Canine Parietal Cell Research Articles

Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits secretagogue- stimulated gastric acid secretion via an EGF receptor located on parietal cells. The aim of this study was to examine whether this growth factor inhibited carbachol-stimulated acid secretion through a protein kinase C-dependent mechanism. METHODS: The effect of EGF on carbachol-stimulated aminopyrine uptake, inositol trisphosphate formation, and intracellular Ca2+ ([Ca2+]i) in purified cultured parietal cells was studied. The ability of protein kinase A and C inhibitors to alter the inhibitory action of EGF was assessed. EGF- mediated translocation and activation of protein kinase C in parietal cells were determined. RESULTS: EGF dose dependently inhibited carbachol-stimulated aminopyrine uptake in a pertussis toxin- insensitive, genistein (tyrosine kinase inhibitor)--sensitive manner, with a maximal inhibitory effect (37.5% +/- 6.8%) achieved at 10(-7) mol/L. EGF did not significantly inhibit carbachol-stimulated inositol trisphosphate formation and did not alter the initial transient increase or sustained plateau in [Ca2+]i stimulated by this secretagogue. The protein kinase C inhibitors H-7 and staurosporine dose dependently reversed the inhibitory action of EGF, whereas H-89 (protein kinase A inhibitor) failed to alter the effect of EGF. EGF pretreatment increased the translocation of alpha and beta 1 isoforms of protein kinase C and stimulated kinase activity in parietal cells. EGF did not down-regulate the parietal cell muscarinic receptor. CONCLUSIONS: The inhibitory action of EGF on carbachol-stimulated parietal cell activity seems to involve protein kinase C. (Gastroenterology 1996 Feb;110(2):469-77)

Canine Parietal Cell Binding by Antibodies to the Complementary Peptide of Somatostatin

Using antibodies to a complementary peptide of somatostatin, putative somatostatin binding proteins were characterized on canine parietal cells. A synthetic peptide (S-C1) was derived from the complementary mRNA sequence for somatostatin-14. Antiserum containing antibodies to S-C1 inhibited competitively 125I-Tyr11-somatostatin binding to canine oxyntic mucosal membranes. Canine parietal cell preparations were incubated with carbachol in the presence or absence of somatostatin and antisera to S-C1. Antibodies to S-C1 produced a decrease in carbachol-stimulated 14C-aminopyrine uptake comparable with that produced by 10(-6) M somatostatin. In immunocytochemical studies by light microscopy, antibodies to S-C1 produced positive staining of parietal cells throughout the oxyntic gland area. By electron microscopy using immunogold techniques, binding by antibodies to somatostatin C-1 was localized ultrastructurally to basolateral and intracellular membranes and to secretory canalicular membranes of parietal cells. These studies support the conclusion that antibodies to the somatostatin complementary peptide demonstrate properties similar to those of somatostatin in that they inhibit carbachol-stimulated aminopyrine uptake and 125I-somatostatin binding. Furthermore, these antibodies localize to specific regions on plasma membranes of parietal cells, which may represent somatostatin binding sites.

Cloning and characterization of gastrin receptor from ECL carcinoid tumor of [formula omitted

We report here the cDNA cloning of a putative gastrin receptor from enterochromaffin-like (ECL) carcinoid tumor of Mastomys natalensis . For this study, we used the polymerase chain reaction technique to amplify transmembrane domain sequences related to rat pancreatic cholecystokinin (CCK)-A receptor from the ECL tumor cDNA library. The amino acid sequence deduced from the cloned cDNA showed 85.7% and 49.0% identity to canine parietal cell gastrin receptor and rat pancreatic CCK-A receptor, respectively. Ligand binding studies using COS-7 cells transfected with the cDNA showed the same binding specificity for gastrin and CCK-8 as the gastrin receptor on the Mastomys carcinoid tumor membrane. Both gastrin and CCK-8 elevated free cytosolic calcium concentration in COS-7 cells expressing the cloned receptor. RNA blot analysis revealed the expression of the gastrin receptor in both Mastomys stomach and brain.

Expression of gastric antisecretory and prostaglandin E receptor binding activity of misoprostol by misoprostol free acid

In enriched canine parietal cell preparations, misoprostol, an analog of prostaglandin E1 methyl ester, was rapidly deesterified to misoprostol free acid. Under this circumstance, misoprostol and misoprostol free acid exhibited equal antisecretory potency against histamine-stimulated acid secretion and bound equally well to prostaglandin E receptors. When the deesterification of misoprostol was inhibited by paraoxon, an esterase inhibitor, the antisecretory and receptor binding activity of misoprostol was markedly reduced, with potency much less than misoprostol free acid. These results indicate that misoprostol free acid is the active biological form of misoprostol that binds to prostaglandin E receptors and mediates the antisecretory action of misoprostol.

