On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187. II. Ca++-cyclic AMP interactions

The possibility of interactions between calcium and cyclic AMP (cAMP) in the mechanism of stimulation of H+ transport by A23187 was studied in the isolated gastric mucosa of the toad Bufo marinus. A23187 stimulated H+ secretion and histamine release. The amount of histamine released by A23187 did not explain the degree of stimulation. Metiamide partially inhibited the response to A23187. Ca++ ionophore produced an overstimulation of secretion after H+ transport had been induced by supramaximal effective concentrations of histamine (10-4 M). In the presence of metiamide, IMX potentiated the response to A23187. Also, in the same condition (metiamide treated) the effects of db-cAMP and A23187 were additive. The results are consistent with an interaction between Ca++ and ionophore-released histamine at the oxyntic cell in the stimulation by A23187. The stimulatory response may be the result of a potentiation between calcium and cAMP at the intracellular level.     

Inhibition of gastric acid secretion by human calcitonin gene-related peptide with picomolar potency in guinea-pig parietal cell preparations

The effect of CGRP on [14C]-aminopyrine accumulation in isolated parietal cell preparations from guinea-pig fundic mucosa was studied. Parietal cells consisted of 60% of the preparations. [14C]-Aminopyrine accumulation was used as an index of physiological response of parietal cells to secretagogues. CGRP dose-dependently (10(-12)-10(-9) M) inhibited parietal cell aminopyrine accumulation stimulated by histamine (10(-4) M), carbachol (10(-4) M), and pentagastrin (5 X 10(-6) M). The concentration of CGRP exerting half-maximal inhibition of [14C]-aminopyrine accumulation was 8.7 X 10(-11) M for histamine, 9.1 X 10(-11) M for carbachol, and 4.7 X 10(-11) M for pentagastrin. The inhibitory effect was much more potent than cimetidine, pirenzepine or benzotript. CGRP but not cimetidine inhibited DBcAMP stimulated aminopyrine accumulation (IC50 = 7.5 X 10(-11) M). These results suggest that CGRP may exert its inhibitory action on gastric acid secretion by a direct action on the parietal cell or the somatostatin-producing D cell.     

Participation of protein kinase C in hormonal regulation of pepsin secretion in the rat stomach]

It was shown that pentagastrin (0.5 micrograms/100 g of body mass) increases the activity of Ca2+ and phospholipid-dependent protein kinase C in the membrane fraction of rat gastric mucosa cells. This effect of pentagastrin is accompanied by a decrease of the protein kinase C activity in the cytosolic fraction. Chromatography of the membrane fraction revealed an additional peak of the enzyme activity. Analysis of isolated gastric mucosa cells demonstrated that pentagastrin (10(-8)-10(-6) M) (but not 10(-4) M histamine) added to the incubation mixture increased the protein kinase C concentration in the membranes. The pentagastrin effect was directly correlated with the amount of pepsin-producing chief cells in the cellular pools. Carbacholine, another well-known pepsin secretion stimulator, was able to activate, similar to pentagastrin, the protein kinase C activity. It is concluded that protein kinase C plays a prominent role in hormonal regulation of the chief gastric cell function.     

Endothelium-dependent relaxations to histamine in the rat common carotid, renal and cranial mesenteric artery

The effect of histamine on the isolated rat common carotid, renal and cranial mesenteric arteries was examined. Histamine (10(-8)-10(-4) M) caused concentration-dependent relaxations of the arteries during contractions induced with phenylephrine (10(-8)-10(-7) M). Removal of the vascular endothelium inhibited the histamine-induced relaxations. Pyrilamine (6 X 10(-6) M), but not metiamide (10(-6) M), abolished the relaxant effect of histamine. Moreover, pyrilamine (6 X 10(-6) M) did not affect endothelium-dependent relaxations of the arteries produced with acetylcholine. These results indicate that histamine causes endothelium-dependent relaxations of the rat peripheral large conduit arteries, which appeared to be mediated via H1-histaminergic receptors.     

