l-Type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca2+) or trivalent (Gd3+) ions stimulates acid production in the absence of secretagogues by increasing H+,K+-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by l-amino acids in the presence of physiological concentrations of extracellular Ca2+ (Cao2+; 1.5–2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by l-amino acids, we examined the effect of the amino acids l-phenylalanine (l-Phe), l-tryptophan, and l-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to l-Phe resulted in gastric luminal pH significantly lower than controls. Studies using d-Phe (inactive isomer) failed to elicit a response on gastric pH. H+-K+-ATPase activity was monitored by measuring the intracellular pH (pHi) of individual parietal cells in isolated rat gastric glands and calculating the rate of H+ extrusion. We demonstrated that increasing Cao2+ in the absence of secretagogues caused a dose-dependent increase in H+ extrusion. These effects were amplified by the addition of amino acids at various Cao2+ concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system l-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H+ extrusion in the presence of l-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Cao2+ concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00571.2004 ◽

2005 ◽

Vol 289 (6) ◽

pp. G1084-G1090 ◽

Cited By ~ 56

Author(s):

Matthias M. Dufner ◽

Philipp Kirchhoff ◽

Christine Remy ◽

Patricia Hafner ◽

Markus K. Müller ◽

...

Keyword(s):

Atpase Activity ◽

Acid Secretion ◽

Gastric Acid Secretion ◽

Gastric Acid ◽

Divalent Cations ◽

Parietal Cells ◽

Trivalent Cation ◽

Calcium Sensing Receptor ◽

Gastric Glands ◽

Calcium Sensing

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 μM) and inhibited by omeprazole (100 μM). Reduction of the extracellular divalent cation concentration (0 Mg2+, 100 μM Ca2+) inactivated the CaSR and reduced histamine-induced activation of H+-K+-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd3+ 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 H2 receptor antagonist cimetidine (100 μM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd3+ led to a sustained increase in intracellular Ca2+ 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.

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Stimulatory Pathways of the Calcium-Sensing Receptor on Acid Secretion in Freshly Isolated Human Gastric Glands

Cellular Physiology and Biochemistry ◽

10.1159/000099190 ◽

2007 ◽

Vol 19 (1-4) ◽

pp. 33-42 ◽

Cited By ~ 28

Author(s):

Christine Remy ◽

Philipp Kirchhoff ◽

Patricia Hafner ◽

Stephanie Busque ◽

Markus Mueller ◽

...

Keyword(s):

Acid Secretion ◽

Calcium Sensing Receptor ◽

Gastric Glands ◽

Calcium Sensing

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00283.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. G145-G153 ◽

Cited By ~ 24

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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Functionally distinct pools of actin in secretory cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.2.c407 ◽

2001 ◽

Vol 281 (2) ◽

pp. C407-C417 ◽

Cited By ~ 26

Author(s):

David A. Ammar ◽

Phuong N. B. Nguyen ◽

John G. Forte

Keyword(s):

Parietal Cell ◽

Cultured Cells ◽

Parietal Cells ◽

Secretory Cells ◽

Resting Cells ◽

Gastric Glands ◽

Gastric Parietal Cell ◽

Actin Organization ◽

Isolated Gastric Glands ◽

High Concentrations

Acid secretion by the gastric parietal cell is controlled through movement of vesicles containing the proton pump, the H+-K+-ATPase (HK). We have used latrunculin B (Lat B), which binds to monomeric actin, to investigate actin turnover in the stimulated parietal cell. In isolated gastric glands, relatively high concentrations of Lat B were required to inhibit acid accumulation (ED50∼70 μM). Cultured parietal cells stimulated in the presence of low Lat B (0.1–1 μM) have reduced lamellipodia formation and some aberrant punctate phalloidin-stained structures, but translocation of HK and vacuolar swelling appeared unaffected. High Lat B (10–50 μM) resulted in gross changes in actin organization (punctate phalloidin-stained structures throughout the cell and nucleus) and reduced translocation of HK and vacuolar swelling. Resting parietal cells treated with high Lat B showed minor effects on morphology and F-actin staining. If resting cells treated with high Lat B were washed immediately before stimulation, they exhibited a normal stimulated morphology. These data suggest distinct pools of parietal cell actin: a pool highly susceptible to Lat B primarily involved in motile function of cultured cells; and a Lat B-resistant pool, most likely microvillar filaments, that is essential for secretion. Furthermore, the stimulation process appears to accentuate the effects of Lat B, most likely through Lat B binding to monomer actin liberated by the turnover of the motile actin filament pool.

