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C S Chew

Publications and source records attributed to C S Chew.

At least 19 recordsLinked to original sources
PubMed2002

Regulation of protein and vesicle trafficking at the apical membrane of epithelial cells.

The characterization of endocytotic and post-endocytotic trafficking pathways at the apical membrane of epithelial cells presents a potential avenue for the identification of targets to modulate the initial stages of absorption and transepithelial transport of macromolecules. In addition, it is becoming increasingly clear that the activity of a number of apical membrane transporters is acutely regulated by vesicular trafficking. The gastric HCl-secreting parietal (oxyntic) cell is a model system to characterize an apical membrane vesicular trafficking pathway and its relationship to the regulation of the function of the gastric proton pump. The subapical tubulovesicular compartment of the parietal cell is highly enriched in the H,K-ATPase and is a key endosomal-like system in the apical membrane recycling pathway. In the process of cataloging the proteins that interact with the H,K-ATPase and tubulovesicles, we have identified novel components that may regulate protein sorting through this compartment and candidate linker proteins between the vesicular trafficking machinery and the cytoskeleton. One protein associated with H,K-ATPase-rich tubulovesicles is the nonreceptor tyrosine kinase c-src, identified by a screen for dynamin-binding proteins. The tyrosine kinase is active, as it can tyrosine-phosphorylate tubulovesicular proteins in vitro. One of the tyrosine-phosphorylated proteins of M(r) 100 kDa may be the H,K-ATPase itself, or a protein in a complex with the H,K-ATPase that is stable to dissociation by nonionic detergents. By virtue of its association with tubulovesicular membranes, c-src may regulate the trafficking and/or activity of the H,K-ATPase. A second protein identified by a screen for dynamin-binding proteins is the protein lasp-1. Lasp-1, through its modular protein structure, may bind to dynamin and to the actin cytoskeleton, thus linking the vesicular trafficking machinery with the cytoskeleton. These two examples illustrate the utility of the parietal cell in the biochemical characterization of components potentially involved in the regulation of apical membrane trafficking pathways.

Animals↗
PubMed2000

The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia.

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.

Actins↗
PubMed1999

Isolation, cloning, and characterization of a new mammalian coronin family member, coroninse, which is regulated within the protein kinase C signaling pathway.

In order to understand the regulatory role of protein kinase C (PKC) in secretory epithelia, it is necessary to identify and characterize specific downstream targets. We previously identified one such protein in studies of gastric parietal cells. This protein was referred to as pp66 because it migrated with an apparent molecular mass of 66 kDa on SDS-polyacrylamide gels. The phosphorylation of pp66 is increased by the cholinergic agonist, carbachol, and by the PKC activator, phorbol-12-myristate-13-acetate, in a calcium-independent manner. In this study, we have purified pp66 to homogeneity and cloned the complete open reading frame. GenBankTM searches revealed a 45% homology with the Dictyostelium actin-binding protein, coronin, and approximately 67% homology with the previously cloned human and bovine coronin-like homologue, p57. pp66 appears to be most highly expressed in the gastrointestinal mucosa and in kidney and lung. Confocal microscopic studies of an enhanced green fluorescent protein fusion construct of pp66 in cultured parietal cells and in Madin-Darby canine kidney cells indicate that pp66 preferentially localizes in F-actin-rich regions. On the basis of our findings, we propose that pp66 may play an important, PKC-dependent role in regulating membrane/cytoskeletal rearrangements in epithelial cells. We have tentatively named this protein coroninse, because it appears to be highly expressed in secretory epithelia.

Amino Acid Sequence↗
PubMed1998

Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells.

Previous investigations in several systems have demonstrated that Rab3 family members redistribute to soluble fractions on fusion of secretory granules with target plasma membranes. Rab proteins are then recycled back onto mature secretory vesicles after reinternalization of the membrane. Although this cycle is well established for Rab3, far less is known about redistribution of other Rab proteins during vesicle fusion and recycling. In the gastric parietal cell, Rab11a is associated with H-K-ATPase-containing tubulovesicles, which fuse with the apical plasma membrane (secretory canaliculus) in response to agonists such as histamine. We have analyzed distribution of Rab11a and other tubulovesicle proteins in resting and histamine-stimulated rabbit parietal cells. Stimulation of isolated gastric glands in the presence of 100 microM histamine and 100 microM 3-isobutyl-1-methylxanthine did not cause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a, Rab25, syntaxin 3, and SCAMPs increased immunoreactivity in stimulus-associated vesicles prepared from rabbits treated with histamine compared with those from ranitidine-treated animals. The large GTPase dynamin was found in both vesicle preparations, but there was no change in amount of immunoreactivity. Immunofluorescence staining of resting and histamine-stimulated primary cultures of parietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate that Rab11a does not cycle off the membrane during the process of tubulovesicle fusion with the secretory canaliculus. Thus Rab11a may remain associated with recycling apical membrane vesicle populations.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell.

Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Calcium signaling in cultured human and rat duodenal enterocytes.

Vagal stimuli increase duodenal mucosal HCO-3 secretion and may provide anticipatory protection against acid injury, but duodenal enterocyte (duodenocyte) responses and cholinoceptor selectivity have not been defined. We therefore developed a stable primary culture model of duodenocytes from rats and humans. Brief digestion of scraped rat duodenal mucosa or human biopsies with collagenase/dispase yielded cells that attached to the extracellular matrix Matrigel within a few hours of plating. Columnar cells with villus enterocyte morphology that exhibited spontaneous active movement were evident between 1 and 3 days of culture. Rat duodenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+]i), with an apparent EC50 of approximately 3 microM. In a first type of signaling pattern, [Ca2+]i returned to basal or near basal values within 3-5 min. In a second type, observed in cells with enlarged vacuoles characteristic of crypt cell morphology, the initial transient increase was followed by rhythmic oscillations. Human duodenocytes responded with a more sustained increase in [Ca2+]i, and oscillations were not observed. Rat as well as human duodenocytes also responded to CCK-octapeptide but not to vasoactive intestinal polypeptide. Equimolar concentrations (100 nM) of the subtype-independent muscarinic antagonist atropine and the M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 microM carbachol, whereas the M1 antagonist pirenzepine and the M2 antagonists methoctramine and AF-DX 116BS had no effect at similar concentrations. Responses in rat and human duodenocytes were similar. A new agonist-sensitive primary culture model for rat and human duodenocytes has thus been established and the presence of enterocyte CCK and muscarinic M3 receptors demonstrated.

Animals↗
PubMed1997

Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2).

The mechanism by which H+ alters the kinetics of the H+-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H+ on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H+ influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H+ on the IC50 values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H+ did not change the IC50 value for Ala-Lys but did decrease the IC50 value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H+ binding and translocation in H+-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K(t) (Michaelis-Menten constant) or the IC50 values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H+ stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.

Animals↗
PubMed1997

Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion.

Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Gö 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-alpha and PKC-beta1/beta2, respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.

Actins↗
PubMed1996

Purification, cloning, and expression of a novel, endogenous, calcium-sensitive, 28-kDa phosphoprotein.

In gastric parietal cells, cholinergically induced increases in intracellular free calcium concentrations have been well characterized, but little is known about the signaling events beyond the initial rise in intracellular calcium. In the present study, we report the isolation of a 28-kDa protein, which is rapidly phosphorylated in intact, enriched parietal cells in response to both the cholinergic agonist, carbachol, and the calcium ionophore, ionomycin. A combination of in situ 32P labeling and one- and two-dimensional gel electrophoresis was used to acquire sufficient quantities of protein to obtain partial amino acid sequence. Cloning of the pp28 cDNA revealed a novel protein which we have named CSPP28 based on its calcium-sensitive phosphorylation. There are three CSPP28 mRNA species (1.7, 2.2, and 3.3 kilobases) that are widely distributed throughout a variety of rabbit tissues. Recombinant CSPP28 was phosphorylated by both crude parietal cell homogenate and purified CaM kinase II in a calcium/calmodulin-dependent manner. We propose that CSPP28 may play an important and ubiquitous role in the calcium signaling pathway.

Amino Acid Sequence↗
PubMed1996

Parietal cell MAP kinases: multiple activation pathways.