Thiocyanate and nitrite inhibit proton translocation in gastric mucosa

Isolated frog gastric mucosa was used to study the separation of formation of protons (or their precursors) from proton translocation by using various inhibitors. Both thiocyanate (SCN-) and nitrite (NO2-) inhibit the acid secretion in spontaneously secreting mucosa. The inhibition is reversed when the inhibitor is removed such that the excess acid secreted above baseline in the 'off'-period compensates for the amount inhibited in the 'on'-period. Both agents also inhibit the effect on acid secretion of pulse stimulation with histamine though to a lesser extent. Upon removal of the inhibitor, the total amount of acid secreted in excess of basal is equal to that observed with histamine alone. Likewise, metiamide, an H2-antagonist, also inhibits acid secretion with or without histamine. However, in contrast to SCN- and NO2-, removal of this inhibitor is without effect on the acid-secretion rate. These results indicate that both SCN- and NO2- inhibit the proton translocation rather than the formation of protons or their precursors as is the case with metiamide.     

Comparing binding of histamine and H2-antagonist with their effects on gastric acid secretion

A microsomal fraction from isolated frog gastric mucosa was used to study the binding of labeled histamine, labeled metiamide (a histamine H2-antagonist), and competition between labeled histamine and unlabeled metiamide. The separation of free from bound ligand was done by gel chromatography. The acid secretion was studied in frog gastric mucosa in vitro by a pH-stat method. The binding data could be interpreted in terms of two independent binding sites for both histamine and metiamide. However, the competition between histamine and metiamide does not support the independence of the sites. Moreover, the dissociation kinetics of labeled metiamide in the presence of unlabeled metiamide is non-monotone and, thus, indicates cooperativity. In the physiological studies, the dependence of the rate of acid secretion on histamine stimulation occurs within very narrow limits, which is the result of characteristics other than related to binding. However, the total amount of acid secreted caused by a pulse of histamine does indicate two sites, of which the high-affinity site is the more effective. Metiamide inhibition of acid secretion can be interpreted as an interaction between high-affinity sites of histamine and metiamide. Overall, studies involving physiological effects provide less precise data than the direct binding studies.     

Effect of beta-adrenoreceptor stimulation on gastric secretion stimulated by histamine, acetylcholine, and pentagastrin

In chronic experiments on dogs with gastric and duodenal fistulas and catheters implanted into the jugular vein, it was established that the beta-adrenoagonist novodrin inhibits gastric secretion stimulated with acetylcholine or pentagastrin but does not alter secretion stimulated with histamine. The inhibitory effect of novodrin on gastric secretion is a consequence of its direct action on beta-adrenoreceptors of the gastric mucosa. The scheme demonstrating interrelations of beta-adrenoreceptors to acetylcholine, gastrin and histamine is offered.     

Necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and cholecystokinin2 receptors on parietal cells in isolated mouse stomach

The existence of a direct action of acetylcholine and gastrin on muscarinic M3 and cholecystokinin2 (CCK2) receptors on gastric parietal cells has not yet been convincingly established because these stimulated acid secretions are remarkably inhibited by histamine H2 receptor antagonists. In the present study, we investigated the necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and CCK2 receptors on parietal cells using an isolated mouse stomach preparation. Bethanechol (10-300 microM) produced a marked increase in acid output and this increase was completely blocked by famotidine (10 microM). In the presence of famotidine, bethanechol (1-30 microM) augmented the acid secretory response to dibutyryl AMP (200 microM) in a concentration-dependent manner. The augmentation was blocked by atropine (1 microM), 4-DAMP (0.1 microM), a muscarinic M3-selective antagonist, and by Ca2+ exclusion from the serosal nutrient solution. Pentagastrin (0.3-3 microM) also concentration-dependently stimulated gastric acid secretion, but the effect was completely inhibited by famotidine. In the presence of famotidine, pentagastrin (0.1-0.3 microM) elicited a definite potentiation of the acid secretory response to dibutyryl cyclic AMP (200 microM). This potentiation was inhibited by YM022 (1 microM), a CCK2 receptor antagonist, and by exclusion of Ca2+ from the serosal nutrient solution. The present results suggest that gastric acid secretion via the activation of muscarinic M3 and CCK2 receptors on the parietal cells is induced by activation of the cyclic AMP-dependent secretory pathway.     

