, W.J. and M.M. Appleman, 1971, Multiple cyclic nucleotide phosphodiesterase activities from rat brain, Biochemistry 10,311. Volicer, L. and B.I. Gold, 1973, Effect of ethanol on cyclic AMP levels in the rat brain, Life Sci. 13, 269. Weisbrodt, N.W., M. Kienzle and A.R. Cooke, 1973, Comparative effects of aliphatic alcohols on the gastric mucosa, Proc. Soc. Exptl. Biol. Med. 142, 450. Woodward, E.R., C. Robertson, H.D. Ruttenberg and H. Schapiro, 1957, Alcohol as a gastric secretory stimulant, Gastroenterology 32,727. Woodward, E.R., D.S. Slotten and V.C. Tillmans, 1955, Mechanism of alcoholic stimulation of gastric secretion, Proc. Soc. Exptl. Biol. Med. 89, 428.
European Journal of Pharmacology, 38 (1976) 275--279 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
EFFECTS OF ETHANOL ON THE CYCLIC AMP SYSTEM OF THE DOG GASTRIC MUCOSA JUHANI PUURUNEN, HEIKKI KARPPANEN, MATTI KAIRALUOMA and TEUVO LARMI
Departments of Pharmacology and Surgery, University of Oulu, SF-90 220 Oulu 22, Finland Received 9 Septemper 1975, revised MS received 27 January 1976, accepted 3 April 1976
J. PUURUNEN, H. KARPPANEN, M. KAIRALUOMA and T. LARMI, Effects of ethanol on cyclic AMP system of the dog gastric mucosa, European J. Pharmacol. 38 (1976) 275--279. The effect of ethanol on the cyclic AMP system of the dog fundic mucosa was studied in vitro. The gastric mucosal content of cyclic AMP was increased by 2.5% ethanol, whereas 10 and 20% ethanol decreased the mucosal content of cyclic AMP. The activity of adenylate cyclase was increased by 2.5 and 5% ethanol, whereas 10% ethanol did not significantly affect it. The activity of cyclic AMP phosphodiesterase was inhibited by ethanol in a competitive manner. The increase in the gastric mucosal content of cyclic AMP, induced by low concentrations of ethanol, is apparently due to the stimulation of adenylate cyclase and inhibition of phosphodiesterase. Changes in the phosphodiesterase or adenylate cyclase activities do not explain the decrease of the mucosal content of cyclic AMP by higher concentrations of ethanol. The mechanism of the decrease is discussed. Gastric acid secretion Adenylate cyclase
Gastric mucosa
Cyclic AMP
1. Introduction Low concentrations of ethanol in the gastric lumen increase the secretion of gastric acid in various species, including man and dog (Beazell and Ivy, 1940; Goodman and Gilman, 1970). However, in anaesthetized rat, orally administered ethanol inhibits the secretion of gastric acid at concentrations down to 1% (Puurunen and Karppanen, 1975). Also in the dog, higher ethanol concentrations decrease the content of hydrogen ions in the gastric juice {Beazell and Ivy, 1940; Davenport, 1967; Weisbrodt et al., 1973; Sernka et al., 1974). In the dog, the stimulatory effect of ethanol on the output of acid may be partly due to the release of gastrin from the antral mucosa (Woodward et al., 1955; Woodward et al., 1957}. However, ethanol also increases the secretion of acid when applied directly into the Heidenhain pouch {Davenport, 1967; Weisbrodt et al., 1973). These findings suggest
Phosphodiesterase
Ethanol
that ethanol also stimulates the parietal cells locally. Cyclic AMP may be an intracellular mediator of the hormone-stimulated gastric acid secretion in various species (Harris et al., 1965; Domschke et al., 1972; Bieck et al., 1973; Karppanen and Westermann, 1973; Ruoff and Sewing, 1973; Karppanen et al., 1974). Ethanol affects the cyclic AMP system in many tissues (Gorman and Bitensky, 1970; Greene et al., 1971; Volicer and Gold, 1973). Ethanol inhibits the activity of adenylate cyclase in the rat gastric mucosa (Tague and Shanbour, 1974; Puurunen and Karppanen, 1975) and decreases the content of cyclic AMP both in vivo and in vitro (Puurunen and Karppanen, 1975). Low ethanol concentrations stimulate the adenylate cyclase and increase the content of cyclic AMP in the human gastric mucosa in vitro (Karppanen et al., in press). The findings in man and rat suggest that ethanol might influence the secretion of gastric acid by chang-
276
ing the mucosal content of cyclic AMP (Puurunen and Karppanen, 1975; Karppanen et al., in press). The involvement of cyclic AMP in the canine gastric acid secretion has been challenged (Mao et al., 1972). However, other studies suggest that cyclic AMP may also regulate the secretion of gastric acid in the dog (Robertson et al., 1950; Bieck et al., 1973). In the present work we have studied the effects of ethanol on the cyclic AMP content of the isolated canine gastric mucosa. The effects of ethanol on adenylate cyclase and phosphodiesterase, the enzymes which control the intracellular level of the nucleotide, were also studied.
