Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

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Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00177.2011 ◽

2011 ◽

Vol 301 (3) ◽

pp. G537-G546 ◽

Cited By ~ 10

Author(s):

Michael K. Jones ◽

Ercheng Zhu ◽

Edna V. Sarino ◽

Oscar R. Padilla ◽

Takamune Takahashi ◽

...

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Acid Secretion ◽

Parietal Cell ◽

Nitrosative Stress ◽

Cell Function ◽

Acute Injury ◽

Gastric Injury ◽

Gastric Parietal Cell ◽

Deficient Mice

Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% ( P < 0.05) reduction in total gastric SOD activity and a 93% ( P < 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% ( P < 0.0001) and 44% ( P < 0.005), respectively. Gastric tissue ATP content was reduced by 34% ( P < 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% ( P < 0.0001) and 40% ( P < 0.0005), respectively. There was a fourfold ( P < 0.02) increase in gastric mucosal apoptosis and 41% ( P < 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.

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Parietal cell MAP kinases: multiple activation pathways

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.271.4.g640 ◽

1996 ◽

Vol 271 (4) ◽

pp. G640-G649 ◽

Cited By ~ 10

Author(s):

K. Nakamura ◽

C. J. Zhou ◽

J. Parente ◽

C. S. Chew

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Parietal Cell ◽

Intracellular Signaling ◽

Map Kinases ◽

Sodium Dodecyl ◽

Cell Types ◽

Parietal Cells ◽

Gastric Parietal Cell ◽

Cell Acid

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.

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A Morphometric Study of Changes in Dolichos Biflorus Agglutinin (DBA)-binding Pattern of the Human Gastric Parietal Cell in Association with Acid Secretion

Okajimas Folia Anatomica Japonica ◽

10.2535/ofaj1936.66.5_277 ◽

1989 ◽

Vol 66 (5) ◽

pp. 277-282 ◽

Cited By ~ 4

Author(s):

Masataka ITO ◽

Hayato KAWAKAMI ◽

Shozo SAITO ◽

Toshio AOYAGI ◽

Hiroshi HIRANO

Keyword(s):

Acid Secretion ◽

Parietal Cell ◽

Morphometric Study ◽

Binding Pattern ◽

Gastric Parietal Cell ◽

Dolichos Biflorus ◽

Dolichos Biflorus Agglutinin

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Acid secretion and membrane reorganization in single gastric parietal cell in primary culture

Biology of the Cell ◽

10.1016/0248-4900(90)90349-8 ◽

1990 ◽

Vol 69 (3) ◽

pp. 223-232 ◽

Cited By ~ 20

Author(s):

Paul Mangeat ◽

Tutus Gusdinar ◽

Alain Sahuquet ◽

David K Hanzel ◽

John G Forte ◽

...

Keyword(s):

Acid Secretion ◽

Primary Culture ◽

Parietal Cell ◽

Gastric Parietal Cell ◽

Membrane Reorganization

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Modulatory role of phosphoinositide 3-kinase in gastric acid secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00138.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. G532-G543 ◽

Cited By ~ 16

Author(s):

S. E. Mettler ◽

S. Ghayouri ◽

G. P. Christensen ◽

J. G. Forte

Keyword(s):

Acid Secretion ◽

Parietal Cell ◽

Phosphodiesterase Inhibitors ◽

Dominant Negative ◽

Gastric Glands ◽

Gastric Parietal Cell ◽

Pi3k Inhibitor Ly294002 ◽

Akt Phosphorylation ◽

Phosphoinositide 3 Kinase

The gastric parietal cell is responsible for the secretion of HCl into the lumen of the stomach mainly due to stimulation by histamine via the cAMP pathway. However, the participation of several other receptors and pathways have been discovered to influence both stimulation and inhibition of acid secretion (e.g., cholinergic). Here we examine the role of phosphoinositide 3-kinase (PI3K) in the modulation of acid secretion. Treatment of isolated gastric glands and parietal cells with the PI3K inhibitor, LY294002 (LY), potentiated acid secretion in response to histamine to nearly the maximal secretion obtained with histamine plus phosphodiesterase inhibitors. As cAMP levels were elevated in response to histamine plus LY, but other means of elevating cAMP (e.g., forskolin, dbcAMP) were not influenced by LY, we posited that the effect might require activation of G-protein-coupled histamine H2 receptors, possibly through the protein kinase B pathway (also known as Akt). Study of downstream effectors of PI3K showed that histaminergic stimulation increased Akt phosphorylation, which in turn was blocked by inhibition of PI3K. Expression studies showed that high expression of active Akt decreased acid secretion, whereas dominant-negative Akt increased acid secretion. Taken together, these data suggest stimulation with histamine increases the activity of PI3K leading to increased activity of Akt and decreased levels of cAMP in the parietal cell.

