Canine Parietal Cells Research Articles

Effect of omeprazole on gene expression in canine gastric parietal cells

Stimulation of gastric parietal cells by carbachol induces coordinate expression of the genes for two enzymes involved in the process of acid secretion, H(+)-K(+)-ATPase and carbonic anhydrase II (CA II). The basis of this coordinate expression was examined in experiments using parietal cells that had been pretreated with omeprazole. We observed a twofold increase in the steady-state mRNA levels of both H(+)-K(+)-ATPase and CA II after cells were treated with the inhibitor. The induction of CA II mRNA by carbachol followed the same kinetics in omeprazole-pretreated cells as in those that were not pretreated, suggesting that the induction of CA II gene expression by carbachol was not dependent on activation of the gastric H(+)-K(+)-ATPase. In addition, carbachol stimulation of omeprazole-pretreated cells resulted in an induction of one or more larger mRNA species that hybridized with the H(+)-K(+)-ATPase probe. The observation that carbachol-induced increases in steady-state levels of beta-actin mRNA in parietal cells could be inhibited by omeprazole pretreatment suggests a possible linkage between increased beta-actin gene expression and the process of acid secretion.

<b>ACTIVATION OF PROTEIN KINASE C INHIBITS NaF-INDUCED INOSITOLPHOSPHOLIPID TURNOVER IN ISOLATED CANINE PARIETAL CELLS </b>

Effects of protein kinase C (PKC) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on NaF-ionduced inositolphospholipid metabolism were investigated in canine parietal cells. NaF dose-dependently stimulated the formation of [ 3 H]inositolphosphates, which was characterized by a rapid generation of [ 3 H]inositolphosphate (IP 3 ) within 3 min. Further-more, NaF elicited a rapid decrease in 32 P-labeling of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) with a simultaneous increase in [ 32 P]phosphatidic acid (PA)

Muscarinic receptors in isolated canine gastric parietal cells involved in cyclic AMP formation and inositol phospholipid breakdown

Muscarinic receptors in isolated canine gastric parietal cells involved in cyclic AMP formation and inositol phospholipid breakdown

Differences in the Antisecretory Actions of the Proton Pump Inhibitor AG-1749 (Lansoprazole) and the Histamine H2-Receptor Antagonist Famotidine in Rats and Dogs

Antisecretory effects of a substituted benzimidazole, (±)-2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl]sulfinyl]-lH-benzimidazole (AG-1749) were compared with those of a histamine H2-receptor antagonist, famotidine. AG-1749 inhibited acid formation regardless of the stimulant in isolated canine parietal cells, while famotidine inhibited the histamine-stimulated acid formation selectively. In pylorus-ligated rats, AG-1749 suppressed basal acid secretion, histamine-, bethanechol-, pentagastrin-, 2-deoxy-D-glucosc- and stress (restraint and waterimmersion)-induced acid secretion; ID50 values were 1.0-6.0mg/kg. On the other hand, famotidine only partially inhibited the acid secretion induced by 2-deoxy-D-glucose or stress, although it suppressed the acid secretion stimulated by other secretagogues several times more potently than AG-1749. The antisecretory effect of AG-1749 lasted longer than that of famotidine, especially in the case of bethancchol-stimulated acid secretion. In Heidenhain pouch dogs, both AG-1749 and famotidine potently inhibited histamine-, bethanechol-, pentagastrin- and peptone meal-stimulated acid secretion, but the inhibitory effect of famotidine was short-lived in the case of bethanechol- and pentagastrin-stimulated acid secretion. These results suggest that AG-1749 persistently inhibits acid secretion induced by both peripheral and central stimuli and suggest that the antisecretory effect of famotidine depends on the nature of the stimuli.

Differences in the Antisecretory Actions of the Proton Pump Inhibitor AG-1749 (Lansoprazole) and the Histamine H2-Receptor Antagonist Famotidine in Rats and Dogs.

Antisecretory effects of a substituted benzimidazole, (+/-)-2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl] sulfinyl]-1H-benzimidazole (AG-1749) were compared with those of a histamine H2-receptor antagonist, famotidine. AG-1749 inhibited acid formation regardless of the stimulant in isolated canine parietal cells, while famotidine inhibited the histamine-stimulated acid formation selectively. In pylorus-ligated rats, AG-1749 suppressed basal acid secretion, histamine-, bethanechol-, pentagastrin-, 2-deoxy-D-glucose- and stress (restraint and water-immersion)-induced acid secretion; ID50 values were 1.0-6.0 mg/kg. On the other hand, famotidine only partially inhibited the acid secretion induced by 2-deoxy-D-glucose or stress, although it suppressed the acid secretion stimulated by other secretagogues several times more potently than AG-1749. The antisecretory effect of AG-1749 lasted longer than that of famotidine, especially in the case of bethanechol-stimulated acid secretion. In Heidenhain pouch dogs, both AG-1749 and famotidine potently inhibited histamine-, bethanechol-, pentagastrin- and peptone meal-stimulated acid secretion, but the inhibitory effect of famotidine was short-lived in the case of bethanechol- and pentagastrin-stimulated acid secretion. These results suggest that AG-1749 persistently inhibits acid secretion induced by both peripheral and central stimuli and suggest that the antisecretory effect of famotidine depends on the nature of the stimuli.