Hormones and Neurotransmitters Control Cyclic AMP Metabolism in Choroid Plexus Epithelial Cells

The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 micrograms/ml cholera toxin, a 50-fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10(-5) M isoproterenol, 10(-4) M prostaglandin E1, 10(-5) M histamine, and 10(-5) M serotonin caused increases of intracellular cyclic concentrations of 100-, 50-, 20-, and 4-fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol-mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin-dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.     

A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaffin-like cell secretion and proliferation

Gastrin regulates ECL cell histamine release and is a critical determinant of acid secretion. ECL cell secretion and proliferation is inhibited by gastrin antagonists and somatostatin but little is known about the role of dopamine agonists in this process. Since the ECL cell exhibits all three classes of receptor we evaluated and compared the effects of the gastrin receptor antagonist, (YF476), lanreotide (SST agonist) and novel dopaminergic agents (BIM53061 and BIM27A760) on ECL cell histamine secretion and proliferation. Highly enriched (>98%) ECL cell preparations prepared from rat gastric mucosa using a FACS approach were studied. Real-time PCR confirmed presence of the CCK2, SS2 and SS5 and D1 receptors on ECL cells. YF476 inhibited histamine secretion and proliferation with IC(50)s of 1.25 nM and 1.3 x 10(-11) M respectively, values 10-1000x more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 x 10(-10) M) and increased YF476-inhibited proliferation a further 5-fold. The dopamine agonist, BIM53061, inhibited gastrin-mediated ECL cell secretion and proliferation (17 nM, 6 x 10(-10) M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 x 10(-10) M). Our studies demonstrate that the gastrin receptor antagonist, YF476, is the most potent inhibitor of ECL cell histamine secretion and proliferation. Lanreotide, a dopamine agonist and a dopamine/somatostatin chimera inhibited ECL cell function but were 10-1000x less potent than YF476. Agents that selectively target the CCK2 receptor may provide alternative therapeutic strategies for gastrin-mediated gastrointestinal cell secretion and proliferation such as evident in the hypergastrinemic gastric carcinoids associated with low acid states.     

Cholinergically stimulated gastric acid secretion is mediated by M(3) and M(5) but not M(1) muscarinic acetylcholine receptors in mice

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M(1)-M(5)) remains unclear. This study examined the involvement of M(1), M(3), and M(5) receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M(3) KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M(1) KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulated by carbachol, histamine, and gastrin. Pirenzepine, known as an M(1)-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M(1) KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M(1)-receptor antagonism. Notably, M(5) KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M(5)-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M(3) (on parietal cells) and M(5) receptors (conceivably in the submucosal plexus), but not M(1) receptors.     

Stimulatory effects of endogenous and exogenous nitric oxide on gastric acid secretion in anesthetized rats

We previously reported the stimulatory effect of endogenous nitric oxide (NO) on gastric acid secretion in the isolated mouse whole stomach and histamine release from gastric histamine-containing cells. In the present study, we investigated the effects of endogenous and exogenous NO on gastric acid secretion in urethane-anesthetized rats. Acid secretion was studied in gastric-cannulated rats stimulated with several secretagogues under urethane anesthesia. The acid secretory response to the muscarinic receptor agonist bethanechol (2 mg/kg, s.c.), the cholecystokinin(2) receptor agonist pentagastrin (20 microg/kg, s.c.) or the centrally acting secretagogue 2-deoxy-D-glucose (200 mg/kg, i.v.) was dose-dependently inhibited by the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 or 50 mg/kg, i.v.). This inhibitory effect of L-NNA was reversed by a substrate of NO synthase, L-arginine (200 mg/kg, i.v.), but not by D-arginine. The histamine H(2) receptor antagonist famotidine (1 mg/kg, i.v.) completely inhibited the acid secretory response to bethanechol, pentagastrin or 2-deoxy-D-glucose, showing that all of these secretagogues induced gastric acid secretion mainly through histamine release from gastric enterochromaffin-like cells (ECL cells). On the other hand, histamine (10 mg/kg, s.c.)-induced gastric acid secretion was not inhibited by pretreatment with L-NNA. The NO donor sodium nitroprusside (0.3-3 mg/kg, i.v.) also dose-dependently induced an increase in acid secretion. The sodium nitroprusside-induced gastric acid secretion was significantly inhibited by famotidine or by the soluble guanylate cyclase inhibitor methylene blue (50 mg/kg, i.v.). These results suggest that NO is involved in the gastric acid secretion mediated by histamine release from gastric ECL cells.     

