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oai:null:10339/374232026-09-02T08:47:34Zcom_10339_14934col_10339_38132

Fisher, Kimberly Day 2012-09-05T08:35:12Z 2013-09-05T08:30:10Z 2012 https://wakespace.lib.wfu.edu/handle/10339/37423 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 well as H+,K+-ATPase-mediated pHi recovery in mOMCD1 cells. In addition, inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H+-ATPase-mediated pHi recovery but not phosphorylation of p38. We conclude that Pyk2 is required for ERK1/2 and p38 signaling pathways that stimulate H+-ATPase and H+,K+-ATPase activity, respectively, in response to acute acidosis in mOMCD1 cells. en Wake Forest University ATPase BCECF intracellular pH MAPK Pyk2 signaling Regulation of acid-stimulated H+ secretion by Pyk2 and MAPK signaling pathways in the outer medullary collecting duct Dissertation DuBose, Thomas D Loeser, Richard Penn, Raymond Petrovic, Snezana Molecular Medicine and Translational Science 2013-09-05 restricted