Regulation of acid-stimulated H+ secretion by Pyk2 and MAPK signaling pathways in the outer medullary collecting duct
type: CreativeWork
description: Acid-secreting intercalated cells of the outer medullary collecting duct respond to changes in systemic pH through regulation of apical H+ transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pHo). Pyk2 is a non-receptor tyrosine kinase activated by auto-phosphorylation at Tyr402 by cell-specific stimuli, including decreased pH and is involved in the regulation of MAPK signaling pathways and transporter activity. Therefore we examined whether the Pyk2 and MAPK up-regulate ATPase mediated proton secretion in response to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 siRNA to knockdown Pyk2 expression levels, the Src kinase inhibitor PP 1 to inhibit Src phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. Furthermore, we used adenoviral-gene transfer of AdCRNK, a dominant-interfering truncated Pyk2 construct, to block Pyk2 phosphorylation at Tyr402 as well as the p38-specific inhibitor, SB203580. The pH-sensitive fluorescent probe BCECF-AM was used to assay H+ transporter activity. The activity of H+ transporters was measured as the rate of intracellular pH recovery after an NH4Cl pre-pulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media (pHo 6.7) and reduction in pHi induced by an NH4Cl pre-pulse increased the phosphorylation of Pyk2, ERK1/2 and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H+-ATPase and H+,K+-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 and ERK1/2 phosphorylation as well as H+-ATPase-mediated recovery. Pyk2 inhibition by AdCRNK prevented Pyk2 and p38 phosphorylation as we…
abstract: Acid-secreting intercalated cells of the outer medullary collecting duct respond to changes in systemic pH through regulation of apical H+ transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pHo). Pyk2 is a non-receptor tyrosine kinase activated by auto-phosphorylation at Tyr402 by cell-specific stimuli, including decreased pH and is involved in the regulation of MAPK signaling pathways and transporter activity. Therefore we examined whether the Pyk2 and MAPK up-regulate ATPase mediated proton secretion in response to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 siRNA to knockdown Pyk2 expression levels, the Src kinase inhibitor PP 1 to inhibit Src phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. Furthermore, we used adenoviral-gene transfer of AdCRNK, a dominant-interfering truncated Pyk2 construct, to block Pyk2 phosphorylation at Tyr402 as well as the p38-specific inhibitor, SB203580. The pH-sensitive fluorescent probe BCECF-AM was used to assay H+ transporter activity. The activity of H+ transporters was measured as the rate of intracellular pH recovery after an NH4Cl pre-pulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media (pHo 6.7) and reduction in pHi induced by an NH4Cl pre-pulse increased the phosphorylation of Pyk2, ERK1/2 and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H+-ATPase and H+,K+-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 and ERK1/2 phosphorylation as well as H+-ATPase-mediated recovery. Pyk2 inhibition by AdCRNK prevented Pyk2 and p38 phosphorylation as we…
author: Fisher, Kimberly Day
language: en
keywords: ATPase
date published: 2012
date modified: 2026-07-27T22:01:27Z
provider: Wake Forest University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Wake Forest University

PropertyValue
property ID: Handle
value: http://hdl.handle.net/10339/37423