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The H + ,K + -ATPase was identified as the primary proton secretory pathway in the gastric parietal cell and is the pharmacological target of agents suppressing acid secretion. Recently, we identified a second acid secretory protein expressed in the parietal cell, the vacuolar H + -ATPase (V-type ATPase). The aim of the present study was to further characterize H + -ATPase activation by modulations in extracellular calcium via the calcium sensing receptor (CaSR). Isolated gastric glands were loaded with the pH indicator dye BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester] to measure intracellular pH. Experiments were conducted in the absence of sodium and potassium to monitor H + -ATPase-specific transport activity. CaSR was activated with the calcimimetic R568 (400 nM) and/or by modulations in extracellular Ca 2+ . Elevation in calcium concentrations increased proton extrusion from the gastric parietal cell. Allosteric modification of the CaSR via R568 and calcium increased vacuolar H + -ATPase activity significantly (ΔpH/min lowCa 2+ (0.1mM)  = 0.001 ± 0.001, ΔpH/min normalCa 2+ (1.0mM)  = 0.033 ± 0.004, ΔpH/min highCa 2+ (5.0mM)  = 0.051 ± 0.005). Carbachol significantly suppressed calcium-induced gastric acid secretion via the H + -ATPase under sodium- and potassium-free conditions. We conclude that the V-type H + -ATPase is tightly linked to CaSR activation. We observed that proton pump inhibitor (PPI) exposure does not modulate H + -ATPase activity. This elevated blood calcium activation of the H + -ATPase could provide an explanation for recurrent reflux symptoms while taking a PPI therapy. NEW & NOTEWORTHY This study emphasizes the role of the H + -ATPase in acid secretion. We further demonstrate the modification of this proton excretion pathway by extracellular calcium and the activation of the calcium sensing receptor CaSR. The novelty of this paper is based on the modulation of the H + -ATPase via both extracellular Ca (activation) and the classical secretagogues histamine and carbachol (inactivation). Both activation and inactivation of this proton pump are independent of PPI modulation.

Caffeine, generally known as a stimulant of gastric acid secretion (GAS), is a bitter-tasting compound that activates several taste type 2 bitter receptors (TAS2Rs). TAS2Rs are expressed in the mouth and in several extraoral sites, e.g., in the gastrointestinal tract, in which their functional role still needs to be clarified. We hypothesized that caffeine evokes effects on GAS by activation of oral and gastric TAS2Rs and demonstrate that caffeine, when administered encapsulated, stimulates GAS, whereas oral administration of a caffeine solution delays GAS in healthy human subjects. Correlation analysis of data obtained from ingestion of the caffeine solution revealed an association between the magnitude of the GAS response and the perceived bitterness, suggesting a functional role of oral TAS2Rs in GAS. Expression of TAS2Rs, including cognate TAS2Rs for caffeine, was shown in human gastric epithelial cells of the corpus/fundus and in HGT-1 cells, a model for the study of GAS. In HGT-1 cells, various bitter compounds as well as caffeine stimulated proton secretion, whereby the caffeine-evoked effect was ( i ) shown to depend on one of its cognate receptor, TAS2R43, and adenylyl cyclase; and ( ii ) reduced by homoeriodictyol (HED), a known inhibitor of caffeine's bitter taste. This inhibitory effect of HED on caffeine-induced GAS was verified in healthy human subjects. These findings ( i ) demonstrate that bitter taste receptors in the stomach and the oral cavity are involved in the regulation of GAS and ( ii ) suggest that bitter tastants and bitter-masking compounds could be potentially useful therapeutics to regulate gastric pH., Competing Interests: Conflict of interest statement: J.H., J.P.L., S.W., and G.K. are employees of Symrise, Holzminden, Germany.

Parietal cell atrophy is considered to cause metaplasia in the stomach. We developed mice that express the diphtheria toxin receptor specifically in parietal cells to induce their death, and found this to increase proliferation in the normal stem cell zone and neck but not to cause metaplastic reprogramming of chief cells. Furthermore, the metaplasia-inducing agents tamoxifen or DMP-777 still induced metaplasia even after previous destruction of parietal cells by diphtheria toxin. Atrophy of parietal cells alone therefore is not sufficient to induce metaplasia: completion of metaplastic reprogramming of chief cells requires mechanisms beyond parietal cell injury or death., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type.

Secretion from the gastric gland involves the activation of various types of cells in a coordinated manner. In order to elucidate the mechanisms underlying the coordination of secretion, we studied live fluorescence images of guinea pig gastric glands stained with acridine orange (AO). On 2 μM AO staining, individual cells were characterized by metachromatic colors and various intensities of fluorescence. When the gland was stimulated with 100 μM of histamine, green fluorescence was transiently increased in parietal cells and intermediate cells and propagated along the gland for a long distance over many cells. Local stimulation in a couple of cells with histamine in the presence of suramin also induced propagation. However, the fluorescence response was suppressed by the addition of H-89, a protein kinase A inhibitor. These findings suggest that a cAMP-dependent signal propagates intercellularly through a variety of cells to induce coordinated secretion in the entire gastric gland., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Atrophic gastritis caused by infection with Helicobacter pylori is characterized by parietal cell loss, which is a main risk factor for gastric cancer. Parietal cells play a crucial role in the regulation of cell lineage maturation and proliferation in the gastric units. Among the classical cadherins, E-cadherin plays an important role not only in epithelial cell-cell connections, but also in the maintenance of epithelial polarity and gastric glandular architecture and regulation of cell proliferation. The aim of this study is to elucidate how parietal cells and E-cadherin are altered in gastritis with Helicobacter pylori infection. We studied the effects of Helicobacter pylori on gastric mucosal E-cadherin 2 weeks after inoculation and investigated the relationship between parietal cell loss and the amount of E-cadherin on parietal cells in Mongolian gerbils. The number of parietal cells and amount of staining of E-cadherin below the isthmus were investigated by immunohistochemistry. It was shown that a reduction in intercellular E-cadherin preceded the disappearance of parietal cells. The gastric glands where parietal cells were lost were replaced by mucus secreting cells without E-cadherin. These results suggest that Helicobacter pylori damaged E-cadherin on parietal cells and caused massive parietal cell loss, leading to the deregulation of gastric morphology.