Cellular mechanisms of somatostatin action in the gut

We have used isolated canine parietal cells to examine the receptor and postreceptor events mediating the inhibitory effects of somatostatin on acid secretion. Somatostatin-14 (S14) and somatostatin-28 (S28) dose dependently inhibited parietal cells stimulated by secretagogues that activate both the adenylate cyclase/cyclic adenosine monophosphate and the inositol phospholipid/protein kinase C cascades. The inhibitory action was mediated via a specific cell surface receptor that consists of a single subunit protein (molecular weight 99,000 d). This receptor recognized S14 and S28 equally well. Somatostatin inhibited parietal cell activity via mechanisms that are both dependent on and independent of a pertussis toxin-sensitive inhibitory guanine nucleotide binding protein.

Effect of a new H2-receptor antagonist (Roxatidine acetate) on the acid secretion by isolated canine parietal cells

Effect of a new H2-receptor antagonist (Roxatidine acetate) on the acid secretion by isolated canine parietal cells

Carbonic anhydrase II gene expression in isolated canine gastric parietal cells

The participation of carbonic anhydrase (CA II) in the gastric acid secretory process has been the subject of considerable controversy. We utilized a cDNA probe for CA II to measure CA II gene expression in canine gastric parietal cells that had been stimulated with the three principal acid secretagogues, carbachol, gastrin, and histamine. Hybridization to dot blots of total parietal cell RNA showed a significant increase in specific CA II mRNA content within minutes of secretagogue addition: carbachol stimulation led to an increase of 52 +/- 8.9%, reaching a maximum within 20 min; gastrin stimulation led to an increase within 60 min of 104 +/- 10.6%; stimulation with histamine was followed within 20 min by an increase of 30 +/- 7.2%. We also measured transcription rates for the CA II gene in cells stimulated by each agent and found an increase within 15 min. Our results show that CA II gene expression is regulated by agents that stimulate the parietal cell to secrete acid and that the accumulation of CA II mRNA subsequent to the initial interaction of stimulant appears to result from new transcription of the CA II gene. These data suggest the participation of CA II in the acid secretory response of the parietal cell to secretagogues.

Prostanoid inhibition of canine parietal cells: Mediation by the inhibitory guanosine triphosphate-binding protein of adenylate cyclase

We have investigated the mechanisms underlying prostaglandin inhibition of histamine-stimulated parietal cell function. Enzyme-dispersed canine parietal cells were enriched by elutriation. The accumulation of the weak base [14C]aminopyrine was used as an index of parietal cell function and cyclic adenosine monophosphate content was measured by radioimmunoassay. Step density gradients of the elutriator-enriched parietal cell fractions indicated that parietal cells accounted for the histamine stimulation of cyclic adenosine monophosphate production and inhibition by the prostaglandin E analogue Enprostil. Pertussis toxin adenosine diphosphate-ribosylates a subunit with a molecular weight of 41,000, thereby inactivating the inhibitory guanine nucleotide-binding protein of adenylate cyclase. Pertussis toxin treatment of parietal cells in over-night suspension culture was used to determine if inhibitory guanosine triphosphate-binding protein mediated prostanoid inhibition. In control cultured cells, prostaglandin E2 and Enprostil markedly inhibited forskolin- and histamine-stimulated aminopyrine accumulation. In parietal cells treated with pertussis toxin (300 ng/ml) for 18 h, stimulation of parietal cell function by histamine, isobutylmethyl-xanthine, and forskolin was unaltered compared with control cells, whereas prostaglandin E2 and Enprostil inhibition was markedly reduced. In pertussis toxin-treated cells, histamine-stimulated cyclic adenosine monophosphate generation was unaltered, whereas Enprostil inhibition of histamine-stimulated cyclic adenosine monophosphate production was markedly reduced. Pertussis toxin treatment of membranes from control, but not from pertussis toxin-treated, cells induced the [32P]adenosine diphosphate-ribosylation of a membrane protein with a molecular weight of 41,000, presumably the α-subunit of inhibitory guanosine triphosphate-binding protein. We conclude that prostanoids inhibit parietal cell function by receptor-mediated interaction with the inhibitory guanine nucleotidebinding protein of adenylate cyclase.