Epidermal growth factor (EGF) is a potent mitogen for many cell types; however, the best known effect of EGF on gastric parietal cell HCl secretion is inhibition of this response. Using rabbit parietal cells in primary culture, we recently showed that the effect of EGF is biphasic with acute inhibition followed by sustained enhancement of acid secretory-related responses. We hypothesized that EGF might activate a mitogen-activated protein (MAP) kinase signaling pathway in parietal cells, and this pathway might play a role in mediating sustained and/or acute effects of EGF on parietal cell acid secretory-related functions [C. S. Chew, K. Nakamura, and A. C. Petropolous. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G818-G826, 1994]. We used several methodological approaches to demonstrate the presence of MAP kinase (MAPK) isoforms, extracellular signal-regulated kinases (ERKs) 1 and 2, in parietal cells and to begin to characterize their mechanisms of activation in this highly differentiated cell type. In acutely isolated, 90-98% enriched parietal cells, EGF biphasically activated ERK-1 and ERK-2, with peak response occurring at approximately 5 min followed by a sustained lower level of activation for at least 2 h. The EC50 for EGF (1.2 +/- 0.4 nM) was similar to the previously determined EC50 for the stimulatory effect of EGF on acid secretory responses. In contrast to EGF, the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a sustained activation of ERK-1 and ERK-2 for at least 2 h. Carbachol also activated ERK-1 and ERK-2; however, this response was weaker and monophasic. Neither the Ca2+ ionophore ionomycin nor the adenylyl cyclase activator forskolin altered basal or stimulated ERK activity. Carbachol, but not EGF or TPA, also activated an unidentified 70-kDa protein kinase as detected with in-gel myelin basic protein (MBP) kinase renaturation assays. Parietal cell MAPK activation was not correlated to a shift in apparent relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that basal phosphorylation of ERK isoforms may be higher in parietal cells compared with actively proliferating cell lines. Also, in contrast to observations in neutrophils, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor, wortmannin (0.3-3 microM), failed to inhibit ERK activation in response to EGF, carbachol, or TPA. The combined data indicate that 1) EGF, TPA, and carbachol activate overlapping as well as distinct intracellular signaling pathways in gastric parietal cells, 2) EGF activates ERKs and enhances parietal cell acid secretory related functions via receptors with similar affinities, and 3) in contrast to some cell types, the parietal cell ERK-signaling cascade does not appear to be directly modulated by the PtdIns 3-kinase pathway or by elevated intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate concentrations.

Androstadienes↗
PubMed1994

Multiple actions of epidermal growth factor and TGF-alpha on rabbit gastric parietal cell function.

Parietal cells in primary culture and freshly isolated parietal cells were used to compare acute and chronic effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on acid-secretory related activity, measured as accumulation of the weak base, [14C]aminopyrine (AP). EGF and TGF-alpha chronically enhanced basal and agonist-stimulated AP accumulation (mean effective concentration 0.6-0.8 nM) but acutely inhibited responses to histamine and carbachol (half-maximal inhibitory concentration approximately 4 nM). Pertussis toxin (250 ng/ml, 4 h) suppressed acute EGF inhibition of histamine-stimulated AP accumulation but not the chronic enhancement. A subclass of tyrosine kinase inhibitors suppressed chronic EGF effects (genistein > tyrphostin B56 >>> tyrphostin B42), whereas tyrphostin A25, lavendustin A, and the inactive genistein analogue, daidzein, had no significant effect. In contrast, histamine-stimulated AP accumulation was acutely potentiated by genistein, daidzein, and tyrphostin B42, but not tyrphostin B56. Reduced phosphorylation of a 44- to 45-kDa protein with an isoelectric point of approximately 7 [phosphoprotein (pp) 44] was correlated with chronic inhibition but not with acute potentiation by specific tyrosine kinase inhibitors. Preliminary data indicate that pp44 is a member of the mitogen-activated protein kinase family of tyrosine/threonine kinases (also known as extracellular signal-related kinases). We propose that 1) EGF and/or TGF-alpha modulates parietal cell function by multiple signaling pathways, 2) a soluble tyrosine kinase may be involved in the mediation of the chronic effects of EGF, and 3) acute potentiation of histamine-stimulated AP accumulation by certain tyrosine kinase inhibitors and daidzein is probably not mediated by receptor-associated tyrosine kinases.

Aminopyrine↗
PubMed1993

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

Actins↗
PubMed1991

Thapsigargin potentiates histamine-stimulated HCl secretion in gastric parietal cells but does not mimic cholinergic responses.

