- Email: [email protected]
Histamine-Sensitive Adenylate Cyclase of Human Gastric Mucosa
Vol. 73 , No.2
73:429-431, 1977 Copyright © 1977 by the American Gastroenterological Association
GASTROENTEROLOGY
Printed in U .S A .
HISTAMINE-SENSITIVE ADENYLATE CYCLASE OF HUMAN GASTRIC MUCOSA Bernd Simon and Horst Kather Medizinische Universitatsklinik Heidelberg und Gastroenterologische Abteilung der Medizinischen Universitatsklinik Heidelberg,F.R.G. ABSTRACT. The adenylate cyclase system of human fundic gastric mucosa was found to respond to histamine, prostaglandin E 2 and the non-hormonal activators NaF and 5~guanylyl-imidodiphosphate (GMP(PNP)). Half maximal stimulation of enzyme activity was observed at a histamine concentration of 5o pM. Maximal stimulation (about 25o%) occurred at a histamine concentration of 1 rnM. The stimulatory effect of histamine was competitively inhibited by cimetidine. The stimulatory effect of prostaglandin E 2 was found to be dose-dependent over a concentration ran~e from 0.1 pM to 1 mM exerting maximal effects at 0.3 mM. NaF and GMP(PNP) by inducing an about 3.5-fold increase of enzyme activity were more potent in stimulating the human enzyme system than histamine and prostaglandin E 2. Maximal stimulatory doses of prostaglandin E 2 and histamine had an additive effect on the adenylate cyclase activity from fundic gastric mucosa. This implies that histamine acts on an individual adenylate cyclase system. Our results are suggestive for the existence of an adenylate cyclase system in human gastric mucosa coupled to histamine H 2-receptor sites. ethanol and 3 mM ATP. The homogenate was filtered through a nylon mesh and assayed for enzyme activity by the method of Salomon et al. s .only freshly prepared membranes were used. The protein content of the samples was determined according to Lowry et al. 6 using bovine serum albumin as standard. Unless otherwise stated, data are given in nmol of cAMP formed per mg protein per 15 min. (oc,- JJ.p )ATP( 2-6 counts/mmol) and ( 3 H) cAMP (27 counts/mmol) were purchased from the Radiochemical Centre Amersham Bucks,U.K. Histamine and NaF were obtained from Merck AG,Darmstadt, GMPP(NP) was from Boehringer Mannheim. Cimetidine was kindly given to us by the Research Institute,Smith Kline and French Laboratories Ltd.,Welwyn Garden City,Hertfordshire,England. Prostaglandin E 2 was kindly given to us by Upjohn GmbH,Heppenheim,F.R.G.
The adenylate cyclase system plays a central role in hormone action. In amphibia and guinea pig, stimulation of adenylate cyclase from gastric mucosa by histamine has been shown to be an important step in the regulation of gastric secretion ",-z • Data concerning the role of cAMP in rat gastric secre• tion are still conflicting 3,"" • Activation of the human enzyme system by this secretagogue has not yet been documented. The present study shows that human gastric mucosa contains adenylate cyclases sensitive to histamine, prostaglandin E 2 and to non-hormonal activators such as NaF and GMP(PNP) METHODS Stomach tissue was obtained by subtotal gastric resection from 7 patients suffering from peptic ulcer. Scrapings of the fundus gastric mucosa (approximately 24 gr) were homogenized in a teflon glass homogenizer (Zell-Homogenisator,Colora-Messtechnik GmbH, Lorch,Wtirttemb,F.R.G)after addition of 2o ml of 5 mM Tris-HCl buffer, pH 7.6, containing 3 mM MgCl , 1 mM EDTA, 3 mM mercapto-
RESULTS Basal adenylate cyclase activity of human gastric mucosa averaged 0.8 0.3 nmol cAMP formed per mg protein per 15 min. Ascending concentrations of histamine ( 1 pM to 1o mM ) caused a dose-dependent increase of enzyme activity (figure 1). Half maximal stimulation
Address reprint requests to Dr. SIMON, Dr. KATHER 429
Table I shows the effects of maximally effective concentrations of histamine, prostaglandin E 2 and the non-hormonal activators NaF and GMP(PNP) on the adenylate cyclase system from human gastric mucosa. Additions of the hormones and the non-hormonal activators resulted in up to 2.5-4-fold stimulation of adenylate cyclase. TABLE 1 The maximal stimulatory effect of histamine, prostaglandin E 2 and non-hormonal activators on the human gastric mucosal adenylate cyclase
I
I
._.. Histomme
1.-----A
Histamine +CJmetidme
II xi0-5M)
A-- -A
Histamme +
C1metidine
rsx w- 5 ;,1)
Histamine [-tog molar]
Fig. 1 Effect of histamine on the adenylate cyclase activity _of human gastric mucosa in the presence or absence of 1 o )lM and 5o pM cimetidine. It shows a representative experiment out of 6 individual preparations. , of enzyme activity was observed at a histamine concentration of about 5o )lM. ~atur~tin~ concentrations of hJ.stamJ.ne ( 1 mM ) increased enzyme activity by about 25o%. The stimulatory effect of histamine was competitively inhibited by the histamine H 2-receptor antagonist cimetidine ( 1 o )lM and 5o }lM ) • This compound caused a dosedependent parallel right-ward shift of the histamine dose-response curve. Cimetidine up to 5o pM had no effect on basal activity of the enzyme system.