Effect of misoprostol on histamine-stimulated acid secretion and cyclic AMP formation in isolated canine parietal cells.

Isolated canine parietal cells were used to study the ability of misoprostol to inhibit acid secretion in the presence of a number of acid secretagogues. Misoprostol inhibited histamine-stimulated acid secretion in a dose-dependent and noncompetitive manner. A concentration of 2-3 X 10(-9) M misoprostol inhibited maximal histamine-stimulated acid secretion by one half. Misoprostol had little to no effect on acid secretion stimulated by carbachol and dibutyryl cAMP, had no effect on the acid secretion directly stimulated by pentagastrin, and only modestly inhibited acid secretion stimulated by forskolin. Misoprostol noncompetitively inhibited cAMP formation in response to histamine, with an IC50 value similar to that for the inhibition of histamine-stimulated acid secretion. These results indicate that: (1) misoprostol specifically inhibits histamine-stimulated acid secretion in parietal cells, and (2) the antisecretory action of misoprostol is closely related to the reduction of histamine-stimulated cAMP formation with the site of major action most likely in the coupling process between histamine H2 receptor sites and histamine-sensitive adenylate cyclase.

Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells.

We applied affinity cross-linking methods to label the gastrin receptor on isolated canine gastric parietal cells in order to elucidate the nature of its chemical structure. 125I-labeled Leu15-gastrin and 125I-labeled gastrin2(-17) bound to intact parietal cells and their membranes with equal affinity, and half-maximal inhibition of binding was obtained at an incubation concentration of 3.2 X 10(-10) M unlabeled gastrin. 125I-gastrin2(-17) was cross-linked to plasma membranes or intact parietal cells by incubation in disuccinimidyl suberate. The membrane pellets were solubilized with or without dithiothreitol and applied to electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. Autoradiograms revealed a band of labeling at Mr 76,000 and labeling of this band was inhibited in a dose-dependent fashion by addition of unlabeled gastrin to the incubation mixture. Dithiothreitol in concentrations as high as 100 mM did not alter the electrophoretic mobility of the labeled band. After taking into account the molecular weight of 125I-gastrin2(-17), our results suggest that the gastrin receptor on parietal cells is a single protein of Mr 74,000 without disulfide-linked subunits.

Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin.

A gastrin binding protein (GBP) has been identified in detergent extracts of porcine gastric mucosal membranes by covalent cross-linking to 125I-[Nle15]gastrin with disuccinimidyl suberate. The apparent molecular weight of the cross-linked complex (80,000) is uneffected by reduction suggesting that the GBP is not composed of disulfide-bonded subunits. Subtraction of the molecular weight of 125I-gastrin indicates that the molecular weight of the GBP is 78,000. A similar molecular weight has been observed previously for the gastrin receptor (74,000) on intact canine parietal cells and plasma membranes therefrom, and for the receptor for the related hormone cholecystokinin (76,000-85,000) on pancreatic acinar membranes under reducing conditions. The similarity in molecular weight between the gastrin receptor and the solubilized GBP suggests that the latter protein is probably the gastrin receptor. However, the concentration (2 microM) of [Nle15]gastrin required for 50% inhibition of cross-linking of gastrin to the GBP solubilized in 0.1% Triton X-100 is 200-fold greater than the value (10 nM) observed for the gastrin receptor on isolated canine gastric parietal cells. A lower concentration (0.3 microM) of [Nle15]gastrin was required to inhibit cross-linking in a milder detergent (0.4% digitonin, 0.08% cholate). Thus, the reduced affinity for gastrin of the putative solubilized form of the gastrin receptor appears to be a result of detergent extraction.

Prostanoid inhibition of canine parietal cells

Cellular mechanisms underlying the anti-secretory actions of the prostaglandin E2 analogue enprostil were studied using enzyme-dispersed, elutriator-enriched canine parietal cells and the accumulation of the weak base 14C-labeled aminopyrine as a functional index. Enprostil inhibited the accumulation of aminopyrine stimulated by histamine and the phosphodiesterase inhibitor isobutylmethyl, but not by carbachol, gastrin, or dibutyryl cyclic adenosine monophosphate. Inhibition by enprostil was dose-dependent (0.1 nM to 1 microM), with maximal inhibition ranging from 65 to 95 percent. Over the same concentration range, enprostil inhibited the histamine-stimulated generation of cyclic adenosine monophosphate. This selective inhibition of histamine activation of parietal cell function was comparable to that found for prostaglandin E2. Forskolin, a diterpene that directly activates the catalytic subunit of adenylate cyclase, was also markedly inhibited by nanomolar concentrations of prostaglandin E2 and enprostil. We conclude that at least a component of the secretory inhibition by enprostil reflects direct interference with histamine stimulation of parietal cell adenylate cyclase.

