{"id":{"repo_id":"wfu","oai_identifier":"oai:wakespace.lib.wfu.edu:10339/37423"},"canonical_url":"https://search.dev.ndltd.org/etd/wfu/oai:wakespace.lib.wfu.edu:10339/37423","repository":{"repo_id":"wfu","name":"Wake Forest University","base_url":"https://wakespace.lib.wfu.edu/oai/request"},"display":{"title":"Regulation of acid-stimulated H+ secretion by Pyk2 and MAPK signaling pathways in the outer medullary collecting duct","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 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.","abstract_html":"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.","abstract_has_math":false,"creators":["Fisher, Kimberly Day"],"institution":"Wake Forest University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012","date_published":"2012","updated_at":"2026-07-27T22:01:27Z","subjects":["ATPase"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10339/37423","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Fisher, Kimberly Day"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2012-09-05T08:35:12Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2013-09-05T08:30:10Z"]},{"key":"dc:date.issued","label":"Date","values":["2012"]},{"key":"dc:publisher","label":"Institution","values":["Wake Forest University"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["ATPase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10339/37423"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["Regulation of acid-stimulated H+ secretion by Pyk2 and MAPK signaling pathways in the outer medullary collecting duct"]}]}],"canonical_facts":{"dc:creator":["Fisher, Kimberly Day"],"dc:date.accessioned":["2012-09-05T08:35:12Z"],"dc:date.available":["2013-09-05T08:30:10Z"],"dc:date.issued":["2012"],"dc:description.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 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."],"dc:identifier.uri":["http://hdl.handle.net/10339/37423"],"dc:language.iso":["en"],"dc:publisher":["Wake Forest University"],"dc:subject":["ATPase"],"dc:title":["Regulation of acid-stimulated H+ secretion by Pyk2 and MAPK signaling pathways in the outer medullary collecting duct"],"dc:type":["Dissertation"]},"updated_at":"2026-07-27T22:01:27Z"}