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Illuminating the allosteric modulation of the calcium-sensing receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922231117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21711-21722

Author(s):

Hongkang Liu ◽

Ping Yi ◽

Wenjing Zhao ◽

Yuling Wu ◽

Francine Acher ◽

...

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Membrane Receptors ◽

Allosteric Modulators ◽

Active Conformation ◽

Calcium Sensing Receptor ◽

Single Receptor ◽

Calcium Sensing

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

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The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule

Kidney International ◽

10.1038/ki.2013.137 ◽

2013 ◽

Vol 84 (2) ◽

pp. 277-284 ◽

Cited By ~ 36

Author(s):

Giovambattista Capasso ◽

Peter J. Geibel ◽

Sara Damiano ◽

Philippe Jaeger ◽

William G. Richards ◽

...

Keyword(s):

Acid Secretion ◽

Proximal Tubule ◽

Calcium Sensing Receptor ◽

Fluid Reabsorption ◽

Calcium Sensing

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Differential effects of GTP gamma S on acid and pepsinogen secretion by permeable gastric glands

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.6.g962 ◽

1996 ◽

Vol 270 (6) ◽

pp. G962-G968 ◽

Cited By ~ 1

Author(s):

M. D. Miller ◽

S. J. Hersey

Keyword(s):

Acid Secretion ◽

Parietal Cells ◽

Tetraacetic Acid ◽

Alpha Toxin ◽

Gastric Glands ◽

Differential Effects ◽

Pepsinogen Secretion ◽

Stimulus Secretion Coupling ◽

Gamma S ◽

Coupling Mechanisms

Gastric glands isolated from rabbit stomach were permeabilized with Staphylococcus aureus alpha-toxin. Acid secretion by parietal cells, as measured by the accumulation of weak base, was inhibited by incubation with alpha-toxin but could be restored by addition of exogenous ATP (1 mM). The permeable glands were found to retain acid secretory responses to receptor-linked secretagogues, histamine and carbachol, as well as to intracellular mediators, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate, indicating the presence of intact, functional intracellular coupling mechanisms. Both basal and stimulated acid secretion by the permeable glands were blocked by the Mg2+ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA; 5 mM), whereas CDTA had no effect on nonpermeabilized glands. These results are interpreted to show that alpha-toxin permeabilizes parietal cells to moderate sized molecules without causing a loss of critical intracellular components. The acid secretory responses to histamine and carbachol persisted in media containing low ( < 50 nM) levels of free Ca2+ buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (0.5 mM), indicating that changes in bulk Ca2+ are not required for these responses. Inclusion of the nonhydrolyzable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM), resulted in inhibition of spontaneous acid secretion, blocked responses to all agents tested, and inhibited stimulated acid secretion. GTP gamma S had no effect on nonpermeabilized glands. No effects on acid secretion by either permeable or nonpermeable glands were observed with GTP, guanosine diphosphate, or guanosine 5'-O-(2-thiodiphosphate). GTP gamma S had no effect on H+ gradient formation by gastric membrane vesicles, showing that it does not inhibit the gastric H(+)-K(+)-adenosinetriphosphatase directly. These results are interpreted to show that GTP gamma S interacts at a postreceptor site to inhibit or reverse a critical step in stimulus-secretion coupling in parietal cells. In contrast to the effect on parietal cells, GTP gamma S was found to stimulate pepsinogen secretion by alpha-toxin-permeabilized chief cells. The differential effects of GTP gamma S on acid and pepsinogen secretions suggest unique roles for GTP binding proteins in these two secretory processes. The use of alpha-toxin-permeabilized gastric glands should prove useful in defining the stimulus-secretion coupling mechanisms involved in both acid and pepsinogen secretions.

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