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Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

Molecular Biology of the Cell ◽

10.1091/mbc.8.3.399 ◽

1997 ◽

Vol 8 (3) ◽

pp. 399-407 ◽

Cited By ~ 69

Author(s):

X R Peng ◽

X Yao ◽

D C Chow ◽

J G Forte ◽

M K Bennett

Keyword(s):

Membrane Protein ◽

Acid Secretion ◽

Parietal Cell ◽

Membrane Trafficking ◽

Parietal Cells ◽

Gastric Parietal Cell ◽

Protein Receptors ◽

Alternative Function ◽

Target Membrane ◽

Syntaxin 3

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

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Computer modeling of gastric parietal cell: significance of canalicular space, gland lumen, and variable canalicular [K+]

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00431.2015 ◽

2016 ◽

Vol 310 (9) ◽

pp. G671-G681 ◽

Cited By ~ 5

Author(s):

James M. Crothers ◽

John G. Forte ◽

Terry E. Machen

Keyword(s):

Acid Secretion ◽

Parietal Cell ◽

Driving Forces ◽

Short Circuit ◽

Open Circuit ◽

Cell Secretion ◽

Gastric Parietal Cell ◽

Atpase Inhibitors ◽

Long Time ◽

Cellular Components

A computer model, constructed for evaluation of integrated functioning of cellular components involved in acid secretion by the gastric parietal cell, has provided new interpretations of older experimental evidence, showing the functional significance of a canalicular space separated from a mucosal bath by a gland lumen and also shedding light on basolateral Cl− transport. The model shows 1) changes in levels of parietal cell secretion (with stimulation or H-K-ATPase inhibitors) result mainly from changes in electrochemical driving forces for apical K+ and Cl− efflux, as canalicular [K+] ([K+]can) increases or decreases with changes in apical H+/K+ exchange rate; 2) H-K-ATPase inhibition in frog gastric mucosa would increase [K+]can similarly with low or high mucosal [K+], depolarizing apical membrane voltage similarly, so electrogenic H+ pumping is not indicated by inhibition causing similar increase in transepithelial potential difference ( Vt) with 4 and 80 mM mucosal K+; 3) decreased H+ secretion during strongly mucosal-positive voltage clamping is consistent with an electroneutral H-K-ATPase being inhibited by greatly decreased [K+]can (Michaelis-Menten mechanism); 4) slow initial change (“long time-constant transient”) in current or Vt with clamping of Vt or current involves slow change in [K+]can; 5) the Na+-K+-2Cl− symporter (NKCC) is likely to have a significant role in Cl− influx, despite evidence that it is not necessary for acid secretion; and 6) relative contributions of Cl−/HCO3− exchanger (AE2) and NKCC to Cl− influx would differ greatly between resting and stimulated states, possibly explaining reported differences in physiological characteristics of stimulated open-circuit Cl− secretion (≈H+) and resting short-circuit Cl− secretion (>>H+).

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Modulations in extracellular calcium lead to H+-ATPase-dependent acid secretion: a clarification of PPI failure

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00132.2017 ◽

2018 ◽

Vol 315 (1) ◽

pp. G36-G42 ◽

Cited By ~ 2

Author(s):

Alice Miriam Kitay ◽

Marie-Therese Schneebacher ◽

Anne Schmitt ◽

Katharina Heschl ◽

Sascha Kopic ◽

...

Keyword(s):

Atpase Activity ◽

Acid Secretion ◽

Parietal Cell ◽

Proton Pump ◽

Extracellular Calcium ◽

Calcium Sensing Receptor ◽

Gastric Parietal Cell ◽

Blood Calcium ◽

Calcium Sensing ◽

Sodium And Potassium

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 Ca2+. 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/minlowCa2+(0.1mM) = 0.001 ± 0.001, ΔpH/minnormalCa2+(1.0mM) = 0.033 ± 0.004, ΔpH/minhighCa2+(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.

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