Background and Aims: Enterochromaffin-like (ECL) cells are central in the regulation of acid secretion. G cells release gastrin and activate ECL cell histamine secretion which stimulates parietal cell H(2) receptors initiating acid secretion. It is unclear whether histamine-mediated parietal cell activation is via a vascular or paracrine pathway. To assess this, we utilized immunohistochemistry (IHC) and electron microscopy to examine gastric tissue and used visualization of formalin fixed dispersed gastric cells and glands to investigate and define the anatomical relationship between ECL and parietal cells., Material and Methods: Sprague-Dawley rat stomachs were instilled with formalin. Thereafter fixed mucosal cells and whole gastric glands were dispersed by mechanical and chemical dissolution and enzymatic digestion. Smears with fixed isolated cells and whole glands were stained by IHC with histidine decarboxylase (HDC) and H+/K+-ATPase antibodies. Whole tissue samples of Sprague-Dawley and cotton rat oxyntic mucosa were investigated with IHC using HDC, VMAT2 and H+/K+-ATPase antibodies, and electron microscopy was performed to further delineate the precise anatomic relationship between ECL cells and parietal cells., Results: Each ECL cell generated a network of HDC- and VMAT2-positive dendritic-like elongations that were in direct contact with several parietal cells. Thus, ECL cells at the base of the gland were in communication with parietal cells in the middle of the gland. Electron microscopy confirmed that the cytoplasmic ECL cell elongations containing secretory vesicles were in direct juxtaposition to parietal cells., Conclusions: These findings indicate that ECL cells directly regulate parietal cell function in a neurocrine manner via slender neuron-like elongations.

The Ca(2+) activated K(+) channel K(ca)3.1 is expressed in a variety of tissues. In the gastric gland it is expressed in the basolateral cell membrane. To determine the functional significance of K(ca)3.1 activity for gastric acid secretion, gastric acid secretion was determined in isolated glands from gene targeted mice lacking functional K(ca)3.1 (K(ca)3.1(-/-)) and from their wild type littermates (K(ca)3.1(+/+)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. Na(ca)-independent pH recovery (ΔpH/min) following an ammonium pulse, a measure of H(ca)/K(ca) ATPase activity, was, however, significantly faster in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. Accordingly, the luminal pH was significantly lower and the acid content significantly higher in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. The abundance of mRNA encoding H(ca)/K(ca) ATPase and KCNQ1 was similar in both genotypes. Increase of extracellular K(ca) concentrations to 35 mM (replacing Na(ca)/NMDG) and treatment with histamine (100 μM) significantly increased ΔpH/min to a larger extent in K(ca)3.1(+/+) than in K(ca)3.1(-/-) mice and dissipated the differences between the genotypes. Carbachol (100 μM) increased ΔpH/min in both genotypes but did not abolish the difference between K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. In K(ca)3.1(+/+) mice the K(ca)3.1 opener DCEBIO (100 μM) did not significantly alter basal ΔpH/min but significantly blunted ΔpH/min in the presence of carbachol. In conclusion, K(ca)3.1 activity suppresses carbachol stimulated gastric acid secretion., (Copyright © 2011 S. Karger AG, Basel.)

Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.

Gastric pathology is a common complication in diabetes mellitus. The aim of the study was to evaluate the functions and morphological changes of the parietal cells of the rat stomach after streptozotocin-induced diabetes. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). The rats were weighed weekly and sacrificed after 6 months. The glandular portion of the stomach was removed and processed for H(+)-K(+)-ATPase immunohistochemistry and light and electron microscopy studies. Acid secretion was measured in vivo. After 6 months of diabetes, the mean weight of the rats was significantly lower (P < 0.001) compared to control. The mean weight of the stomach to body weight percentage increased significantly (P < 0.001) compared to control. The blood glucose level in diabetic rats was significantly higher (P < 0.001) than in normal control. Diabetic rats showed significant (P < 0.001) decrease in basal and stimulated acid secretion when compared to control. Electron micrographs of the parietal cells of glandular stomach of diabetic rats revealed significant (P < 0.0002) reduction in the number of mitochondria and a small though not significant increase in the number of canaliculi in the parietal cells compared with normal. Immunohistochemistry showed reduced H(+)-K(+)-ATPase (P < 0.00001) compared to control. Long-term diabetes induces morphological as well as functional changes in gastric parietal cells. The decrease in the number of mitochondria accompanied by reduced in H(+)-K(+)-ATPase in parietal cells may explain the reduced acid secretion observed in diabetics.

