VIP inhibits histamine-induced ultrastructural changes related to acid secretion by parietal cells

We have studied the in vitro effect of VIP and histamine on ultrastructure of the parietal cells in isolated guinea pig fundic glands. The morphological changes induced by histamine in the parietal cells can be compared to those observed after histamine stimulation in vivo or in vitro on gastric mucosa preparations. In contrast, VIP incubation did not produce the ultrastructural changes related to gastric acid secretion, in resting parietal cells. Pretreatment of the glands by VIP resulted in a remarkable suppression of the histamine effect, since the parietal cells assumed an almost resting state. The data (1) indicate that the parietal cells in isolated gastric glands of the guinea pig retain in vitro the capacity to undergo the ultrastructural changes that are related to acid secretion in vivo after histamine or cAMP and (2) suggest that VIP is an inhibitor of histamine-induced gastric acid secretion in the guinea pig. It is proposed that VIP could act directly on the parietal cell via cAMP-phosphodiesterase activation, or indirectly via gastric somatostatin and/or prostaglandin secretions, inhibiting the H2 receptor-cAMP system of the parietal cell.     

Epidermal growth factor inhibits cytoskeleton-related changes in the surface of parietal cells

The effect of epidermal growth factor (EGF) on gastric acid secretion was correlated with the morphological changes of the apical pole of rat parietal cells studied by transmission electron microscopy. Gastric acid secretion was stimulated by histamine, carbachol, pentagastrin, and insulin-induced hypoglycemia, and estimated by continuous recording of pH variations of gastric luminal perfusate. EGF inhibits acid secretion in these conditions. The action of the hormone also results in the arrest or reversal of the changes in shape undergone by parietal cells as they go into secretion. In view of the evidence involving cytoskeletal elements in the generation of these structural alterations, our observations suggest that the action of EGF on gastric acid secretion may be a consequence of a general effect of this hormone on cytoskeletal function.     

Properties of isolated gastric enterochromaffin-like cells.

The gastric enterochromaffin-like cell (ECL) has been studied in gastric fundic glands by confocal microscopy and as a purified cell preparation by video imaging of calcium signaling and measurements of histamine release. Regulation of gastric acid secretion is largely due to alterations of histamine activation of the H2 receptor on the parietal cell and can be divided into central neural regulation, with direct actions of neuronally released mediators and into peripheral regulation by substances released from other endocrine cells. Gastric neuronal stimulation of acid secretion by alteration of ECL cell function is probably mediated by pituitary adenylate cyclase activating peptide (PACAP) receptors on the ECL cell, which activate calcium signaling and histamine release. Peripheral stimulation of acid secretion via the ECL cell is largely mediated by gastrin stimulation of calcium signaling and histamine release. Gastric neuronal inhibition of ECL cell function is probably mediated by galanin inhibition of calcium signaling, and histamine release and peripheral inhibition of ECL cell function is mainly due to somatostatin release from D cells.     

Participation of cyclic nucleotides and phospholipidis in signal transduction of histamin receptor H3 of the gastric mucosa parietal cells in rats with experimental ulcer

The goal of the work was to evaluate plasma membrane phospholipid composition in rat gastric parietal cells under the histamine H3 receptor activation. The content of cyclic nucleotides was also studied. It was shown that H3-receptor agonist (R)-alpha-methylhistamine increases the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) level and decreases the phoshatidylinositole level in plasma membrane of rat gastric parietal cells and leads to attenuation of the cGMP production and enhancement of the cAMP production under the experimental stress--the induced stomach ulcer formation. The histamine H3-receptor antagonist, thioperamide, insignificantly increases the PC and PE level and increases the phoshatidylinositole level in the plasma membrane of rat gastric parietal cells and leads to cAMP production attenuation, and cGMP contents decreases in the above-stated cells. Thus it was shown that histamine H3 receptor activation causes different effects on polyphosphoinositide and adenylatcyclase cascades in parietal cells under stomach ulcer conditions.     