Agent
Adenylate Cyclase Activ. nmol cAMP/mg prot./15min
Basal Histamine (1 mM) Prostagl.E 2 (0.3 mM) NaF (2o mM) GMP(PNP~
(0.1 mM Histamine plus prostagl. E 2 p
< 0.02
-
0.80 + 0.30 2.00 + 0.40 2.40
-+ 0.40
-
2.80 + 0.50 3.00 + 0.45
-
- o.6o
4.40 + , n=7
Both non-hormonal activators were more potent in stimulating the enzyme activity than histamine and prostaglandin E 2. 1o mM NaF and. 0.1 mM GMP(PNP) caused an approxJ.mately 3.5-4.0-fold increase of enzyme activity. The parent nucleotide GTP had no effect on basal and hormonestimulated enzyme activities when applied in concentrations ranging from 0.1 pM to 1 mM. Maximally stimulatin~ concentrations of histamine ( 1 mM ) and prostaglandin E 2 had an additive effect on the adenylate cyclase activity from human gastric mucosa.
430
REFERENCES 1. Nakajima SB, Hirschowitz BI, Sachs G: Studies on adenyl cyclase in Necturus gastric mucosa Arch.Biochem.Biophys. 143: 123126, 1971 2. Dousa ThP, Code ChF: Effect of histamine and its methyl derivatives on cyclic AMP metabolism in gastric mucosa and its blockade by an H 2-receptor antagonist. J.clin.Invest. 53: 334-337, 1974 3. Thompson WJ, Chang LK, Rosenfeld GC, Jacobson ED: Activation of rat gastric mucosal adenylyl cyclase by secretory inhibitors Gastroenterology 72:251-254,1977 4. McNeill JH,Verma SC: Stimulation of rat gastric adenylate cyclase by histamine and histamine analogues and blockade by burimamide. Br.J.Pharmacol. 52: 1o4-1o6, 1974 5. Salomon Y, Londos C, Rodbell M: A highly sensitive adenylate cyclase assay. Anal.Biochem. 58: 541-548, 1974 6. Lowry OH, Rosebrough JJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J. Biol.Chem. 193: 265275, 1951 7. Kather H, Simon B: Catecholamine-sensitive adenylate cyclase of human fat cell ghosts. Characteristics of the GMP(PNP)liganded state. Clin.Chim.Acta 73: 497-5o4,1976 8. Way L, Durbin RP: Inhibition of gastric acid secretion in vitro by prostaglandin E 1. Nature (Lond.) 221: 874-875,1969 9. Kimberg DV: Cyclic Nucleotides and their role in gastrointestinal secretion. Gastroenterology 67: 1o23-1o64, 1974 1o. Wollin A, Code ChF, Dousa ThP: Interaction of prostaglandins and histamine with enzymes of cyclic AMP metabolism from guinea pig gastric mucosa. J.clin.Invest. 57: 1548-1553, 1976.
DISCUSSION stimulation of adenylate cyclase activity by both GMP(PNP) and NaF has been reported to occur in a variety of animal tissues irrespective of the hormone 'freceptors coupled to the system · • The stimulatory effects of these non-hormonal acti vators, therefore, suggest that the properties of the adenylate cyclase in human gastric mucosa closely resembles those of adenylate cyclases from various sources. The demonstration of an histaminesensitive adenyl ate cyclase system in human gastric mucosa supports the view that cyclic AMP is involved in the gastric secretagogue effect of histamine. The stimulatory action of histamine was competitively inhibited by the selective H 2~ceptor antagonist cimetidine. This finding supports the contention that histamine activates the adenylate cyclase system of human gastric mucosa via interaction with histamine H 2-receptor sites. ~ostaglandins are inhibitors of gastric acid secretion 8,9 • The additive effects of prostaglandin E 2 and histamine on the human enzyme system suggest that the crude homogenate of fundic gastric mucosa contains at least two different hormone-sensitive adenylate cyclases. Similar results have been reported from guinea pig gastric mu·Cosa '10 • Since crude membrane preparations were used in this study, we cannot finally discern whether the two hormones are acting on the same or on different cell types.