The parietal cell is responsible for secreting concentrated hydrochloric acid into the gastric lumen. To fulfill this task, it is equipped with a broad variety of functionally coupled apical and basolateral ion transport proteins. The concerted scientific effort over the last years by a variety of researchers has provided us with the molecular identity of many of these transport mechanisms, thereby contributing to the clarification of persistent controversies in the field. This article will briefly review the current model of parietal cell physiology and ion transport in particular and will update the existing models of apical and basolateral transport in the parietal cell.

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2-/- mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2-/-, GHKAalpha-/-, and NHE4-/- mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

Gastrulation in vertebrate embryos results in the formation of the primary germ layers: ectoderm, mesoderm and endoderm, which contain the progenitors of the tissues of the entire fetal body. Extensive studies undertaken in Xenopus, zebrafish and mouse have revealed a high degree of conservation in the genes and cellular mechanisms regulating endoderm formation. Nodal, Mix and Sox gene factor families have been implicated in the specification of the endoderm across taxa. Considerably less is known about endoderm development in marsupials. In this study we review what is known about the molecular aspects of endoderm development, focusing on evolution and development of the stomach and parietal cells and highlight recent studies on parietal cells in the stomach of Tammar Wallaby, Macropus eugenii. Although the regulation of parietal cells has been extensively studied, very little is known about the regulation of parietal cell differentiation. Intriguingly, during late-stage forestomach maturation in M. eugenii, there is a sudden and rapid loss of parietal cells, compared with the sharp increase in parietal cell numbers in the hindstomach region. This has provided a unique opportunity to study the development and regulation of parietal cell differentiation. A PCR-based subtractive hybridization strategy was used to identify candidate genes involved in this phenomenon. This will allow us to dissect the molecular mechanisms that underpin regulation of parietal cell development and differentiation, which have been a difficult process to study and provide markers that can be used to study the evolutionary origin of these cells in vertebrates.

Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain approximately 60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H(+)/K(+) ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H(+)/K(+) ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H(+)/K(+) ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in approximately 30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H(+)/K(+) ATPase which underpin the regulation of acid secretion.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

It is clinicopathologically important to elucidate the cell kinetics for the maintenance of normal gastric epithelium. In a rat gastric mucosa isolated after stimulation, a number of cells were exfoliated into the gastric lumen of the pit region. The present study was undertaken to clarify the origin of exfoliated cells and their histochemical profiles by taking the advantages of cryotechniques. As results, most of the exfoliated cells were identified as pit-parietal cells labeled with both peanut-lectin and anti-H+/K+-ATPase antibody. Quantitative analysis verified a time-dependent increase in the number of exfoliated cells in the gastric mucosa isolated after stimulation. The exfoliated cells exhibited a diffuse intracellular staining for E-cadherin, suggesting a dissociation of the adhesion molecule prior to the cell exfoliation. It should be noted that most of the exfoliated cells were negative to the apoptotic markers (TUNEL staining and caspase-3). Ultrastructurally, autophagosome-like structures consisting of H+/K+-ATPase positive membranes were frequently seen in the exfoliated pit-parietal cells. In addition, the pit-parietal cell exfoliation was accompanied by sealing of their basal portion with the cytoplasmic processes of adjacent surface mucous cells. The present morphological findings provide a new insight into the cell kinetics in the gastric epithelium in vitro.

Glucocorticoids stimulate gastric acid secretion, an effect favoring the development of peptic ulcers. Putative mechanisms involved include the serum- and glucocorticoid-inducible kinase (SGK1), which stimulates a variety of epithelial channels and transporters. The present study explored the contribution of SGK1 to effects of glucocorticoids on gastric acid secretion. In isolated gastric glands from gene-targeted mice lacking functional SGK1 (sgk1 (-/-)) and their wild-type littermates (sgk1 (+/+)), H(+)-secretion (DeltapH/min) was determined utilizing 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-fluorescence, SGK1 transcript levels by in situ hybdridization, and expression of KCNQ1 channels by immunohistochemistry and real-time polymerase chain reaction. SGK1 transcript levels were enhanced by a 4-day treatment with 10 mug/g body weight (BW)/day dexamethasone (DEX). Before treatment, DeltapH/min was similar in sgk1 (-/-) and sgk1 (+/+)mice. DEX increased DeltapH/min approximately fourfold in sgk1 (+/+)mice and approximately twofold in sgk1 (-/-)mice, effects abolished in the presence of K(+)/H(+)ATPase-inhibitor omeprazole (50 microM). Increase in local K(+) concentrations to 35 mM (replacing Na(+)) enhanced DeltapH/min, which could not be further stimulated by DEX and was not significantly different between sgk1 (-/-) and sgk1 (+/+)mice. Carbachol (100 microM) and forskolin (5 microM) stimulated gastric acid secretion to a similar extent in sgk1 (-/-) and sgk1 (+/+)mice. In conclusion, SGK1 is not required for basal and cyclic AMP-stimulated gastric H(+) secretion but participates in the stimulation of gastric H(+) secretion by glucocorticoids. The effects of glucocorticoids and SGK1 are not additive to an increase in extracellular K(+) concentration and may thus involve stimulation of K(+) channels.