Demonstration of specific E-type prostaglandin receptors using enriched preparations of canine parietal cells and [ 3H] misoprostol free acid

High-affinity, E-type prostaglandin binding sites in enriched canine parietal cell preparations were identified with [ 3H] misoprostol free acid, a prostaglandin E 1 analogue. Saturable, reversible, and highly stereospecific binding was identified, with approximately 8,000 binding sites per cell. Prostaglandin I and F bound weakly, and cimetidine and histamine did not bind. The results indicate that [ 3H] misoprostol free acid binds to E-type prostaglandin receptors, which suggests that the ulcer-healing inhibition of gastric acid secretion by misoprostol results from its interaction with a specific E-type prostaglandin receptor.

Review: antisecretory drugs: cellular mechanisms of action.

Parietal cell secretory function may be inhibited by three mechanisms. (1) Receptors for gastrin, histamine and acetylcholine are present on the canine parietal cell, and parietal cell function may be directly inhibited by specific antagonists for each of these receptors. (2) Receptor activation of parietal cell function is mediated by cyclic AMP-dependent (histamine) and calcium-dependent (cholinergic agents and gastrin) mechanisms. The antisecretory action of prostaglandins reflect interference with histamine activation of adenylate cyclase. The current generations of calcium channel blockers have only weak antisecretory actions in vivo and are unlikely to be useful in clinical practice. (3) A third mechanism of inhibition is blockade of H+/K(+)-ATPase by substituted benzimidazoles, such as omeprazole. Each of these three mechanism provides modalities of potential clinical usefulness for treating acid-peptic disease. Gastrin and acetylcholine receptors are present on other fundic cells, in addition to the parietal cell. These other cells include the somatostatin cell in the dog fundic mucosa and the histamine-containing enterochromaffin-like (ECL) cell present in the fundic mucosa of several species. The relative impact of these receptors on different cell types on the regulation of acid secretion remains uncertain, and is probably variable among different species. One gastrin receptor of considerable importance is the gastrin receptor that exerts a trophic effect on the ECL cell in the fundic mucosa. Sustained hypergastrinaemia in response to profound hypochlorhydria is associated with hyperplasia of this cell type; the elucidation of the conditions that promote this hyperplasia and the clinical consequences of this association are pressing challenges.

Antisecretory activity of misoprostol, its racemates, stereoisomers and metabolites in isolated parietal cells

Inhibition of histamine-stimulated acid secretion by misoprostol was compared to its racemates, stereoisomers and metabolites in isolated canine parietal cell preparations. The concentration of misoprostol required to inhibit 50% of maximal histamine-stimulated acid secretion (IC50) was 3.8 +/- 0.3 nM. One racemate of misoprostol was at least 1000 times more potent than the other. Of the four misoprostol stereoisomers, the 11R, 16S isomer exhibited the most potent activity against histamine with an IC50 value of 1.4 +/- 0.1 nM. The acid metabolite of misoprostol was equally potent as misoprostol. In contrast to the acid metabolite, the beta-oxidation metabolites of misoprostol lacked significant activity at 1 microM. The results indicate that: 1) the acid metabolite of misoprostol may play a significant role in the antisecretory activity of misoprostol, and 2) the high degree of stereo-specificity associated with the antisecretory effects indicates that the activity of misoprostol may be receptor mediated.

Mechanisms of action of antisecretory drugs. Studies on isolated canine fundic mucosal cells.

Cellular mechanisms underlying the actions of antisecretory agents were studied with dispersed canine fundic cells; aminopyrine accumulation monitored parietal cell (PC) function. Canine PC have pharmacologically typical histamine (H) H2 and muscarinic (M) receptors. PC also have gastrin (G) receptors, which were selectively blocked by gastrin/CCK antagonists. Potentiating interactions occurred between secretagogues, one of the components of the interdependency between regulatory pathways. Prostaglandins (PG) E2 inhibited H-stimulated PC function. Treatment of PC with pertussis toxin (PT), which inactivates the inhibitory GTP-binding protein of adenylate cyclase (Gi), markedly reduced PG inhibition, indicating PG action via Gi. PC function can also be directly inhibited by H+/K+-ATPase inhibitors, such as omeprazole. When canine mucosal cells were studied, stimulatory G and inhibitory M receptors were present on fundic somatostatin (S) cells. Histamine was localized to canine fundic mast cells, which lacked G or M receptors, a conclusion that may not pertain to fundic histamine cells in other species. Nonparietal cell receptors may be important modulators of the regulation of acid secretion.

Acid secretion and mucosal blood flow in the distal and proximal parts of the canine parietal cell mass in response to cholinergic stimulation.