ACKNOWLEDGEMENTS Thanks are due to Miss Th. Fromm for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft,Bad Godesberg, WestGermany. 431
73:429-431, 1977 Copyright © 1977 by the American Gastroenterological Association
GASTROENTEROLOGY
Printed in U .S A .
HISTAMINE-SENSITIVE ADENYLATE CYCLASE OF HUMAN GASTRIC MUCOSA Bernd Simon and Horst Kather Medizinische Universitatsklinik Heidelberg und Gastroenterologische Abteilung der Medizinischen Universitatsklinik Heidelberg,F.R.G. ABSTRACT. The adenylate cyclase system of human fundic gastric mucosa was found to respond to histamine, prostaglandin E 2 and the non-hormonal activators NaF and 5~guanylyl-imidodiphosphate (GMP(PNP)). Half maximal stimulation of enzyme activity was observed at a histamine concentration of 5o pM. Maximal stimulation (about 25o%) occurred at a histamine concentration of 1 rnM. The stimulatory effect of histamine was competitively inhibited by cimetidine. The stimulatory effect of prostaglandin E 2 was found to be dose-dependent over a concentration ran~e from 0.1 pM to 1 mM exerting maximal effects at 0.3 mM. NaF and GMP(PNP) by inducing an about 3.5-fold increase of enzyme activity were more potent in stimulating the human enzyme system than histamine and prostaglandin E 2. Maximal stimulatory doses of prostaglandin E 2 and histamine had an additive effect on the adenylate cyclase activity from fundic gastric mucosa. This implies that histamine acts on an individual adenylate cyclase system. Our results are suggestive for the existence of an adenylate cyclase system in human gastric mucosa coupled to histamine H 2-receptor sites. ethanol and 3 mM ATP. The homogenate was filtered through a nylon mesh and assayed for enzyme activity by the method of Salomon et al. s .only freshly prepared membranes were used. The protein content of the samples was determined according to Lowry et al. 6 using bovine serum albumin as standard. Unless otherwise stated, data are given in nmol of cAMP formed per mg protein per 15 min. (oc,- JJ.p )ATP( 2-6 counts/mmol) and ( 3 H) cAMP (27 counts/mmol) were purchased from the Radiochemical Centre Amersham Bucks,U.K. Histamine and NaF were obtained from Merck AG,Darmstadt, GMPP(NP) was from Boehringer Mannheim. Cimetidine was kindly given to us by the Research Institute,Smith Kline and French Laboratories Ltd.,Welwyn Garden City,Hertfordshire,England. Prostaglandin E 2 was kindly given to us by Upjohn GmbH,Heppenheim,F.R.G.
The adenylate cyclase system plays a central role in hormone action. In amphibia and guinea pig, stimulation of adenylate cyclase from gastric mucosa by histamine has been shown to be an important step in the regulation of gastric secretion ",-z • Data concerning the role of cAMP in rat gastric secre• tion are still conflicting 3,"" • Activation of the human enzyme system by this secretagogue has not yet been documented. The present study shows that human gastric mucosa contains adenylate cyclases sensitive to histamine, prostaglandin E 2 and to non-hormonal activators such as NaF and GMP(PNP) METHODS Stomach tissue was obtained by subtotal gastric resection from 7 patients suffering from peptic ulcer. Scrapings of the fundus gastric mucosa (approximately 24 gr) were homogenized in a teflon glass homogenizer (Zell-Homogenisator,Colora-Messtechnik GmbH, Lorch,Wtirttemb,F.R.G)after addition of 2o ml of 5 mM Tris-HCl buffer, pH 7.6, containing 3 mM MgCl , 1 mM EDTA, 3 mM mercapto-
RESULTS Basal adenylate cyclase activity of human gastric mucosa averaged 0.8 0.3 nmol cAMP formed per mg protein per 15 min. Ascending concentrations of histamine ( 1 pM to 1o mM ) caused a dose-dependent increase of enzyme activity (figure 1). Half maximal stimulation
Address reprint requests to Dr. SIMON, Dr. KATHER 429
Table I shows the effects of maximally effective concentrations of histamine, prostaglandin E 2 and the non-hormonal activators NaF and GMP(PNP) on the adenylate cyclase system from human gastric mucosa. Additions of the hormones and the non-hormonal activators resulted in up to 2.5-4-fold stimulation of adenylate cyclase. TABLE 1 The maximal stimulatory effect of histamine, prostaglandin E 2 and non-hormonal activators on the human gastric mucosal adenylate cyclase
I
I
._.. Histomme
1.-----A
Histamine +CJmetidme
II xi0-5M)
A-- -A
Histamme +
C1metidine
rsx w- 5 ;,1)
Histamine [-tog molar]
Fig. 1 Effect of histamine on the adenylate cyclase activity _of human gastric mucosa in the presence or absence of 1 o )lM and 5o pM cimetidine. It shows a representative experiment out of 6 individual preparations. , of enzyme activity was observed at a histamine concentration of about 5o )lM. ~atur~tin~ concentrations of hJ.stamJ.ne ( 1 mM ) increased enzyme activity by about 25o%. The stimulatory effect of histamine was competitively inhibited by the histamine H 2-receptor antagonist cimetidine ( 1 o )lM and 5o }lM ) • This compound caused a dosedependent parallel right-ward shift of the histamine dose-response curve. Cimetidine up to 5o pM had no effect on basal activity of the enzyme system.