Gastric parietal cells possess an amplified apical membrane recycling system dedicated to regulated apical recycling of H-K-ATPase. While amplified in parietal cells, apical recycling is critical to polarized secretory processes in most epithelial cells. To clarify putative regulators of apical recycling, we prepared immunoisolated parietal cell H-K-ATPase-containing recycling membranes from human stomachs and analyzed protein contents by tryptic digestion and mass spectrometry. We identified and validated by Western blots many of the proteins previously identified on immunoisolated rabbit tubulovesicles, including Rab11, Rab25, syntaxin 3, secretory carrier membrane proteins (SCAMPs), and vesicle-associated membrane protein (VAMP)2. In addition, we detected several previously unrecognized proteins, including Rab10, VAMP8, syntaxin 7, and syntaxin 12/13. We also identified the K(+) channel component KCNQ1. Immunostaining of human gastric mucosal sections confirmed the presence of each of these proteins in parietal cells and their colocalization with H-K-ATPase on tubulovesicles. To investigate the role of the identified soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in apical recycling, we transfected them as DsRed2 fusions into an enhanced green fluorescent protein (EGFP)-Rab11a-expressing Madin-Darby canine kidney (MDCK) cell line. Syntaxin 12/13 and VAMP8 caused a collapse of the EGFP-Rab11a compartment, whereas a less dramatic effect was observed in cells transfected with syntaxin 3, syntaxin 7, or VAMP2. The five DsRed2-SNARE chimeras were also transfected into a MDCK cell line overexpressing Rab11-FIP2(129-512). All five of the chimeras were drawn into the collapsed apical recycling system. This study, which represents the first proteomic analysis of an immunoisolated vesicle population from native human tissue, demonstrates the diversity of putative regulators of the apical recycling system.

We have previously shown that stimulation of acid secretion in parietal cells causes rapid initial cell shrinkage, followed by Na(+)/H(+) exchange-mediated regulatory volume increase (RVI). The factors leading to the initial cell shrinkage are unknown. We therefore monitored volume changes in cultured rabbit parietal cells by confocal measurement of the cytoplasmic calcein concentration. Although blocking the presumably apically located K(+) channel KCNQ1 with chromanol 293b reduced both the forskolin- and carbachol-induced cell shrinkage, inhibition of Ca(2+)-sensitive K(+) channels with charybdotoxin strongly inhibited the cell volume decrease after carbachol, but not after forskolin stimulation. The cell shrinkage induced by both secretagogues was partially inhibited by blocking H(+)-K(+)-ATPase with SCH28080 and completely absent after incubation with NPPB, which inhibits parietal cell anion conductances involved in acid secretion. The subsequent RVI was strongly inhibited with the Na(+)/H(+) exchanger 1 (NHE1)-specific concentration of HOE642 and completely by 500 muM dimethyl-amiloride (DMA), which also inhibits NHE4. None of the above substances induced volume changes under baseline conditions. Our results indicate that cell volume decrease associated with acid secretion is dependent on the activation of K(+) and Cl(-) channels by the respective secretagogues. K(+), Cl(-), and water secretion into the secretory canaliculi is thus one likely mechanism of stimulation-associated cell shrinkage in cultured parietal cells. The observed RVI is predominantly mediated by NHE1.

The stimulation of gastric acid secretion from parietal cells involves both intracellular calcium and cAMP signaling. To understand the effect of increased cAMP on parietal cell function, we engineered transgenic mice expressing cholera toxin (Ctox), an irreversible stimulator of adenylate cyclase. The parietal cell-specific H(+),K(+)-ATPase beta-subunit promoter was used to drive expression of the cholera toxin A1 subunit (CtoxA1). Transgenic lines were established and tested for Ctox expression, acid content, plasma gastrin, tissue morphology, and cellular composition of the gastric mucosa. Four lines were generated, with Ctox-7 expressing approximately 50-fold higher Ctox than the other lines. Enhanced cAMP signaling in parietal cells was confirmed by observation of hyperphosphorylation of the protein kinase A-regulated proteins LASP-1 and CREB. Basal acid content was elevated and circulating gastrin was reduced in Ctox transgenic lines. Analysis of gastric morphology revealed a progressive cellular transformation in Ctox-7. Expanded patches of mucous neck cells were observed as early as 3 mo of age, and by 15 mo, extensive mucous cell metaplasia was observed in parallel with almost complete loss of parietal and chief cells. Detection of anti-parietal cell antibodies, inflammatory cell infiltrates, and increased expression of the Th1 cytokine IFN-gamma in Ctox-7 mice suggested that autoimmune destruction of the tissue caused atrophic gastritis. Thus constitutively high parietal cell cAMP results in high acid secretion and a compensatory reduction in circulating gastrin. High Ctox in parietal cells can also induce progressive changes in the cellular architecture of the gastric glands, corresponding to the development of anti-parietal cell antibodies and autoimmune gastritis.

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK)., (Copyright (c) 2005 Wiley-Liss, Inc.)

Gastric acid secretion is a complex process that requires hormonal, neuronal, or calcium-sensing receptor activation for insertion of pumps into the apical surface of the parietal cell. Activation of any or all these pathways causes the parietal cell to secrete concentrated acid with a pH at or close to 1. This acidic fluid combines with enzymes that are secreted from neighbouring chief cells and passes out of the gland up through a mucous gel layer covering the surface of the stomach producing a final intragastric pH of less than 4 during the active phase of acid secretion. Defects in either the mucosal barrier or in the regulatory mechanisms that modulate the secretory pathways will result in erosion of the barrier and ulcerations of the stomach or esophagus. The entire process of acid secretion relies on activation of the catalytic cycle of the gastric H+,K+-ATPase, resulting in the secretion of acid into the parietal cell canaliculus, with K+ being the important and rate-limiting ion in this activation process. In addition to K+ as a rate limiter for acid production, Cl- secretion via an apical channel must also occur. In this review we present a discussion of the mechanics of acid secretion and a discussion of recently identified transporter proteins and receptors. Included is a discussion of some of the recent candidates for the apical K' recycling channel, as well as two recently identified apical proteins (NHE-3, PAT-1), and the newly characterized calcium-sensing receptor (CaSR). We hope that this review will give additional insight into the complex process of acid secretion.