Histamine-mediated hyposmotic stimulation of gastric acid secretion

Gastric glands incubated in hyposmotic medium (200 mOsm) accumulated aminopyrine, a measure of acid secretion, to the same extent as that of paired glands in isomotic medium containing histamine (10(-4) M). These maximal responses to hyposmolality and histamine were not additive. The hyposmotic response peaked earlier than the histamine response. Hyposmotic stimulation was nearly abolished by preincubation of the glands with metiamide and cimetidine, H-2 histamine antagonists. In the presence of histaminase, no hyposmotic stimulation occurred. The response to forskolin, a stimulant of adenylate cyclase, was equivalent in hyposmotic and isosmotic media. These results indicate that hyposmolality releases histamine from a paracrine cell in the gastric gland and that histamine binds to H-2 receptors on the parietal cell to initiate a cyclic AMP-mediated stimulation of acid secretion.     

Modulation of acetylcholine-induced secretion of gastric bombesin-like immunoreactivity by cholinergic and histamine H2-receptors, somatostatin and intragastric pH

Recently we have shown the release of bombesin-like immunoreactivity (BLI) from the isolated perfused rat stomach. In these experiments we have shown that BLI secretion is stimulated by acetylcholine. Gastric inhibitory peptide (GIP) exerts an inhibitory effect which is dependent on the intraluminal pH. The present study was designed to examine further the exact cholinergic mechanisms and to study the interaction between cholinergic and histaminergic mechanisms as well as the effect of the intraluminal pH. Acetylcholine elicited a dose-dependent increase in BLI and gastrin secretion (10(-6) M and 2 X 10(-6)M), whereas somatostatin release was suppressed at luminal pH 7. Blockade of muscarinic cholinergic receptors by atropine (10(-5)M) and nicotinic cholinergic receptors by hexamethonium (10(-5) M) abolished the effect of acetylcholine on all three peptides. Reduction of the intraluminal pH to 2 also abolished acetylcholine-induced stimulation of BLI and gastrin secretion and the inhibition of somatostatin secretion. Changes of intraluminal pH per se had no effect on the secretion of either peptide. Somatostatin (10(-7) M) reduced both BLI and gastrin secretion during stimulation with acetylcholine. The addition of the H2-receptor antagonist cimetidine (10(-5) M) abolished the effect of both doses of acetylcholine on BLI and somatostatin secretion and also the effect of the lower dose of acetylcholine (10(-6) M) on gastrin secretion during luminal pH 7. At luminal pH 2 cimetidine did not alter BLI and somatostatin secretion in response to acetylcholine, however, gastrin release was augmented in the presence of cimetidine. These data demonstrate that the effect of acetylcholine on BLI, gastrin, and somatostatin secretion is mediated by muscarinic and nicotinic cholinergic receptors and also by histamine H2-receptors. Somatostatin inhibits cholinergically induced BLI secretion. The cholinergic effects on BLI, somatostatin and gastrin secretion are abolished during an acidic intragastric pH. In this isolated perfused rat stomach model the inhibitory effect of intraluminal acid on gastrin secretion is, at least in part, mediated by H2-receptors. This suggests that the secretion of bombesin, a potential peptidergic neurotransmitter is modulated by neural, endocrine and local tissue factors and also by alterations of intragastric pH.     