Histochemical study of carbonic anhydrase and HCO3(-)-stimulated adenosine triphosphatase in the rat stomach during hydrochloric acid secretion]

By means of histochemical methods it was established that carboanhydrase of the parietal cells of fundal glands and HCO-3 stimulated ATP-ase of the rat's gastric mucosa capillaries disposed next to the parietal cells were activated by food and histamine. The obtained data confirm the idea of the multicellular functional essembly sustaining HCL secretion (R. I. Salganik, 1974). Should gastrin induce the formation of histamine in endocrinous cells and the histamine activate carboanhydrase in parietal cells, our data confirm the supposition that HCO-3-ions stimulate ATP-ase sustaining the exchange between HCO-3-cells and C1- of blood to form HCl in the endothelial cells of blood capillaries adjacent to the parietal cells.     

Localization of (3H)histamine and (3H)prostaglandin E2 receptors in isolated cells of rat gastric mucosa

Isolated cells of rat gastric mucosa were obtained by treatment of rat stomach with pronase. Two fractions were isolated, one of which was rich (up to 90%) and the second one poor (to 25%) of parietal cells. Using specific antagonists and agonists of H1- and H2-receptors of histamine (diphenhydramine, metiamide, cimetidine, impromidine, dimaprit) the H2-receptors of histamine were shown to be localized in parietal cells. A preferential binding of (3H)prostaglandin E2 by the receptor proteins of plasma membranes of non-parietal (presumably mucoid) cells was found. The data obtained indicate that rat gastric mucosa contains receptors of histamine and PGE2 which differ in their intracellular localization and strictly selectively bind (3H)histamine and (3H)PGE2. It is assumed that the starting point in the mechanism of action of these intercellular regulators on gastric secretion is probably the process of their specific recognition by the protein receptors localized in functionally different cells.     

Effects of chemical transmitters on function of isolated canine parietal cells

In view of the complexity of the regulation of gastric acid secretion, isolated parietal cells offer the appealing prospect of studying the receptors and mechanisms activating this cell after it has been removed from the confusing milieu of the intact mucosa. Histamine and cholinergic agents stimulate the function of canine parietal cells by interacting with typical H2 and muscarinic receptors. Gastrin produces only a small stimulation, interacting with a third, presumably specific, receptor. Combinations of histamine and carbachol and of histamine and gastrin produce potentiating interactions. When isolated parietal cells are treated with these combinations of agents, cimetidine and atropine display and apparent lack of specificity, reminiscent of that found in vivo, and probably resulting from interference with the histamine and cholinergic components of these potentiating interactions. The action of histamine, but not of carbachol or gastrin, is linked to stimulation of cyclic AMP production by parietal cells. Two potential inhibitors of acid secretion, secretin and prostaglandin E2, also stimulate cyclic AMP production, but these later effects appeared to occur largely in nonparietal cells. PGE2 however specifically inhibits histamine-stimulated parietal cell function, apparently by blocking activation of adenylate cyclase. Cholinergic action on the other hand is closely linked to enhanced influx of extracellular calcium.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.     

Blocking of histamine H2 receptors enhances parietal cell degeneration in the mouse stomach

The effects of the histamine H2 receptor antagonist, ranitidine, on parietal cell lineage was studied in the mouse stomach by using light and electron microscopy techniques. Mice were continuously infused for 15, 30, and 42 hr with ranitidine. Semithin sections examined under the light microscope revealed spherical light areas in the cytoplasm of parietal cells which in thin sections under the electron microscope appeared to be vacuoles. Cells were categorized as normal, altered and damaged. While altered cells were characterized by dilated canaliculi and vacuoles, the damaged cells showed signs of necrosis or apoptosis. In control mice, altered and damaged parietal cells were consistently few and only found in the pit or base regions of the epithelial units. After 15-hr-treatment with ranitidine, 40% of the parietal cells were altered. After 30 hr infusion, altered parietal cells became 53% of the examined population, and after 42 hr, 72% of the parietal cells were affected (42% altered and 30%, damaged). The gradual increase in parietal cell vacuolation was associated with an increase in the census of pre-parietal cells. Some mice were allowed to recover from treatment for 4 days. The appearance of normal parietal cells and disappearance of damaged cells was observed and the gastric glands became morphologically normal. In conclusion, inhibiting acid secretion by blocking the histamine H2 receptors, enhanced not only the degenerative elimination of parietal cells but also the production of pre-parietal cells and thus, the recovery of the population was prompt.     

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.     