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.

Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH(2)- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH(2) terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH(2)- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells.

Polarized attenuated total reflection Fourier transform infrared spectra were recorded on multilayer stacks of native gastric tubulovesicle membranes. The spectral intensity and linear dichroism were measured for average thicknesses ranging between 0 and 100 bilayers. Atomic force microscopy was used to investigate the orientation of the membranes at the top of the stack. Height profiles were obtained along randomly drawn lines and slopes were computed over various distances. Orientation distribution functions were obtained from the slopes and decomposed into Legendre polynomials. It was found that the second Legendre polynomials coefficient characterizing the membrane orientation was always larger than 0.9. It could therefore be concluded that the membrane tilt does not significantly contribute to the infrared dichroism, even for the largest thicknesses tested.

Reg (regenerating gene product) was originally identified as a growth factor involved in pancreatic regeneration. During the healing course of gastric erosion, Reg expression is highly increased in the enterochromaffin-like (ECL) cells surrounding the ulcer crater, suggesting its role as a regulator of gastric mucosal regeneration. However, there has been no direct in vivo evidence of a growth-promoting role of Reg for the gastric mucosal cells. In the current study, Reg-transgenic mice were created and gastric mucosa were analysed for histological changes. Transgenic mice showed a marked increase in the thickness of the fundic mucosa. Anti-proliferating cell nuclear antigen (PCNA) staining of the fundic mucosa demonstrated the enlargement of the proliferating neck zone and the lower PCNA-negative zone. Histological analysis employing antibodies against cell-type markers revealed expansion of the chief cell and parietal cell populations and no change in the number of surface mucus-producing cells, ECL cells, or G cells. In conclusion, Reg has a growth-promoting effect on gastric progenitor cells and an activity to direct the differentiation of the cells into chief cell and parietal cell lineages. This was in contrast to other factors, all of which had been shown to drive differentiation towards mucus producing cells in vivo. In the injured gastric mucosa, Reg may play a unique and important part in the reconstruction of the properly organized mucosal architecture.

The field of acid suppression has been advanced by therapeutics of increasing specificity for inhibiting gastric acid secretion. Particularly important are proton pump inhibitors (PPIs), which inhibit the activity of the gastric acid pump (H+,K(+)-adenosine triphosphatase), the final common step in gastric acid production. Histamine2-receptor antagonists, which act at an early stage of the acid secretion pathway, are less effective and are subject to intolerance. The PPIs are weak bases that undergo accumulation in the acidic space of the secreting parietal cell and are converted in acid to the active thiophilic form, which then forms disulfide bonds with key cysteines of the gastric acid pump. Pantoprazole differs from other PPIs in terms of its reaction with cysteine 822 in the pump and with cysteine 813, a common binding site for all PPIs. Both cysteines are in the sixth transmembrane segment, which is part of the ion transport pathway. This selective binding may have an impact on the dwell time of pantoprazole versus other PPIs because it is inaccessible to reducing agents, in contrast to cysteine 813. Pantoprazole is also very stable (has a slow rate of activation) at neutral pH values compared with other PPIs and has a relatively robust plasma concentration-time curve. These agents are important in the management of duodenal ulcers, nonsteroidal antiinflammatory drug-induced ulcers, gastroesophageal reflux disease, and dyspepsia, but basic pharmacokinetic and pharmacodynamic differences among them may affect clinical utility.

In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20-43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini-granule cells. Their function was not clarified. The second class consisted of pre-pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre-pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre-neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre-zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre-neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre-parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre-parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini-granule cell itself, or from the mitotic activity of pre-pit and pre-neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages.

Acid secretion by the gastric parietal cell is regulated by paracrine, endocrine, and neural pathways. The physiological stimuli include histamine, acetylcholine, and gastrin via their receptors located on the basolateral plasma membranes. Stimulation of acid secretion typically involves an initial elevation of intracellular calcium and/or cAMP followed by activation of a cAMP-dependent protein kinase cascade that triggers the translocation and insertion of the proton pump enzyme, H,K-ATPase, into the apical plasma membrane of parietal cells. Whereas the H,K-ATPase contains a plasma membrane targeting motif, the stimulation-mediated relocation of the H,K-ATPase from the cytoplasmic membrane compartment to the apical plasma membrane is mediated by a SNARE protein complex and its regulatory proteins. This review summarizes the progress made toward an understanding of the cell biology of gastric acid secretion. In particular we have reviewed the early signaling events following histaminergic and cholinergic activation, the identification of multiple factors participating in the trafficking and recycling of the proton pump, and the role of the cytoskeleton in supporting the apical pole remodeling, which appears to be necessary for active acid secretion by the parietal cell. Emphasis is placed on identifying protein factors that serve as effectors for the mechanistic changes associated with cellular activation and the secretory response.

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5'-bromo-2'-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10-30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.