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Mucosal mast cells. II. Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells

Functional mast cells have been isolated from the lamina propria of the small intestine of rats infected with the nematode Nippostrongylus brasiliensis. The cells released histamine on challenge with specific antigen, anti-rat IgE, concanavalin A, and calcium ionophores but were less responsive than peritoneal mast cells (MMC) from the same animals. Intestinal mucosa mast cells (PMC) were refractory to the action of the basic secretagogues peptide 401 from bee venom and compound 48/80. The anti-allergic compounds disodium cromoglycate (less than or equal to 10(-3) M), AH 9679 (less than or equal to 10(-4) M), and theophylline (less than or equal to 10(-2)) did not inhibit antigen-induced histamine secretion by MMC, although these compounds were effective against PMC. In contrast, doxantrazole (10(-5) to 10(-3) M) inhibited the secretion of histamine from both MMC and PMC in a comparable dose-dependent fashion. Thus, we have established that mast cells from different sites are functionally heterogeneous not only in their response to various stimuli for histamine secretion, but also in their responses to different pharmacologic modulators of secretion. It cannot be assumed that anti-allergic compounds effective against mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. The extent of this functional heterogeneity must be established, and its investigation may provide new insights into the biochemical events involved in mast cell secretion.     

Histamine H2-receptors in guinea pig peripheral airway smooth muscle.

The presence and function of histamine H₂-receptors in guinea pig lung was studied using lung strips as an in vitro model of peripheral airway smooth muscle. The lung strips were incubated in Krebs-Henseleit solution in the absence or presence of specific antagonists for 20 min prior to the addition of either histamine or dimaprit added in a half-log cumulative fashion. Changes in isometric tension were recorded. Histamine at low concentrations (10^?7?10^?6M) caused a slight relaxation which was potentiated by the histamine H₁-antagonist chlorpheniramine (10^?7 or 10^?6M) and abolished by the histamine H₂-antagonist metiamide (10^?4M). Higher concentrations of histamine produced a dose-related contraction which was antagonized competitively by chlorpheniramine or potentiated by metiamide. Dimaprit, a histamine H₂-agonist, produced only a relaxant response over the concentration range of 10^?7 ? 10^?3M. This relaxation was reduced by metiamide but not by the beta adrenergic antagonist propranolol. These results indicate the presence of both histamine H₂ and H₁-receptors in guinea pig peripheral airway smooth muscle which mediate the relaxant and contractile effects of histamine respectively.     

Mechanisms for direct inhibition of canine gastric parietal cells by somatostatin

To examine the potential mechanisms by which somatostatin inhibits gastric acid secretion we studied its effects on isolated canine gastric parietal cells. Using 125I-[Leu8-D-Trp22-Tyr25]somatostatin-28 as ligand, we identified somatostatin-binding sites in parietal cell-enriched fractions of fundic mucosa. Two binding sites with respective dissociation constants of 3.2 X 10(-9) and 2.1 X 10(-7) M were identified. Somatostatin-14 and -28 were equally potent both in displacing bound ligand and in inhibiting parietal cell activity as measured by [14C]aminopyrine uptake. Pertussis toxin reversed the ability of somatostatin to inhibit the uptake of [14C]aminopyrine and production of cAMP by parietal cells stimulated with histamine and forskolin but not with dibutyryl cAMP or pentagastrin. Furthermore, somatostatin had no effect on parietal cell membrane inositol phospholipid turnover or changes in protein kinase C (Ca2+/phospholipid-dependent enzyme) activity induced by carbachol or pentagastrin. These data indicate that somatostatin directly inhibits parietal cell activity via mechanisms both dependent on and independent of the pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein.     

Modification by histamine H2-receptor blockade of acid secretion stimulated by histamine, pentagastrin and methacholine in the isolated whole mouse stomach.

The direct influences of the blockade of the gastric histamine H2-receptors on the secretory actions induced by histamine, pentagastrin and methacholine, have been studied on the isolated perfused whole mouse stomach. According to the results cimetidine did not modify the spontaneous basal acid secretion. The interactions of cimetidine with the secretagogues were of a competitive nature with histamine and non-competitive with pentagastrin, while no modification of methacholine stimulated acid secretion.