Effects of cholecystokinin on acid formation in glands and cells isolated from rabbit and rat gastric mucosa

Isolated gastric glands and isolated cells prepared from rabbit and rat were studied to analyse the influence of cholecystokinin octapeptide (CCK 8) on histamine stimulated parietal cell acid formation as assessed by [14C]aminopyrine sequestered in acid tissue compartments. In rabbit gastric glands, CCK 8 evoked 32+/-6% (P<0. 01) inhibition of histamine stimulated acid formation, whereas in glands prepared from rat no inhibition was recorded. Instead, CCK 8 seemed to induce a variable increase of the histamine stimulation in rat gastric glands as the aminopyrine accumulation was increased by 110+/-46% (P<0.1). Further studies on cell preparations derived from rabbit gastric mucosa revealed dual properties of CCK 8, eliciting either inhibition or stimulation of the parietal cell depending on the presence of endocrine cells. The results show that paracrine communication may be effective in glandular preparations, but seems to vary depending on species.     

Histamine interaction on surface recognition sites of H2-type in parietal and non-parietal cells isolated from the guinea pig stomach

In gastric cells isolated by pronase digestion from the guinea pig, histamine stimulated cAMP production in 3 fundic cell fractions (EC50 = 1.6--2 x 10(-4) M) enriched in parietal (94%), peptic (63%) and mucous cells (87%) as well as in antral cells (EC50 = 4 x 10(-4) M) that are devoid of parietal cells. Histamine stimulations were completely inhibited by the H2 antagonist cimetidine (Ki = 0.27--0.57 x 10(-6) M) or by the H1 antagonist diphenhydramine, but at 100-times lower potency (Ki = 22--45.7 x 10(-6) M), indicating the presence of histamine H2 receptors in parietal and nonparietal cells of the guinea pig gastric mucosa.     

Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells

Acid secretory activity and respiration in rabbit gastric glands are stimulated by cAMP-dependent and -independent agonists. Potentiation between agonists suggests interaction of the activation pathways. Regulation of secretory response by protein kinase C was investigated with 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA elevated basal respiration, pepsin release, and acid secretion but inhibited histamine and carbachol stimulation of acid secretion by gastric glands, as measured by [dimethylamino-14C]aminopyrine accumulation. The inhibition of histamine response was specific for protein kinase C activators, occurred after a 20-min lag, and was not reversed by removal of TPA after 3 min of preincubation. TPA pretreatment inhibited acid secretory responses to cholera toxin and forskolin but enhanced the response to cAMP analogues. Cholera toxin and pertussis toxin simulated ADP-ribosylation of 45 and 41 kDa proteins, respectively, in parietal cell membranes. Therefore, both stimulatory (Gs) and inhibitory (Gi) GTP binding proteins of adenylyl cyclase appear to be present in parietal cells. Pretreatment with pertussis toxin attenuated PGE2 but not TPA inhibition of histamine stimulation of aminopyrine accumulation. Thus, the inhibitory effect of TPA does not appear to be associated with an action on Gi. The results with histamine and carbachol suggest that protein kinase C may regulate both cAMP-dependent and -independent stimulation of parietal cell acid secretion.     

High-pressure freezing of isolated gastric glands provides new insight into the fine structure and subcellular localization of H+/K+-ATPase in gastric parietal cells.

High-pressure freezing (HPF) is currently the most reliable method to obtain an adequately frozen sample for high-resolution morphological evaluation. Here we applied the HPF technique to isolated rabbit gastric glands to reveal structural evidence that may be correlated with functional activity of gastric parietal cells. This approach provided well-preserved fine structure and excellent antigenicity of several parietal cell proteins. Microtubules were abundant in the cytoplasm and frequently appeared to be associating with tubulovesicles. Interestingly, many electron-dense coated vesicles were apparent around the intracellular canaliculi (IC) of resting parietal cells, consistent with active membrane retrieval from the apical membranes. Immunolabeling of H+/K+-ATPase was evident on the endocytic components (e.g., multivesicular bodies) and tubulovesicles. After histamine stimulation, the parietal cells characteristically showed expanded IC membranes with varied features of their apical microvilli. The labeling density of H+/K+-ATPase was four-fold higher on the IC membrane of stimulated parietal cells than on that of resting parietal cells. Immunolabeling of ezrin was clearly identified on the IC and basolateral membranes of parietal cells, corresponding to their F-actin-rich sites. The present findings provide a new insight into the correlation of cell structure and function in gastric parietal cells.     