Estrogen, if it is produced in the gastrointestinal tract, may overflow into the systemic circulation in the case of increased portal-systemic shunting. This idea is in accord with a significant step-up in serum estradiol (E2) concentration in the portal vein of rats, compared with that in the artery. Gene expression of aromatase, estrogen synthetase, was demonstrated by RT-PCR in the gastric mucosa of male and female adult rats, equivalent to that in the ovary. Aromatase activity and production of E2 in the gastric mucosa were demonstrated by (3)H(2)O assay and gas chromatography-mass spectrometry, and they were inhibited by aromatase inhibitor, 4-hydroxyandrostenedione. Conversion of (14)C-androstenedione to (14)C-E2 through (14)C-testosterone in cultured gastric mucosa was also demonstrated. Parietal cells exhibited strong signals for aromatase mRNA and immunoreactive protein by in situ hybridization histochemistry and immunohistochemistry. Estrogen receptor alpha mRNA and immunoreactive protein were demonstrated in hepatocytes by RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Total gastrectomy reduced portal venous E2 concentration, without changing systemic E2 concentration, together with down-regulation of estrogen receptor alpha mRNA level in the liver. These findings indicate that gastric parietal cells play a potent endocrine role in secreting estrogen that may function as a regulator of the gastro-hepatic axis.

Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)(+)/vacuolating toxin (vacA)(+) or with cagA(-)/vacA(-) H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-kappaB (NF-kappaB) was studied in nuclear extracts of PC by electrophoretic mobility shift assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA(+)/vacA(+) H. pylori strains but not by cagA(-)/vacA(-) negative strains or by the cagE knockout mutant. S. aureus and C. jejuni had no effect. PC showed no IL-8 or CINC-1 secretion on exposure to cagA(+)/vacA(+) H. pylori. cagA(+)/vacA(+) strains induced activation of NF-kappaB complexes in nuclear extracts of PC, which were composed of p65 and p50 subunits. No significant stimulation of NF-kappaB activation was detected by incubation of PC with the cagE knockout mutant. Preincubation of PC with antisense but not missense oligodeoxynucleotides against the p65 subunit significantly reduced DNA binding to the kappaB recognition sequence. The p65 oligonucleotides as well as the proteasome inhibitor N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine completely prevented PC apoptosis induced by cagA(+)/vacA(+) strains. In summary, cagE presence appears to be essential for H. pylori-induced apoptosis of gastric parietal cells, and this effect is dependent on the activation of NF-kappaB and production of nitric oxide.

The parietal cell is capable of secreting high concentrations of hydrochloric acid into the lumen of the stomach. The apical membrane of this cell contains K+H+ ATPase, which is responsible for proton transport into the lumen. Potassium and chloride channels are also present. The basolateral membrane of the parietal cell possesses transporters that maintain intracellular homeostasis. Specifically, large amounts of bicarbonate that are generated by carbonic anhydrase must be removed from the cell to prevent alkalinization. Efflux of bicarbonate into the blood after acid secretion can be detected and is known as the alkaline tide. Determination of the alkaline tide has been used to measure acid secretion. In this review, we summarize the published data.

Background: Interleukin 1beta (IL-1beta) is a potent inhibitor of gastric acid secretion. Regulatory actions at several levels have previously been demonstrated, including direct inhibition of parietal cell acid secretion. Although IL-1beta may activate several intracellular signalling pathways, the mechanisms responsible for inhibition of carbachol stimulated acid secretion have not been determined., Aims: To investigate the roles of protein kinase C (PKC) and the sphingomyelinase signalling pathways in the regulation of acid secretion by IL-1beta., Methods: Rabbit parietal cells were obtained by collagenase-EDTA digestion and centrifugal elutriation. Acid secretion stimulated by carbachol and A23187 (to mimic elevations in intracellular calcium) was assessed by 14C aminopyrine uptake in response to IL-1beta, PKC, and sphingomyelinase manipulation., Results: IL-1beta inhibited carbachol and A23187 stimulated acid secretion in a dose dependent manner. The inhibitory actions were completely reversed by each of three different PKC inhibitors, staurosporine, H-7, and chelerythrine, as well as by PKC depletion with high dose phorbol ester pretreatment. IL-1beta did not downregulate parietal cell muscarinic receptor. IL-1beta significantly increased membrane PKC activity. Activation of the sphingomyelinase/ceramide pathway had no effect on basal or stimulated acid secretion. The inhibitory action of IL-1beta was independent of protein kinase A and protein kinase G activity., Conclusions: IL-1beta directly inhibits parietal cell carbachol stimulated acid secretion. This action occurs distal to muscarinic receptor activation and elevations in intracellular calcium and requires PKC.

Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H+-K+-ATPase at the apical membrane. The trafficking of the H+-K+-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+-K+-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.