J. P U U R U N E N E T AL.
2. Materials and methods
riod, 2 ml of ice cold 10% trichloroacetic acid was added. Thereafter, the samples were homogenized for 40 sec with an ultrasonic homogenizer (Fischer Ultrasonic Probe ®, manufactured by Blackstone Ultrasonics Inc.). The total cyclic AMP in the tissue and the incubation medium was thus measured. After homogenization the samples were centrifuged for 2 min at 8,000 X g. Aliquots (0.2 ml) of the supernatant were washed 3 times with 1 ml of ether in the presence of 20 pl of 1 N HC1, in order to remove excess trichloroacetic acid. After lyophilization, cyclic AMP was determined by the protein binding assay of Gilman (1970). At the concentrations used, alcohol does not interfere with the assay procedure Puurunen and Karppanen, 1975). Protein was determined by the biuret method (Layne, 1954).
2.1. Isolation o f gastric mucosa
2.3. Assay of adenylate cyclase activity
Fundic gastric mucosa of three mongrel dogs was utilized for the in vitro studies. Dogs were fasted for 20 h and anaesthetized with pentobarbital (Nembutal®), 30 mg/kg i.v. at 9.00 a.m. The abdomen was opened with a longitudinal midline incision and the stomach was removed. A piece of the fundic mucosa was dissected from the other layers of the stomach and placed immediately in a carbogen-gassed, ice cold Tyrode solution. Assays were performed within an hour of the resection.
Pieces of fundic mucosa were homogenized manually in 40 mM Tris-HC1 buffer, pH 7.4, by means of a teflon homogenizer. The homogenate was centrifuged for 2 min at 1,000 X g in order to remove mucus from the sample. Then the supernatant was centrifuged for 20 min at 10,000 X g at 4°C. The supernatant was discarded and the pellet was resuspended in 40 mM Tris-HC1 buffer, pH 7.4. The incubation mixture for adenylate cyclase contained 40 mM Tris-HC1 buffer at pH 7.4, 15 mM MgSO4, 1 mM ATP, 2 mM theophylline, 20 mM phosphoenolpyruvate, 6.5 pg pyruvate kinase and tissue homogenate (0.2-0.3 mg protein/sample) in a total volume of 100 pl. The reaction was linear for at least 10 min. The samples were incubated for 10 min at 37°C. The reaction was stopped by placing the tubes in a boiling water bath for 3 min. The samples were then diluted with 400 #l of redistilled water. After centrifuging for 2 min at 8,000 X g duplicate 50 pl aliquots of the supernatant were used for the determination of cyclic AMP by the protein binding assay of Gilman (1970). The calibration standards, blanks and recoveries were done as described
2. 2. Determination o f cyclic AMP The in vitro effects of ethanol on the mucosal content of cyclic AMP were studied by the method previously described in detail by us (Karppanen et al., 1974). In the present study, the fundic mucosal cells were scraped off the muscle layer of mucosa and incubated in a volume of 2 ml. The protein content of the samples was 3--5 mg. The samples were further handled as described in the original method. Immediately after the 5 min incubation pe-
E T H A N O L A N D G A S T R I C M U C O S A L CYCLIC AMP
in detail by Schwabe et al. (1974), but the standards, like the samples, contained 1 mM ATP in order to correct for possible interference with the protein binding assay of cyclic AMP.
277 6
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2.4. Assay o f phosphodiesterase activity Pieces of fundic mucosa was homogenized in 40 mM Tris-HC1 buffer, pH 7.4, by means of a teflon homogenizer at 600 rpm for 1 min. The homogenate was centrifuged for 5 min at 1,000 × g. The activity of the cyclic AMP phosphodiesterase of the supernatant was assayed by the method of Thompson and Appleman (1971) with slight modifications as described by Klotz et al. (1972). 2. 5. Agents used Pent®barbital sodium (Nembutal ®, Abbot Laboratories Ltd., Queenborough, 3H(8)-labeled cyclic adenosine 3',5'-monophosphate, specific activity 30 Ci/mmole (The Radiochemical Centre, Amersham); cyclic adenosine-3',5'-monophosphate (cyclic A M P ) a n d adenosine-5'-triphosphate (ATP) (Boehringer, Mannheim); anion-exchange resin Dowex @ 1 ×2, 200--400 mesh, p.a. quality (Serva, Heidelberg); phosphoenolpyruvate, pyruvate kinase, cyclic AMP dependent protein kinase, protein kinase inhibitor and crotalus atrox (Sigma Chemical Co., St. Louis); theophylline (Merck, Darmstadt) and ethanol, 94% (Oy Alko Ab, Helsinki). All other chemicals were of reagent grade or of the best commercially available grade.
