Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C

doi:10.1053/gast.1996.v110.pm8566594

Gastroenterology

Volume 110, Issue 2, February 1996, Pages 469-477

https://doi.org/10.1053/gast.1996.v110.pm8566594 Get rights and content

Abstract

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits secretagogue- stimulated gastric acid secretion via an EGF receptor located on parietal cells. The aim of this study was to examine whether this growth factor inhibited carbachol-stimulated acid secretion through a protein kinase C-dependent mechanism. METHODS: The effect of EGF on carbachol-stimulated aminopyrine uptake, inositol trisphosphate formation, and intracellular Ca2+ ([Ca2+]i) in purified cultured parietal cells was studied. The ability of protein kinase A and C inhibitors to alter the inhibitory action of EGF was assessed. EGF- mediated translocation and activation of protein kinase C in parietal cells were determined. RESULTS: EGF dose dependently inhibited carbachol-stimulated aminopyrine uptake in a pertussis toxin- insensitive, genistein (tyrosine kinase inhibitor)--sensitive manner, with a maximal inhibitory effect (37.5% +/- 6.8%) achieved at 10(-7) mol/L. EGF did not significantly inhibit carbachol-stimulated inositol trisphosphate formation and did not alter the initial transient increase or sustained plateau in [Ca2+]i stimulated by this secretagogue. The protein kinase C inhibitors H-7 and staurosporine dose dependently reversed the inhibitory action of EGF, whereas H-89 (protein kinase A inhibitor) failed to alter the effect of EGF. EGF pretreatment increased the translocation of alpha and beta 1 isoforms of protein kinase C and stimulated kinase activity in parietal cells. EGF did not down-regulate the parietal cell muscarinic receptor. CONCLUSIONS: The inhibitory action of EGF on carbachol-stimulated parietal cell activity seems to involve protein kinase C. (Gastroenterology 1996 Feb;110(2):469-77)

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Citation Excerpt :
Interestingly, a recent study showed that Y477 of ezrin becomes phosphorylated in a Src-dependent manner [22]. Given the fact that EGF receptor tyrosine kinase phosphorylates ezrin and inhibits acid secretion in parietal cells [23], it would be of great interest to examine whether EGF-mediated inhibition of acid secretion is through a disruption of ezrin–WWOX interaction. In any event, further characterization of PALS1-ezrin-WWOX in the apical membrane cytoskeletal complex will provide detailed information regarding the molecular composition and structure-function relationships of the ezrin-containing apical signaling complex in cAMP-dependent polarized secretion in gastric parietal cells.
The ezrin–radixin–moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies have mapped the PKA-mediated phosphorylation site to Ser⁶⁶ and established its functional role in parietal cell activation [R. Zhou et al., Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651–35659], but the underlying basis for this regulation is not known. Here, we provide the first evidence that PKA-mediated phosphorylation of Ser⁶⁶regulates the interaction of ezrin with WWOX, a WW domain-containing protein. Our biochemical study reveals that ezrin directly binds to the first WW domain of WWOX via its C-terminal tyrosine-containing polyproline sequence ⁴⁷⁰PPPPPPVY⁴⁷⁷. Mutational analyses further demonstrate that tyrosine⁴⁷⁷ is essential for the ezrin–WWOX interaction. In addition, our study shows that PKA-mediated phosphorylation of ezrin is essential and sufficient for the apical localization of WWOX protein as disruption of ezrin–WWOX interaction eliminated the apical localization of WWOX. Finally, our study demonstrates the essential role of ezrin–WWOX interaction in the apical membrane remodeling associated with H,K-ATPase recruitment. Taken together, these results define a novel molecular mechanism underlying phospho-regulation of ezrin function by PKA in parietal cell activation.
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The examination of the physiological role of CaMKII has made substantial progress in non-epithelial systems but little is known about its function in secretory epithelial cells. A prototypic exocrine cell, the acid secreting gastric parietal cell, largely redistributes its cytoplasmic tubulocisternal membranes (TC) to the secretory apical membrane (SA) after stimulation. We here present a dependence of physiological response on partitioned initial CaMKII activities redistributed between TC and SA. Initial acid secretion after cholinergic stimulation has doubled if activities of phosphatases PP1/PP2A and protein kinase PKC-α were inhibited by the inhibitors calyculin A and Gö 6976. CaMKII appears to be integrated in multienzyme complexes each specific for TC and SA. Therein, PP1/PP2A and PKC-α were found to permanently counteract initial CaMKII activities in different modes of transregulation. Remarkably, a dys-transregulated, hyperactive CaMKII at TC did not result in an increased acid secretion to the same extent. A simple mathematical model to estimate the share of TC- and SA-associated CaMKII activities in cholinergically induced acid secretion suggests that TC-associated CaMKII appears to autoregulate its contribution to the physiological response by a negative feedback control. Subcellular distribution and stimulus-dependent redistribution of domain-associated CaMKII signalosomes indicate a fine balanced, adaptive system to guarantee a stable physiological response.
Functional Role of Protein Kinase B/Akt in Gastric Acid Secretion
2001, Journal of Biological Chemistry
Citation Excerpt :
EGF, in particular, is known to interact with specific EGF receptors present on the parietal cells to modulate gastric acid production (2,7). Several studies conducted in isolated and cultured parietal cells have indicated that EGF has a rapid inhibitory effect on gastric acid secretion that appears to be mediated by activation of PKC (3). In contrast, prolonged incubation of the cells (>16 h) with EGF leads to enhancement of the stimulatory action of carbachol, gastrin, and histamine on gastric acid production (2, 4, 5, 6, 9).
Epidermal growth factor (EGF) stimulates gastric acid secretion and H⁺/K⁺-ATPase α-subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H⁺/K⁺-ATPase gene expression by Northern blotting using a canine H⁺/K⁺-ATPase α-subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV-β-gal, which expresses β-galactosidase. Ad.Myr-Akt induced H⁺/K⁺-ATPase α-subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H⁺/K⁺-ATPase production, measured by Western blots with an anti-H⁺/K⁺-ATPase α-subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H⁺/K⁺-ATPase expression.
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