Background: In duodenal ulcer patients, intragastric acidity during omeprazole treatment is significantly lower before Helicobacter pylori eradication than after cure., Aims: To determine if H pylori enhances the acid inhibitory potency of omeprazole in isolated parietal cells and on H(+)/K(+)-ATPase., Methods: Rat parietal cells and pig gastric membrane vesicles enriched in H(+)/K(+)-ATPase activity were incubated with H pylori and the H pylori fatty acid cis 9,10-methyleneoctadecanoic acid (MOA), and the inhibitory effects of omeprazole on parietal cell acid production, H(+)/K(+)-ATPase enzyme activity, and ATPase mediated proton transport were assessed., Results: In isolated parietal cells, H pylori and MOA increased the acid inhibitory potency of omeprazole 1.8 fold. H pylori did not affect the inhibitory potency of omeprazole on H(+)/K(+)-ATPase enzyme activity. In proton transport studies, H pylori (intact bacteria and sonicate) and MOA accelerated the onset of the inhibitory effect of omeprazole and enhanced the proton dissipation rate in response to omeprazole. H. pylori itself increased proton permeability at the vesicle membrane., Conclusion: Our results show that H pylori augments the acid inhibitory potency of omeprazole in parietal cells and enhances omeprazole induced proton efflux rate from gastric membrane vesicles. We suggest that omeprazole unmasks the permanent effect of H pylori on proton permeability at the apical parietal cell membrane, which is counteracted in the absence of a proton pump inhibitor by a reserve H(+)/K(+)-ATPase capacity.

An expression of mRNA coding the calcium-activated Cl- channel-1 (CLCA1) in rabbit gastric parietal cells was examined to verify the possibility that the CLCA1 mediates housekeeping Cl- channels in the basolateral membrane. In whole-cell voltage-clamp experiments of rabbit parietal cells, A23187 (2 microM), a Ca2+ ionophore, activated the basolateral Cl- channels. The partial cDNA fragment of rabbit CLCA1 could be amplified from the total RNA of tracheal epithelium. A Northern blot analysis showed that rabbit CLCA1 mRNA of 3.4 kb is highly expressed in the tracheal epithelium, but not in the gastric parietal cells. Even in a more sensitive detection of rabbit CLCA1 mRNA by RT-PCR, no signal could be observed in the gastric parietal cells. These results suggest that the CLCA1 protein may not be a subunit of the housekeeping Ca2+ -dependent Cl- channel in the basolateral membrane of rabbit gastric parietal cells.

The aim of this study was to determine the cadmium (Cd)-induced functional and structural changes in gastric parietal cells of male rats exposed to high Cd for 30 d. In the present study, control animals were fed with normal food and tap water; the remaining animals received Cd (15 ppm CdCl2) in drinking water for the same period. Receiving Cd for 30 d increased the mean blood Cd level, the mean tissue Cd content, and the mean blood pressure (p < 0.01, p < 0.001, p < 0.01, respectively). The basal acid output fell; however, the increases in stimulated acid output were not statistically significant. Light and electron microscopic examination revealed respectively that (1) Cd decreases the mean parietal cell number per unit from the control value of 23.46 +/- 3.84 to 19.46 +/- 2.12 (p < 0.05) and it affected preferentially the cells located at the distal half of the zymogenic unit and (2) in parietal cells, the Cd-induced alterations were characterized with swollen canalicular profiles, broken-down tubulovesicles, or degenerated mitochondria. We concluded that Cd augments the elimination rate of parietal cells by increasing the alteration rate and reduced basal acid output can be explained easily with the loss of parietal cell population.

The gastric parietal has two characteristic membrane systems. One is the intracellular canaliculus, which is specialized networks of enfolded luminal membrane channels lined with numerous microvilli. The other structures common to all parietal cells are the tubulovesicles or the tubulovesicular membranes, a system of tubules and vesicles. The tubulovesicular compartment is drastically depleted during maximal gastric acid secretion and this is coincident with an increase in the canalicular cell surface membrane. A plausible explanation for this redistribution is the fusion and transfer of tubulovesicular membranes to the plasma membrane. However, for many years there was no convincing evidence of connections between these two membrane systems. The mechanism of the transformation of tubulovesicular membrane into the plasma membrane without demonstrable connections has been an enigma to electron microscopists. Using a recently developed fixation technique for parietal cells [Sugai et al. (1995) Acta Anat Nippon 74:S101], we have investigated the organization of the cytoplasmic membrane systems in the rat resting and tetragastrin stimulated stomachs by ultra-high-resolution scanning electron microscopy (SEM). Gastric mucosae were microwave-fixed in a cacodylate buffer, (334 milliosmoles/kgH(2)O (mOsm)), to which 1.0% glutaraldehyde and 0.5% formaldehyde were added. Specimens examined by TEM of thin sections revealed the cytoplasm packed with tubular membranes similar to images detected by rapid-freeze/freeze-substitution fixation. To render the cytoplasmic membranes visible by SEM, fixed mucosae were treated by the aldehyde-osmium-DMSO-osmium maceration procedure. With much of the cell matrix and filaments removed, SEM revealed numerous 30-60-nm tubules, which formed a meshwork with small cisternae. Vesicles or isolated tubules were not found in adequately macerated parietal cells. The cytoplasmic surface of the intracellular canaliculus was smooth except for round openings representing the bases of macerated microvilli. In favorable sites, connections of the tubular membranes to the canaliculi were clearly visible. Stereo pair views were particularly useful to demonstrate these continuities. Connections between these two membrane compartments suggest the probability of rapid membrane transposition. In this article, the form and distribution of membrane systems of parietal cells in the resting state and after tetragastrin stimulation will be presented and discussed. Special emphasis is made to demonstrate connections between the tubulovesicular system and the intracellular canaliculus., (Copyright 2000 Wiley-Liss, Inc.)

