Histamine increases phosphorylation of 27- and 40-kDa parietal cell proteins

1987 ◽  
Vol 253 (6) ◽  
pp. G823-G829 ◽  
Author(s):  
C. S. Chew ◽  
M. R. Brown
Keyword(s):  
Parietal Cell ◽  
Prolonged Exposure ◽  
Calcium Ionophore ◽  
Parietal Cells ◽  
Free Calcium ◽  
Camp Content ◽  
General Role ◽  
Histamine Stimulation ◽  
Low Speed ◽  
Hcl Secretion

Purified, hormonally responsive parietal cells from rabbit gastric mucosae were used as a model to study intracellular mechanisms controlling parietal cell HCl secretion. Using a high-resolution, two-dimensional electrophoretic technique, we demonstrate that histamine increases phosphorylation of two parietal cell proteins with approximate molecular weights of 27 and 40 kDa and respective pIs of 5.9 and 6.2. The increase in phosphorylation appears to be mediated via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism because cAMP analogues and forskolin stimulate phosphorylation of these proteins, whereas the cholinergic agonist, carbachol, which elevates parietal cell intracellular free calcium concentration but not cAMP content, and the calcium ionophore, ionomycin, do not. Both phosphoproteins are located in low-speed particulate fractions. The 40-kDa phosphoprotein was found in both enriched chief and parietal cells. This phosphoprotein may be cytoskeleton associated, since it is detected in a Triton-insoluble particulate fraction after prolonged exposure of parietal cells to Triton X-100. The 27-kDa phosphoprotein was detected in parietal but not in enriched chief cells and appeared to be localized in a low-speed fraction previously shown to contain increased H+-K+-ATPase activity after histamine stimulation. The location and rapid increase in phosphorylation of the 27-kDa phosphoprotein upon histamine stimulation make this protein an attractive candidate for future studies of intracellular regulation of parietal cell HCl secretion. The 40-kDa phosphoprotein may play a more general role in control of cytoskeletal activity, and perhaps, in morphological transformations associated with stimulus-secretion coupling.

scholarly journals Localization of ClC-2 Cl− channels in rabbit gastric mucosa

2001 ◽  
Vol 280 (6) ◽  
pp. C1599-C1606 ◽  
Author(s):  
Ann M. Sherry ◽  
Danuta H. Malinowska ◽  
Randal E. Morris ◽  
Georgianne M. Ciraolo ◽  
John Cuppoletti
Keyword(s):  
Gastric Mucosa ◽  
Parietal Cell ◽  
Functional Characterization ◽  
Parietal Cells ◽  
Immunogold Electron Microscopy ◽  
Gastric Glands ◽  
Canalicular Membrane ◽  
Isolated Gastric Glands ◽  
Hcl Secretion

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl− channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl− channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.

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

1991 ◽  
Vol 2 (1) ◽  
pp. 27-39 ◽  
Author(s):  
C S Chew ◽  
A C Petropoulos
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Parietal Cell ◽  
Calcium Release ◽  
Parietal Cells ◽  
Kinase C ◽  
Hcl Secretion ◽  
Protein Kinase C Activation ◽  
Cholinergic Response

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.

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody

1993 ◽  
Vol 264 (4) ◽  
pp. G637-G644 ◽  
Author(s):  
J. Cuppoletti ◽  
P. Huang ◽  
M. A. Kaetzel ◽  
D. H. Malinowska
Keyword(s):  
Parietal Cell ◽  
Parietal Cells ◽  
Bee Venom ◽  
Gastric Parietal Cell ◽  
Functional Changes ◽  
Hcl Secretion ◽  
Stimulation Of

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade

1999 ◽  
Vol 112 (16) ◽  
pp. 2639-2646 ◽  
Author(s):  
B.J. Agnew ◽  
J.G. Duman ◽  
C.L. Watson ◽  
D.E. Coling ◽  
J.G. Forte
Keyword(s):  
Parietal Cell ◽  
Apical Membrane ◽  
Parietal Cells ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Culture Model ◽  
Site Of Action ◽  
Hcl Secretion ◽  
Healthy Control ◽  
Activation Cascade

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

2005 ◽  
Vol 288 (2) ◽  
pp. G376-G387 ◽  
Author(s):  
Catherine S. Chew ◽  
Curtis T. Okamoto ◽  
Xunsheng Chen ◽  
Hai Yan Qin
Keyword(s):  
Parietal Cell ◽  
Cell Types ◽  
Parietal Cells ◽  
Differential Staining ◽  
Mucosal Cell ◽  
Pronounced Increase ◽  
General Role ◽  
Iq Motif ◽  
Mucous Neck Cells ◽  
Cell Cell

IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.

Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

1987 ◽  
Vol 58 (02) ◽  
pp. 737-743 ◽  
Author(s):  
Frarnçois Lanza ◽  
Alain Beretz ◽  
Martial Kubina ◽  
Jean-Pierre Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

scholarly journals Gene expression profiling of gastrin target genes in parietal cells

2006 ◽  
Vol 24 (2) ◽  
pp. 124-132 ◽  
Author(s):  
Renu N. Jain ◽  
Cynthia S. Brunkan ◽  
Catherine S. Chew ◽  
Linda C. Samuelson
Keyword(s):  
Gene Expression ◽  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Parietal Cell ◽  
Target Genes ◽  
Adaptor Protein ◽  
Parietal Cells ◽  
Cell Gene Expression ◽  
Cell Gene

Previous studies demonstrated that mice with a null mutation in the gene encoding the hormone gastrin have impaired gastric acid secretion. Hence, the aim of this study was to evaluate changes in the acid-secreting parietal cell in gastrin-deficient (GAS-KO) mice. Analysis of several transcripts encoding parietal cell proteins involved in gastric acid secretion showed reduced abundance in the GAS-KO stomach, including H+,K+-ATPase α- and β-subunits, KCNQ1 potassium channel, aquaporin-4 water channel, and creatine kinase B, which were reversed by gastrin infusion for 1 wk. Although mRNA and protein levels of LIM and SH3 domain-containing protein-1 (LASP-1) were not greatly changed in the mutant, there was a marked reduction in phosphorylation, consistent with its proposed role as a cAMP signal adaptor protein associated with acid secretion. A more comprehensive analysis of parietal cell gene expression in GAS-KO mice was performed using the Affymetrix U74AV2 chip with RNA from parietal cells purified by flow cytometry to >90%. Comparison of gene expression in GAS-KO and wild-type mice identified 47 transcripts that differed by greater than or equal to twofold, suggesting that gastrin affects parietal cell gene expression in a specific manner. The differentially expressed genes included several genes in signaling pathways, with a substantial number (20%) known to be target genes for Wnt and Myc.

Enhanced calcium signaling and acid secretion in parietal cells isolated from gastrin-deficient mice

2003 ◽  
Vol 284 (1) ◽  
pp. G145-G153 ◽  
Author(s):  
Karen L. Hinkle ◽  
Gina C. Bane ◽  
Ali Jazayeri ◽  
Linda C. Samuelson
Keyword(s):  
Acid Secretion ◽  
Parietal Cell ◽  
Mutant Cell ◽  
Flow Cytometric Analysis ◽  
Cell Number ◽  
Parietal Cells ◽  
Gastric Glands ◽  
Deficient Mice ◽  
Cell Defect

Gastrin-deficient mice have impaired basal and agonist-stimulated gastric acid secretion. To analyze whether an intrinsic parietal cell defect contributed to the reduced acid secretion, we analyzed parietal cell calcium responses and acid secretory function in vitro. Parietal cells were purified by light-scatter cell sorting and calcium responses to gastrin, histamine, and carbachol were measured in gastrin-deficient and wild-type mice cell preparations. Surprisingly, basal and histamine-induced calcium concentrations were higher in the mutant cell preparations. [14C]aminopyrine uptake analysis in acutely isolated gastric glands revealed that basal acid accumulation was enhanced in gastrin-deficient cell preparations as well as on treatment with carbachol or histamine. These results suggested that an intrinsic parietal cell defect was not responsible for the reduced acid secretion in gastrin-deficient mice. Flow cytometric analysis of dispersed, H+-K+-ATPase-immunostained gastric mucosal preparations revealed a marked increase in parietal cell number in gastrin-deficient mice, which may have accounted for the enhanced in vitro acid secretion detected in this study. Parietal cells were found to be significantly smaller in the mutant cell preparations, suggesting that gastrin stimulation modulates parietal cell morphology.

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