Dogs provided with one denervated gastric pouch in the distal part of the corpus fundus and another of equal size in the proximal part were studied during stimulation with bethanechol chloride. Dose-response curves were determined for acid output and for mucosal blood flow, which was measured with the neutral red technique. ED50 for acid output was almost identical for the two pouches, giving a value of 1.13 (median) for the ratio of ED50 proximal pouch to ED50 distal pouch, which is between the ratios of 3.54 and 0.65 noted for pentagastrin and histamine, respectively in earlier investigations. The ED50 for blood flow was significantly lower than that for the simultaneous acid output, with a value of 0.28 for the ratio of ED50 blood flow to ED50 acid output, which gives an intermediate position between the ratios for pentagastrin (0.13 and 0.21 for the distal and proximal pouch, respectively) and histamine (0.61 and 0.73) obtained in earlier investigations. It could be hypothesized that the above differences in ratio between these three agonists reflect a difference at the receptor level of the parietal cells and the arterioles.

Difference in kinetics between the distal and proximal parts of the canine parietal cell mass in response to histamine.

Dogs provided with one denervated gastric pouch in the distal part of the corpus fundus and another of equal size in the proximal part, were studied during histamine stimulation. Dose-response curves were determined for acid output and mucosal blood flow, which was measured with the Neutral Red technique. Maximum acid output was significantly higher for the distal (mean 234.0 mumol min-1) than for the proximal pouch (120.0 mumol min-1). ED50 for acid output was significantly higher for the distal (105.0 micrograms kg-1 h-1 of histamine) than for the proximal pouch (57.0 micrograms kg-1 h-1). ED50 for blood flow did not differ significantly between the distal (40.5 micrograms kg-1 h-1) and the proximal pouch (29.0 micrograms kg-1 h-1). For both pouches ED50 for blood flow was significantly lower than that for simultaneous acid output. This indicated that the rise in blood flow precedes that in acid output. Regression analysis showed a significantly higher ratio between maximum mucosal blood flow and maximum acid output for the distal (0.30 ml mumol-1) than for the proximal pouch (0.15 ml mumol-1). The finding that ED50 for acid output in response to histamine was lower for the proximal pouch is contrary to earlier results obtained with pentagastrin, which showed that ED50 was lower for the distal pouch. There is no valid explanation for these differences in ED50 between the pouches, but a difference at receptor level could be hypothesized. The difference in maximum acid output between the two pouches is explained by the known difference between distal and proximal parts in volume fraction of the parietal cells. The possibility that these differences in acid production depend on dissimilarities in mucosal blood flow can be excluded.

Difference in kinetics between the distal and proximal parts of the canine parietal cell mass in response to pentagastrin.

1. Dogs were provided with one denervated gastric pouch in the distal and another of equal size in the proximal part of the corpus fundus. Dose-response curves for pentagastrin were determined for acid output and mucosal blood flow, which was measured with the Neutral Red technique. 2. Maximum acid output was significantly higher for the distal (mean 165.4 mumol min-1) than for the proximal pouch (53.8 mumol). ED50 for acid output was significantly higher for the proximal (5.10 micrograms kg-1 h-1 of pentagastrin) than for the distal pouch (1.98 micrograms). 3. ED50 for blood flow did not differ significantly between the distal (0.48 microgram) and the proximal pouch (1.23 micrograms). For both pouches ED50 for blood flow was significantly lower than that for simultaneous acid output. This indicated that the rise in blood flow precedes that of acid output. 4. Regression analysis indicated similar ratios between maximum mucosal blood flow and maximum acid output for the distal (0.17 ml mumol-1) and the proximal (0.14 ml mumol-1)pouch. 5. The difference in maximum acid output between the two pouches correlates well with the known difference between distal and proximal parts in volume fraction of the parietal cells, while there is no valid explanation for the difference in ED50 for acid output. It can be excluded that these differences in acid production depend on dissimilarities in mucosal blood flow.

The interaction of stimulants on the function of isolated canine parietal cells.

With isolation, the parietal cell is removed from the effects of the many endogenous substances that may modulate its function in intact mucosa, even in the basal state. The isolated canine parietal cell responds to the major endogenous regulators of secretion: histamine, acetylcholine and gastrin. These agents act on specific receptors as evidenced by (1) the specificity of antagonist (H2 antagonists, atropine, and dibutyryl cyclic GMP respectively), (2) the binding of radiolabelled ligands, and (3) the existence of separate second messenger systems (cyclic AMP for histamine, calcium influx for acetylcholine, and an unidentified mechanism for gastrin). Potentiating interactions, which occur between histamine and acetylcholine or histamine and gastrin, do not involve extra production of second messenger. When histamine and acetylcholine are given together, the amounts of cyclic AMP generated and of calcium entering the cell are not greater than when each is acting alone. The apparent non-specific effects of inhibitors acting in vivo, such as the inhibition of all forms of stimulation by H2 antagonists, could reflect withdrawal of the potentiating action of the background histamine always present in the mucosa.

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of 14C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI 2 inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID 50) at 10 nM. 6 β-PGI 1 also inhibited the action of histamine (ID 50 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6 β-PGI 1 inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6 β-PGI 1 stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.