Selective inhibition of pentagastrin- and cholecystokinin-stimulated exocrine secretion by proglumide

The effectiveness and selectivity of proglumide, a putative cholecystokinin/gastrin receptor antagonist in vitro, were examined on gastric acid and pancreatic secretion in vivo. Gastric secretion was measured in conscious dogs in the basal state and during infusion of pentagastrin, histamine, or bethanechol, alone or in combination with proglumide (300 mg/kg · h). Pancreatic secretion was measured in anesthetized rats in response to cholecystokinin-octapeptide or secretin, alone or in combination with proglumide (100 mg/kg). Proglumide inhibited pentagastrin-stimulated secretion but had no effect on basal, histamine-stimulated, or bethanechol-stimulated gastric acid secretion. Inhibition of pentagastrin-stimulated secretion was of the competitive type. An apparent inhibitory constant was calculated to be 300 mg/kg · h; this dose is capable of eliciting plasma concentrations of ~1 mM. This estimate corresponds closely to that derived from measurements in isolated canine parietal cells. Proglumide also inhibited cholecystokinin-stimulated but not secretin-stimulated pancreatic secretion. The lack of effect of proglumide on basal, histamine-stimulated, or bethanechol-stimulated gastric acid secretion implies that background gastrin has no direct or synergistic influence on stimulation by other secretagogues. The selective effect of gastrin receptor antagonists contrasts with the effectiveness of muscarinic and histamine H2-receptor antagonists against secretion induced by all types of stimulants. Accordingly, the antisecretory potential of gastrin receptor antagonists is confined to digestive secretion when the effect of gastrin is optimal. Their potential as antitrophic agents in duodenal ulcer disease, however, has not been explored yet.

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell.

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.

Cholecystokinin potently releases somatostatin from canine fundic mucosal cells in short-term culture.

Previous studies indicated that gastrin-17 (G-17) and the octapeptide of colecystokinin (CCK-8) were equally potent in their interaction with receptors for 125I-[Leu15]G-17 on isolated canine parietal cells. These findings were inconsistent with the poor efficacy of CCK-8 compared with G-17 as stimuli of acid secretion in dogs. The present study examines the effects of G-17 and CCK-8 on the release of somatostatinlike immunoreactivity (SLI) from a fraction of small canine fundic mucosal cells separated by elutriation and placed in short-term culture. CCK-8 was considerably more potent and more effective than G-17 as a stimulant of SLI release from these cultured cells. CCK-8 was slightly more potent than G-17 in inhibiting 125I-[Leu15]G-17 binding to receptors in the same elutriator fraction. Our present findings support the hypothesis that the poor efficacy of CCK compared with G-17 as a stimulant of acid secretion may reflect pronounced activation of somatostatin-mediated acid-inhibitory mechanisms by CCK-8. The present data indicate that differences in affinity between CCK-8 and G-17 at the 125I[Leu15]G-17 receptor probably do not account for the greater efficacy of CCK-8; the receptor or cell activation mechanisms underlying this greater efficacy of CCK-8 on SLI release remain to be elucidated.

Gastrin receptors on isolated canine parietal cells.

The receptors in the fundic mucosa that mediate gastrin stimulation of acid secretion have been studied. Synthetic human gastrin-17-I (G17) with a leucine substitution in the 15th position ( [Leu15]-G17) was iodinated by chloramine T; high saturable binding was found to enzyme-dispersed canine fundic mucosal cells. 127I-[Leu15]-G17, but not 127I-G17, retained binding potency and biological activity comparable with uniodinated G17. Fundic mucosal cells were separated by size by using an elutriator rotor, and specific 125I-[Leu-15]-G17 binding in the larger cell fractions was highly correlated with the distribution of parietal cells. There was, however, specific gastrin binding in the small cell fractions, not accounted for by parietal cells. Using sequential elutriation and stepwise density gradients, highly enriched parietal and chief cell fractions were prepared; 125I-[Leu15]-G17 binding correlated positively with the parietal cell (r = 0.98) and negatively with chief cell content (r = -0.96). In fractions enriched to 45-65% parietal cells, specific 125I-[Leu15]-G17 binding was rapid, reaching a steady state at 37 degrees C within 30 min. Dissociation was also rapid, with the rate similar after 100-fold dilution or dilution plus excess pentagastrin. At a tracer concentration from 10 to 30 pM, saturable binding was 7.8 +/- 0.8% per 10(6) cells (mean +/- SE) and binding in the presence of excess pentagastrin accounted for 11% of total binding. G17 and carboxyl terminal octapeptide of cholecystokinin (26-33) were equipotent in displacing tracer binding and in stimulating parietal cell function ( [14C]aminopyrine accumulation), whereas the tetrapeptide of gastrin (14-17) had a much lower potency. Proglumide inhibited gastrin binding and selectively inhibited gastrin stimulation of parietal cell function. Canine parietal cells have specific receptors for gastrin that mediate stimulation of parietal cell function. Gastrin receptors were undetectable on chief cells, and yet present on another smaller mucosal cell(s).