Agent
Adenylate Cyclase Activ. nmol cAMP/mg prot./15min
Basal Histamine (1 mM) Prostagl.E 2 (0.3 mM) NaF (2o mM) GMP(PNP~
(0.1 mM Histamine plus prostagl. E 2 p
< 0.02
-
0.80 + 0.30 2.00 + 0.40 2.40
-+ 0.40
-
2.80 + 0.50 3.00 + 0.45
-
- o.6o
4.40 + , n=7
Both non-hormonal activators were more potent in stimulating the enzyme activity than histamine and prostaglandin E 2. 1o mM NaF and. 0.1 mM GMP(PNP) caused an approxJ.mately 3.5-4.0-fold increase of enzyme activity. The parent nucleotide GTP had no effect on basal and hormonestimulated enzyme activities when applied in concentrations ranging from 0.1 pM to 1 mM. Maximally stimulatin~ concentrations of histamine ( 1 mM ) and prostaglandin E 2 had an additive effect on the adenylate cyclase activity from human gastric mucosa.
430
REFERENCES 1. Nakajima SB, Hirschowitz BI, Sachs G: Studies on adenyl cyclase in Necturus gastric mucosa Arch.Biochem.Biophys. 143: 123126, 1971 2. Dousa ThP, Code ChF: Effect of histamine and its methyl derivatives on cyclic AMP metabolism in gastric mucosa and its blockade by an H 2-receptor antagonist. J.clin.Invest. 53: 334-337, 1974 3. Thompson WJ, Chang LK, Rosenfeld GC, Jacobson ED: Activation of rat gastric mucosal adenylyl cyclase by secretory inhibitors Gastroenterology 72:251-254,1977 4. McNeill JH,Verma SC: Stimulation of rat gastric adenylate cyclase by histamine and histamine analogues and blockade by burimamide. Br.J.Pharmacol. 52: 1o4-1o6, 1974 5. Salomon Y, Londos C, Rodbell M: A highly sensitive adenylate cyclase assay. Anal.Biochem. 58: 541-548, 1974 6. Lowry OH, Rosebrough JJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J. Biol.Chem. 193: 265275, 1951 7. Kather H, Simon B: Catecholamine-sensitive adenylate cyclase of human fat cell ghosts. Characteristics of the GMP(PNP)liganded state. Clin.Chim.Acta 73: 497-5o4,1976 8. Way L, Durbin RP: Inhibition of gastric acid secretion in vitro by prostaglandin E 1. Nature (Lond.) 221: 874-875,1969 9. Kimberg DV: Cyclic Nucleotides and their role in gastrointestinal secretion. Gastroenterology 67: 1o23-1o64, 1974 1o. Wollin A, Code ChF, Dousa ThP: Interaction of prostaglandins and histamine with enzymes of cyclic AMP metabolism from guinea pig gastric mucosa. J.clin.Invest. 57: 1548-1553, 1976.
DISCUSSION stimulation of adenylate cyclase activity by both GMP(PNP) and NaF has been reported to occur in a variety of animal tissues irrespective of the hormone 'freceptors coupled to the system · • The stimulatory effects of these non-hormonal acti vators, therefore, suggest that the properties of the adenylate cyclase in human gastric mucosa closely resembles those of adenylate cyclases from various sources. The demonstration of an histaminesensitive adenyl ate cyclase system in human gastric mucosa supports the view that cyclic AMP is involved in the gastric secretagogue effect of histamine. The stimulatory action of histamine was competitively inhibited by the selective H 2~ceptor antagonist cimetidine. This finding supports the contention that histamine activates the adenylate cyclase system of human gastric mucosa via interaction with histamine H 2-receptor sites. ~ostaglandins are inhibitors of gastric acid secretion 8,9 • The additive effects of prostaglandin E 2 and histamine on the human enzyme system suggest that the crude homogenate of fundic gastric mucosa contains at least two different hormone-sensitive adenylate cyclases. Similar results have been reported from guinea pig gastric mu·Cosa '10 • Since crude membrane preparations were used in this study, we cannot finally discern whether the two hormones are acting on the same or on different cell types.
ACKNOWLEDGEMENTS Thanks are due to Miss Th. Fromm for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft,Bad Godesberg, WestGermany. 431