KCNQ1 loss-of-function mutation impairs gastric acid secretion in mice

The KCNQ1 channel is abundantly expressed in the gastric parietal cells. Although the functional coupling of KCNQ1 with the H⁺/K⁺-ATPase has already been confirmed on the basis of pharmacological kinetics, the effect of a KCNQ1 loss-of-function mutation on gastric acidification remains unclear. In this study, parietal cells and gastric glands from both C57BL/6 J mice (normal control) and J343 mice (mice with a KCNQ1 loss-of-function mutation) were isolated to study the effects of KCNQ1 on gastric acidification. We found that the mutation limited intracellular acidification of parietal cells and H⁺ secretion of the stomach in response to histamine. Thus, a KCNQ1 loss-of-function mutation may impair gastric acid secretion.     

Acid secretagogue-induced stimulation of gastric parietal cell gene expression

Carbamoylcholine (carbachol), histamine, and gastrin are three principal stimulants of gastric acid secretion. To explore the mechanisms by which these agents exert their actions in parietal cells, we examined their effects on the gene expression of the enzymes responsible for H+ generation. Each secretagogue induced rapid and coordinate increases in steady-state levels of mRNAs encoding carbonic anhydrase II and H+,K+-ATPase in isolated canine gastric parietal cells. Furthermore, pronounced increases, with different kinetics, in expression of beta-actin mRNA were observed. With increasing time after cell isolation, carbonic anhydrase II and H+,K+-ATPase, but not beta-actin, mRNA levels were attenuated, suggesting that parietal cell-specific genes may be dependent upon maintenance of parietal cell contacts within intact mucosal tissue. Pretreatment of the cells with competitive inhibitors of each secretagogue blocked the increases. Our results indicate that acid secretagogue-specific receptor activation in parietal cells triggers coordinate gene expression of the two enzymes involved in H+ ion generation and that beta-actin may be an important regulator of acid secretion.     

Gastric submucosal microdialysis: a method to study gastrin- and food-evoked mobilization of ECL-cell histamine in conscious rats

Rat stomach ECL cells are rich in histamine and chromogranin A-derived peptides, such as pancreastatin. Gastrin causes the parietal cells to secrete acid by flooding them with histamine from the ECL cells. In the past, gastric histamine release has been studied using anaesthetized, surgically manipulated animals or isolated gastric mucosa, glands or ECL cells. We monitored gastric histamine mobilization in intact conscious rats by subjecting them to gastric submucosal microdialysis. A microdialysis probe was implanted into the submucosa of the acid-producing part of the stomach (day 1). The rats had access to food and water or were deprived of food (48 h), starting on day 2 after implantation of the probe. On day 4, the rats received food or gastrin (intravenous infusion), and sampling of microdialysate commenced. Samples (flow rate 1.2 microl min(-1)) were collected every 20 or 60 min, and the histamine and pancreastatin concentrations were determined. The serum gastrin concentration was determined in tail vein blood. Exogenous gastrin (4-h infusion) raised microdialysate histamine and pancreastatin dose-dependently. This effect was prevented by gastrin receptor blockade (YM022). Depletion of ECL-cell histamine by alpha-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, suppressed the gastrin-evoked release of histamine but not that of pancreastatin. Fasting lowered serum gastrin and microdialysate histamine by 50%, while refeeding raised serum gastrin and microdialysate histamine and pancreastatin 3-fold. We conclude that histamine mobilized by gastrin and food intake derives from ECL cells because: 1) Histamine and pancreastatin were released concomitantly, 2) histamine mobilization following gastrin or food intake was prevented by gastrin receptor blockade, and 3) mobilization of histamine (but not pancreastatin) was abolished by alpha-fluoromethylhistidine. Hence, gastric submucosal microdialysis allows us to monitor the mobilization of ECL-cell histamine in intact conscious rats under various experimental conditions not previously accessible to study. While gastrin receptor blockade lowered post-prandial release of ECL-cell histamine by about 80%, unilateral vagotomy reduced post-prandial mobilization of ECL-cell histamine by about 50%. Hence, both gastrin and vagal excitation contribute to the post-prandial release of ECL-cell histamine.     

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.