Muscarinic receptors and guanylate cyclase in mammalian gastric glandular cells.

To investigate the involvement of guanosine 3',5'-cyclic monophosphate (cGMP) in the cholinergic activation of gastric acid and pepsinogen secretion, we studied the subcellular and cellular relation between particulate guanylate cyclase and muscarinic cholinergic receptor sites. Subcellular fractionation of homogenates from rabbit gastric glands showed that particulate guanylate cyclase and muscarinic receptors were distributed in similar patterns, which differed from the pattern found for Na+-K+-ATPase, a marker for basal-lateral plasma membranes. Assuming a basal-lateral membrane localization for particulate guanylate cyclase and cholinergic receptors, these results suggested a heterogeneity of glandular basal-lateral membranes. The distributions of these markers among fractions enriched in isolated canine parietal or chief cells were also followed. Na+-K+-ATPase correlated with parietal cell distribution (r = 0.86) and guanylate cyclase with chief cell distribution (r = 0.76). The distribution of quinuclidinyl benzilate (QNB) binding sites indicated association of muscarinic receptors with both cell types. The similar subcellular and cellular distributions of guanylate cyclase and QNB binding sites may reflect a functional relationship of these markers in muscarinic-activated pepsinogen secretion. As seen in most other tissues, gastric glandular guanylate cyclase was not stimulated by various gastric secretagogues. We found that small changes in Ca2+ concentration, within the micromolar range, can regulate glandular guanylate cyclase activity. These results are discussed in terms of the cholinergic activation of parietal and chief cell function.

Effect of somatostatin, secretin, and glucagon on secretagogue stimulated aminopyrine uptake in isolated canine parietal cells.

Somatostatin, secretin, and glucagon have been shown to inhibit gastric acid secretion in vivo and thus have been postulated to act directly on the parietal cell. To test the hypothesis that these peptides directly influence the acid secretory cells, we studied the effect of the three gastrointestinal hormones using aminopyrine uptake as an index of acid production. The parietal cells were stimulated to increase aminopyrine uptake by submaximal concentrations of histamine (10(-6) mol/l), methacholine (10(-6) mol/l), and pentagastrin (10(-6) mol/l), but in no concentrations did these gastrointestinal hormones affect any of the secretagogues' response. Our data suggest that gastrointestinal peptides do not modulate acid secretion at the parietal cell level.

Potentiating Interactions of Gastric Stimulants on 14C Aminopyrine Accumulation by Isolated Canine Parietal Cells

Interactions between secretagogues have been examined by using [14C]aminopyrine accumulation as an index of the response of isolated canine parietal cells to stimulation. Potentiating interactions were found between histamine and gastrin and histamine and carbachol, but not between carbachol and gastrin. When all three agents were present simultaneously, the response was greater than the sum of the three individual responses and satisfied other criteria for a true three-way potentiation. Against this three-way combination, atropine and cimetidine caused 42 ± 4% and 69 ± 7% inhibition, respectively. When both atropine and cimetidine were added to the three-way combination, the response was inhibited by 84 ± 3%, with the residual response equivalent to that produced by gastrin alone. The phosphodiesterase inhibitor isobutyl methyl xanthine (10 μM) markedly potentiated the actions of histamine. At 10-fold higher concentrations, isobutyl methyl xanthine also potentiated the response to carbachol and gastrin; these effects were inhibited by cimetidine, as had been found previously for. the action of isobutyl methyl xanthine alone. Interference with these potentiating interactions may explain the apparent nonspecificity of atropine and cimetidine in vivo. The striking potency of cimetidine in inhibiting all stimulants of acid secretion in vivo may be explained by the observation that histamine is an essential component of potentiating interactions that determine the parietal cell response to other stimulants.