Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway
| Autor: | Thomas D. DuBose, Kimberly D. Fisher, Juan Codina, Snezana Petrovic |
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| Rok vydání: | 2012 |
| Předmět: |
Physiology
MAP Kinase Signaling System Intracellular pH ATPase Mice Transgenic Biology Ammonium Chloride Cell Line Mice Nitriles Butadienes Animals Secretion Intercalated Cell Enzyme Inhibitors Kidney Tubules Collecting Phosphorylation RNA Small Interfering Mitogen-Activated Protein Kinase Kinases Kidney Medulla Kinase Transporter Articles Hydrogen-Ion Concentration Cell biology Proton-Translocating ATPases Focal Adhesion Kinase 2 Pyrimidines Cell culture Models Animal biology.protein Pyrazoles Protons |
| Zdroj: | American journal of physiology. Renal physiology. 303(9) |
| ISSN: | 1522-1466 |
| Popis: | Acid-secreting intercalated cells 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 nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins 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 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4- d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2′-7′-bis(carboxyethyl)-5( 6 )-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H+transporter activity. The activity of H+transporters was measured as the rate of intracellular pH (pHi) recovery after an NH4Cl prepulse. 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 [extracellular pH (pHo) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pHiinduced by an NH4Cl prepulse also 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 phosphorylation as well as H+-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H+-ATPase-mediated pHirecovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H+-ATPase, but not H+,K+-ATPase, activity at decreased pHiin mOMCD1 cells. |
| Databáze: | OpenAIRE |
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