K.-F. Sewing

Mechanism of gastric antisecretory effect of SCH 28080

1 The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea‐pig and guinea‐pig gastric membranes purified and enriched with K+/H+‐ATPase. 2 In guinea‐pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. 3 SCH 28080 inhibited the purified K+/H+‐ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 μM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+‐ATPase showed that this enzyme was only slightly depressed by SCH 28080. 4 It is concluded that SCH 28080 acts with high selectivity on the parietal cell K+/H+‐ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+‐site of the gastric ATPase.

Inhibition of Acid Formation in Rabbit Parietal Cells by Prostaglandins Is Mediated by the Prostaglandin E2 Receptor

The affinities of seven natural and synthetic prostaglandins [PGE2; 16,16-dimethyl PGE2; iloprost (stable prostacyclin analogue); PGF2 alpha; PGD2; BW245c (stable PGD2 analogue); and U46619 (stable thromboxane analogue)] to the PGE2 binding site of rabbit gastric mucosa were determined by measuring [3H]PGE2 displacement from its high-affinity plasma membrane binding sites. In parallel, the potency of each prostaglandin in inhibiting acid generation in vitro was determined by measuring the inhibition of histamine-stimulated [14C]aminopyrine accumulation in rabbit parietal cells prepared by enzymatic dispersion and enriched by counterflow elutriation. All seven prostaglandins displaced [3H]PGE2 and inhibited histamine-stimulated [14C]aminopyrine accumulation in a concentration-dependent manner. For all tested prostaglandins, the IC50 values were in excellent agreement for both variables measured. It is concluded that (a) a PGE2 receptor is localized on the parietal cell and mediates inhibition of acid formation by all prostaglandins and (b) the different in vitro antisecretory potencies of prostaglandins can be attributed to their different affinities to this PGE2 receptor.

Interaction of Calcium Channel Antagonists with Parietal Cell Acid Production, Adenylate Cyclase, Intracellular-Free Ca2+ and H+/K+-ATPase

The calcium channel antagonists verapamil, gallopamil and nifedipine were tested for their effects on acid secretion stimulated by histamine and dibutyryl-cyclic AMP in isolated and enriched guinea pig parietal cells, on adenylate cyclase activity mediated by histamine H2 receptors, on histamine-stimulated increase in cytosolic-free Ca2+ concentration [Ca2+], on gastric H+/K(+)-ATPase activity and on H+/K(+)-ATPase-mediated proton uptake in intact gastric membrane vesicles. Verapamil and gallopamil impaired all cellular and enzymatic test systems studied. Both drugs affected with highest potency the acid secretion in the parietal cell preparation (IC50: 1-2 mumol/l) and the H+/K(+)-ATPase-mediated H+ uptake in gastric membrane vesicles, whereas their inhibitory action was less pronounced on adenylate cyclase and on histamine-induced increase in cytosolic-free [Ca2+]. The type of interaction found in the gastric membrane vesicle preparation indicates that both drugs act as protonophores. Nifedipine was less effective as an inhibitor of acid secretion in the parietal cell preparation and in reducing proton concentration in isolated gastric membrane vesicles. The drug failed to block adenylate cyclase and H+/K+-ATPase activity. Since nifedipine is a more effective calcium channel blocking agent but a less lipophilic drug than verapamil and gallopamil, we conclude that the antisecretory activity of calcium channel antagonists in vitro is mediated by a nonspecific, i.e. a protonophoric, action. We suggest that verapamil exhibits its antisecretory activity in vivo partially by its protonophoric action at the secretory membrane of the parietal cell, whereas the decrease in acid secretion by nifedipine is not mediated by this mechanism.

Effects of endogenous and exogenous prostaglandins on glycoprotein synthesis and secretion in isolated rabbit gastric mucosa

We studied gastric glycoprotein synthesis and secretion in organ culture before and during cyclooxygenase inhibition and replacement with exogenous prostaglandins (16,16-dimethyl prostaglandin E2 and prostaglandin F2 alpha). Isolated rabbit antral and fundic mucosal explants incorporated [14C]N-acetylglucosamine and [3H]leucine in a linear fashion and steadily secreted labeled proteins and glycoproteins during the 24-h incubation period. On sepharose 4B, greater than 90% of the secreted protein-bound [14C]N-acetylglucosamine was found in the high molecular weight peak. Incorporation of tracer was not influenced by cyclooxygenase inhibition with indomethacin or the addition of exogenous prostaglandins. Secretion of newly formed glycoprotein, however, was significantly inhibited by indomethacin and stimulated by both tested prostaglandins in a concentration-dependent manner. 16,16-Dimethyl prostaglandin E2 caused significant stimulation in concentrations that are well in the physiologic range for endogenous prostaglandin E2, whereas prostaglandin F2 alpha stimulated in 100 times higher concentrations. We conclude that in the isolated gastric mucosa both endogenous and exogenous prostaglandins stimulate mucus secretion. For prostaglandin E2, but not prostaglandin F2 alpha, a role in the physiologic regulation of gastric mucus secretion is probable.