The ECL cells in the oxyntic mucosa of rat stomach produce histamine and chromogranin A-derived peptides such as pancreastatin. The cells respond to gastrin via cholecystokinin-2 (CCK2) receptors. A CCK2 receptor blockade was induced by treatment (for up to 8 weeks) with two receptor antagonists, YM022 and YF476. Changes in ECL-cell morphology were examined by immunocytochemistry and electron microscopy, while changes in ECL cell-related biochemical parameters were monitored by measuring serum pancreastatin and oxyntic mucosal pancreastatin, and histamine concentrations, and histidine decarboxylase (HDC) activity. The CCK2 receptor blockade reduced the ECL-cell density only marginally, if at all, but transformed the ECL cells from slender, elongated cells with prominent projections to small, spherical cells without projections. The Golgi complex and the rough endoplasmic reticulum were diminished. Secretory vesicles were greatly reduced in volume density in the trans Golgi area. Circulating pancreastatin concentration and oxyntic mucosal HDC activity were lowered within a few hours. Oxyntic mucosal histamine and pancreastatin concentrations were reduced only gradually. The CCK2 receptor blockade was found to prevent the effects of omeprazole-evoked hypergastrinaemia on the ECL-cell activity and density. In conclusion, gastrin, acting on CCK2 receptors, is needed to maintain the shape, size and activity of the ECL cells, but not for maintaining the ECL-cell population.

Background & Aims: Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa., Methods: Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting., Results: The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach., Conclusions: The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.

Na-K-2Cl cotransport and Cl/HCO3 exchange are prominent mechanisms for Cl- uptake in Cl--secreting epithelial cells. We used immunofluorescence microscopy to delineate the distributions of Na-K-2Cl cotransporter-1 (NKCC1) and anion exchanger-2 (AE2) proteins in rat gastric mucosa (zymogenic zone). Parietal cells (PCs) above the neck of the gastric gland contained abundant AE2 but little or no NKCC1, whereas those in the neck and base contained high NKCC1 but diminished AE2. Lower levels of NKCC1 were detected in surface mucous cells and in cells comprising the blind ends of all glands. Pulse labeling of proliferating cells with bromodeoxyuridine indicated that new PCs originate in the isthmus with scant NKCC1; the subset of PCs that migrate downward expresses NKCC1 abruptly on entering the neck, within 7 days of cell division. Our results suggest that downwardly migrating PCs replace one mechanism for Cl- entry (Cl/HCO3 exchange) with another (Na-K-2Cl cotransport).

We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.

Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis.

In order to examine some possibly misleading conclusions of the pharmacological analysis of the signal transduction pathways of gastric acid secretion, we evaluated various agents including inhibitors of protein kinase C, cyclic AMP-dependent protein kinase, phospholipase C, phospholipase A2, lipoxygenase, casein kinase, calmodulin, myosin light chain kinase, tyrosine kinase, anion exchanger, and protein phosphatase; and activators of protein kinase C. Among them, the cyclic AMP-dependent protein kinase inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinylsulfonamide (H-89), the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082), three myosin light chain kinase inhibitors (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), and wortmannin), the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), the phospholipase C inhibitor neomycin, and most known calmodulin antagonists strongly inhibited [14C]aminopyrine accumulation, an indicator of acid secretion, in isolated rabbit gastric glands stimulated by N6,2'-O-dibutyryl-cyclic AMP. ONO-RS-082, calmidazolium, and DIDS inhibited H+,K+-ATPase. Most of the chemicals with antisecretory activity showed protonophore-like activity in gastric microsomes as well as in the mitochondria. It is concluded that H-89, ONO-RS-082, ML-7, ML-9, neomycin, and all calmodulin antagonists tested so far should not be used as tools to analyze gastric acid secretion.

1. Mice were subjected to gastrectomy (GX) or food deprivation (24 h). The release of insulin and glucagon in response to different secretagogues was monitored in vivo and in isolated islets 3-4 weeks after surgery. 2. GX animals responded to glucose with an impaired glucose tolerance and a poor increase in plasma insulin. Islets from GX or food-deprived mice displayed impaired insulin release to high glucose and enhanced glucagon release at low glucose. 3. After GX the insulinogenic index, Delta insulin (microU ml-1)/Delta glucose (mg ml-1), was suppressed by 65% after oral glucose and by 59% after i.v. glucose. The integrated insulin response after oral glucose was reduced by 90% in GX mice. After i.v. glucose the reduction was 67%. 4. Carbachol-induced insulin release in vivo was reduced after food deprivation and exaggerated after GX. Carbachol-stimulated glucagon secretion was suppressed after GX and after food deprivation. A similar pattern was found in vitro. 5. Cyclic AMP activation (by the phosphodiesterase inhibitor isobutylmethylxanthine or the adenylate cyclase stimulator forskolin) induced a greater insulin response in GX or food-deprived mice than in sham-operated, fed mice. A similar pattern was found in vitro. The glucagon response was enhanced in vitro but not in vivo. 6. Crude extracts of rat oxyntic mucosa enhanced basal as well as glucose-induced insulin release from isolated islets, whereas glucagon release was markedly inhibited. The effects were dose dependent, the inhibition of glucagon release being achieved at lower concentrations than the potentiation of glucose-induced insulin release. The active principle was inactivated by incubation with trypsin or leucine aminopeptidase. 7. The data suggest that a circulating agent, probably a peptide, from gastric oxyntic mucosa stimulates glucose-induced insulin secretion. It also suppresses glucagon secretion. The GX-evoked impairment of the insulin (and glucagon) response to glucose is partly compensated for by an enhanced insulin response to cholinergic and/or cyclic AMP activation.