The role of calcium in control of HCl secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+]i), an action that appears to be independent of inositol lipid breakdown and protein kinase C activation and to be mediated, at least partially, by selective inhibition of endoplasmic reticulum Ca2(+)-ATPase. Using the calcium-sensitive fluorescent probe, fura-2, in combination with digitized video image analysis of single cells as well as standard fluorimetric techniques, we found that thapsigargin induced sustained elevation of [Ca2+]i in single parietal cells and in parietal cells populations. Chelation of medium calcium led to a transient rise and fall in [Ca2+]i, indicating that the sustained elevation in [Ca2+]i in response to thapsigargin was due to both intracellular calcium release and influx. Although thapsigargin appeared to affect the same calcium pool(s) regulated by the cholinergic agonist, carbachol, and the pattern of thapsigargin-induced increases in [Ca2+]i were similar to the plateau phase of the cholinergic response, thapsigargin did not induce acid secretory responses of the same magnitude as those initiated by carbachol (28 vs 600% of basal). The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]i in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Furthermore, thapsigargin did not fully mimic potentiating interactions between histamine and carbachol, either in magnitude or in the pattern of temporal response. Assuming that the action of thapsigargin is specific for intracellular calcium release mechanisms, these data suggest that 1) sustained influx of calcium is necessary but not sufficient for cholinergic activation of parietal cell HCl secretion and for potentiating interactions between cAMP-dependent agonists and carbachol; 2) mechanisms in addition to elevated [Ca2+]i and protein kinase C activation may be involved in cholinergic regulation; and 3) increases in [Ca2+]i in response to histamine are not directly involved in the mechanism of histamine-stimulated secretion.

Aminopyrine↗
PubMed1991

Calcium oscillations and morphological transformations in single cultured gastric parietal cells.

Calcium is an important regulator of cellular activities including HCl secretion by parietal cells. With cholinergic agonists, a role for calcium is established; however, with histamine, at least two signaling pathways may be involved including calcium and adenosine 3',5'-cyclic monophosphate (cAMP). Because chelation of medium and/or cellular calcium has pronounced inhibitory effects on cholinergic but lesser effects on histamine-stimulated acid secretory responses in cell populations, the calcium pathway may not be of central importance for HCl secretion regulated by histamine. We have used digitized video imaging of fura-2 fluorescence ratios and cellular morphology to determine more precisely the relationship between cellular calcium signaling mechanisms and acid secretion in single cultured rabbit parietal cells. Calcium signaling patterns were found to exhibit striking differences with histamine as compared with the cholinergic agonist carbachol. Maximal doses of histamine initiated repetitive oscillations in intracellular calcium ([Ca2+]i) in approximately 50% of cells, whereas the maximal carbachol response was characterized by a typical initial spike followed by a sustained elevation in [Ca2+]i. Oscillations in response to carbachol were detected only at doses below the half-maximal concentration for initiation of acid secretion. Correlation of gradual expansion of acidic vacuoles with increases in [Ca2+]i in the same cells indicated that approximately 20% of cells increased acid secretory-related activities in response to histamine with no detectable rise in [Ca2+]i. These data suggest two possibilities: 1) a rise in [Ca2+]i is not necessary for histamine-stimulated HCl secretion, or 2) heterogeneous receptor-coupling mechanisms exist in parietal cell populations with either calcium or cAMP mechanisms predominating in different subpopulations. The ability to assess simultaneously acid secretory-related responses and calcium signaling patterns allows, for the first time, correlation of "physiological" and biochemical responses in single parietal cells. This methodology is expected to provide new insight into second messenger control mechanisms that are not possible either in cell populations or acutely isolated parietal cells that do not exhibit morphological transformations detectable at the light microscope level.

Animals↗
PubMed1990

HCl secretion and [Ca2+]i in cultured parietal cells.

Gastric parietal cells in primary culture have been tested to determine their utility as a model for the study of the role of intracellular calcium ([Ca2+]i) in the control of HCl secretion. Changes in [Ca2+]i were measured in single cells on a microscope stage using the cell-permeant form of the fluorescent calcium probe, fura-2. Simultaneous images of cell fluorescence and morphology were acquired using a digitized video image analysis system and two video cameras, one for low-light level fluorescence detection and one for high resolution DIC/transmitted light images. Both histamine and carbachol, which are known stimulants of HCl secretion, increased [Ca2+]i and stimulated dramatic changes in morphology in these cultured cells. Changes in morphology were accompanied by an increased uptake of the weak base, [14C]-aminopyrine (AP), and a shift from green to red fluorescence of another weak base, acridine orange. These results indicate that cultured parietal cells, maintained under controlled conditions on a microscope stage, retain viability and secretagogue responsiveness. Thus, this cellular model appears to be suitable for correlation of changes in [Ca2+]i and activation of HCl secretion.

Animals↗
PubMed1989

Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa.

A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, [14C]aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor "physiological" responses at the same time within the same cell.

Adenosine Triphosphatases↗