Effects of Histamine on Protein and Glycoprotein Production of Isolated Pig Gastric Mucosal Cells

The production of glycoprotein and protein by isolated pig gastric non-parietal cells was measured by incorporation of N-acetyl-[14C]-D-glucosamine [( 14C]GlcNAc) and [3H]-L-leucine [( 3H]Leu), respectively, into acid insoluble material (AIM). Histamine enhanced incorporation of the tracers into cellular and released AIM in a concentration-dependent manner. The H2 receptor antagonist ranitidine completely blocked the effects of histamine (100 mumol/l) on [3H]Leu incorporation into cellular and released AIM (IC50 37 and 32 mumol/l, respectively) but had no inhibitory effect on the 16,16-dimethylprostaglandin-E2 - and forskolin-stimulated tracer incorporation. The H1 receptor antagonist mepyramine did not inhibit the histamine effect. We conclude that histamine is a stimulant of protein, via H2 receptors, and glycoprotein production of isolated pig gastric non-parietal cells.

Isolation, identification and quantitative evaluation of specific cell types from the mammalian gastric mucosa

Functional in vitro studies with isolated gastric mucosal cells require cytological identification of different cell types in suspension or primary culture. Since suitable techniques have not been well established, different staining methods for the discrimination of dispersed pig and guinea pig gastric cells have been developed on the basis of modified previous protocols for enzymatic cell dispersion. Chief and parietal cells were visualized by combined periodic acid-Schiff stains. Surface mucous and mucous neck cells were identified by affinity-labelling, using lectins with selective staining properties in situ. Two of the lectins were found to be specific markers for gastric polymorphonuclear cells. The following vital tests were found to be useful: succinic dehydrogenase for parietal cells, Nile blue/brilliant cresyl blue stains for chief cells, and different phagocytosis assays for endothelial cells and gastric phagocytes. Endocrine cells were characterized by immunocytochemistry using specific antibodies against gastrin, somatostatin, histamine and serotonin. The same technique using a vimentin antibody was performed for the identification of fibroblasts. Proliferation of mucosal cells in primary culture was monitored by the incorporation of bromo-deoxyuridine, which was subsequently detected by a monoclonal antibody.

Cyclic adenosine monophosphate is the second messenger of prostaglandin E2- and vasoactive intestinal polypeptide-stimulated active bicarbonate secretion by guinea-pig duodenum.

In a guinea-pig model we determined the intracellular events mediating the response of duodenal epithelial cells to vasoactive intestinal polypeptide (VIP) and prostaglandin (PG) E2. Intravenous administration of VIP (10(-9) to 10(-7) mol/kg) and PGE2 (10(-9) to 10(-6) mol/kg) dose-dependently increased duodenal epithelial bicarbonate secretion against an HCO3- concentration gradient, measured by a luminal perfusion technique, in anaesthetized guinea-pigs up to 4.5-fold. This secretion could be mimicked by intraduodenal dibutyryl cyclic adenosine monophosphate (dBcAMP; 10(-9) to 10(-7) mol/kg). Secretin (10(-9) mol/kg) and PGF 2 alpha (10(-9) to 10(-7) mol/kg), both given intravenously, were without effect or considerably less efficient. For VIP and PGE2, specific receptors coupled to adenylate cyclase could be demonstrated in homogenates of isolated duodenal epithelial cells. VIP and PGE2 stimulated adenylate cyclase activity up to sixfold, whereas PGF2 alpha and secretin were considerably less potent and efficient. VIP and PGE2 increased intracellular cyclic AMP levels up to fivefold and ninefold, respectively. This was followed by an increase in cytosolic protein kinase A activity. Bicarbonate secretion was maximal at 30 min. Examination of the subcellular distribution of protein kinase A showed a predominant cytosolic location. These data support the notion the PGE2 and VIP cause bicarbonate secretion by the serial activation of adenylate cyclase and protein kinase A in duodenal epithelial cells.

Studies on the Mechanism of Action of Colloidal Bismuth Subcitrate

The effects of colloidal bismuth subcitrate (CBS) on porcine pepsin have been studied in vitro. CBS inhibited pepsin activity in a pH-dependent manner. CBS was not active at pH 4.0 but inhibited pepsi

Effect of Substituted Benzimidazoles on H+K+ATPase of Isolated Guinea-pig Parietal Cells

Omeprazole, a substituted benzimidazole, has been shown to inhibit gastric acid secretion in vitro and in vivo. In vitro, omeprazole inhibits acid secretion stimulated at all cellular levels, including that stimulated by K+, indicating that it is an inhibitor of the parietal cell proton pump. The present paper describes the inhibitory effect of substituted benzimidazoles, such as picoprazole and omeprazole, on the H+K+ATPase isolated from guinea-pig parietal cells, and the reversibility of acid inhibition in isolated guinea-pig parietal cells by the thiol compound, dithiothreitol.

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non‐parietal cells

1 Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N‐acetyl‐[14C]‐d‐glucosamine and [3H]‐l‐leucine, respectively, into cellular and released acid precipitable material. 2 Histamine and activators of the adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 μm), forskolin (10–100 μm), 3‐isobutyl‐1‐methylxanthine (100 μm), and dibutyryl cyclic AMP (1–3 mm), respectively. In the presence of 3‐isobutyl‐1‐methylxanthine (30 μm) histamine stimulation was enhanced. 3 As shown by gel chromatography, stimulation by histamine (100 μm), forskolin (10 μm), 3‐isobutyl‐1‐methylxanthine (100 μm) and dibutyryl cyclic AMP (1 mm) resulted in a release of high molecular weight (≅2 × 106daltons) glycoproteins from the cells. The histamine H2‐receptor antagonist, ranitidine (100 μm), blocked the effect of histamine. 4 We conclude that cyclic AMP‐dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non‐parietal cells and that histamine may be a physiological activator of this system.