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Fig. 1. E f f e c t of e t h a n o l o n t h e c o n t e n t o f cyclic AMP of t h e dog gastric mucosa. E a c h p o i n t represents t h e m e a n ± S.E. o f 1 7 - - 1 9 e x p e r i m e n t s . * p < 0.05 a n d * * p < 0.001 i n d i c a t e t h e statistical significance o f t h e changes w i t h r e s p e c t t o t h e c o n t r o l value.
AMP by about 50% (p < 0.001). With 20% ethanol the decrease of the cyclic AMP content was still more pronounced, approximately 70% (p < 0.001).
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A concentration of 1.25% (v/v) ethanol did not significantly change the mucosal content of cyclic AMP, whereas 2.5% ethanol increased it {p < 0.05). At a concentration of 10%, ethanol decreased the level of cyclic
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3. Results 3.1. Effects of ethanol on gastri c mucosal cyclic AMP (fig. 1.)
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Fig. 2. E f f e c t o f e t h a n o l o n t h e a d e n y l a t e cyclase activity o f t h e dog gastric mucosa. On t h e left, t h e effect o f e t h a n o l o n t h e basal activity o f a d e n y l a t e cyclase is s h o w n . O n t h e right, t h e e f f e c t of e t h a n o l on N a F - s t i m u l a t e d (10 m M N a F ) a d e n y l a t e cyclase is s h o w n . * p < 0.05 a n d ** p < 0.01 i n d i c a t e t h e statistical significance o f t h e increase in the activity o f a d e n y l a t e cyclase w i t h r e s p e c t t o the c o n t r o l value.
278
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1.5
2.0
Fig. 3. I n h i b i t i o n b y e t h a n o l of the cyclic AMP p h o s p h o d i e s t e r a s e activity o f the dog gastric mucosa. T h e e n z y m e activity o f t h e 1,000 x g s u p e r n a t a n t of t h e m u c o s a l h o m o g e n a t e was t e s t e d in t h e p r e s e n c e o f 5 x 10-7.to 2 x 10 -6 M cyclic AMP as substrate. V is expressed as n m o l e s of cyclic AMP h y d r o l y z e d p e r m g protein per •in. Symbols: control • ~, 5% ethanol x - - x , 9.4% e t h a n o l • m. Each value is the m e a n o f 4 e x p e r i m e n t s , p e r f o r m e d in duplicate.
3.2. Effect of ethanol on gastric mucosal adenylate cyclase (fig. 2.) At concentrations of 2.5 and 5% (v/v), ethanol increased the basal activity of adenylate cyclase by 36 and 22% respectively. The NaF-stimulated activity of adenylate cyclase was increased approximately 42% by 2.5% ethanol and 27% by 5% ethanol. However, the latter increase was not statistically significant. 10% ethanol had no effect on the activity of adenylate cyclase.
3.3. Effect of ethanol on gastric mucosal phosphodiesterase (fig. 3.) In the presence of low concentrations (0.5--2 #M) of cyclic AMP, ethanol inhibited the phosphodiesterase activity of the 1,000 X g supernatant in a competitive manner. The Ki-value was 9 X 10 .2 M, which would correspond to an ethanol concentration of 5%
AMP. This effect of ethanol is explained by the stimulation of adenylate cyclase and the inhibition of phosphodiesterase activity. However, at higher concentrations, ethanol decreased the content of the nucleotide in the incubate. The present results do not explain this effect since the activity of adenylate cyclase was not inhibited. Furthermore, the higher ethanol concentrations should be more p o t e n t inhibitors of the breakdown of the nucleotide than the lower concentrations. Our recent findings in the rat suggest that prostaglandins may be involved in the ethanol-induced inhibition of gastric acid secretion (Karppanen and Puurunen, in press). Prostaglandins affect the formation of cyclic AMP in various tissues (Kahn and Lands, 1973). Therefore, the ethanol-induced decrease of cyclic AMP might be secondary to changes in the formation of prostaglandins. Another possibility is that the portion of cyclic AMP which is released into the incubation medium (Karppanen et al., 1973) is further increased by higher concentrations of ethanol, which increase the permeability of the mucosal cells (Davenport, 1967; Kuo et al., 1974). This pool of cyclic AMP would be rapidly destroyed by the high phosphodiesterase activity which is also released from the minced mucosa (Karppanen et al., 1973). We have previously suggested that ethanol may influence the secretion of gastric acid by changing the gastric mucosal content of cyclic AMP (Puurunen and Karppanen 1975, Karppanen et al., in press), or possibly, by increasing the formation of prostaglandins Karppanen and Puurunen, in press). Further studies are needed to show whether the dual effect of local ethanol on the gastric acid secretion in the dog is due to the changes in cyclic AMP.
(v/v). Acknowledgements 4. Discussion In the isolated gastric mucosa of the dog, 2.5% ethanol increased the content of cyclic
T h e skilful technical assistance o f Mrs. Sirpa Rut a n e n and Miss Tiina H e i k k i n e n is gratefully a c k n o w l edged.
ETHANOL AND GASTRIC MUCOSAL CYCLIC AMP
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