Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells

Hdl Handle:
http://hdl.handle.net/10675.2/344401
Title:
Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells
Authors:
Cai, Zheqing
Abstract:
Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
Affiliation:
Department of Internal Medicine
Issue Date:
Jun-2001
URI:
http://hdl.handle.net/10675.2/344401
Additional Links:
http://ezproxy.gru.edu/login?url=https://search.proquest.com/docview/251696925?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorCai, Zheqingen
dc.date.accessioned2015-02-11T22:42:19Z-
dc.date.available2015-02-11T22:42:19Z-
dc.date.issued2001-06-
dc.identifier.urihttp://hdl.handle.net/10675.2/344401-
dc.description.abstractNitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=https://search.proquest.com/docview/251696925?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectNitric Oxideen
dc.subjectEndothelin-1en
dc.subjectMAP Kinasesen
dc.subjectCaveolin-1en
dc.subjectTyrosine Phophorylationen
dc.subjectShear Stressen
dc.subjectInner Medullaen
dc.subjectKidneyen
dc.titleNitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cellsen
dc.typeDissertationen
dc.contributor.departmentDepartment of Internal Medicineen
dc.description.advisorPollock, Jennifer S.en
dc.description.committeeFuchs, Leslie C.; Mivechi, Nahid F.; Barman, Scott A.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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