| Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin. | |
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MedLine Citation:
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PMID: 19794516 Owner: NLM Status: In-Process |
Abstract/OtherAbstract:
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The objective of the present work was to characterize the biochemical activity of the proton pumps present in the C11 clone of Madin-Darby canine kidney (MDCK) cells, akin to intercalated cells of the collecting duct, as well as to study their regulation by hormones like aldosterone and vasopressin. MDCK-C11 cells from passages 78 to 86 were utilized. The reaction to determine H+-ATPase activity was started by addition of cell homogenates to tubes contained the assay medium. The inorganic phosphate (P(i)) released was determined by a colorimetric method modified from that described by Fiske and Subbarow. Changes in intracellular calcium concentration in the cells was determined using the Ca2+-sensing dye fluo-4 AM. Homogenates of MDCK-C11 cells present a bafilomycin-sensitive activity (vacuolar H+-ATPase), and a vanadate-sensitive activity (H+/K+-ATPase). The bafilomycin-sensitive activity showed a pH optimum of 6.12. ATPase activity was also stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 50 mmol x L(-1), with an apparent K(m) for the release of P(i) of 0.13 mmol x L(-1) and Vmax of 22.01 nmol x mg(-1) x min(-1). Incubation of cell monolayers with 10(-8) mol x L(-1) aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10(-5) mol x L(-1) spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10(-11) mol x L(-1) vasopressin, an effect prevented by a V1 receptor-specific antagonist. This dose of vasopressin determined a sustained rise of cytosolic ionized calcium. We conclude that (i) homogenates of MDCK-C11 cells present a bafilomycin-sensitive (H+-ATPase) activity and a vanadate-sensitive (H+/K+-ATPase) activity, and (ii) vacuolar H+-ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors. |
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Authors:
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Priscilla M C Dos Santos; Fabio P Freitas; Jeane Mendes; Ana Lucia Tararthuch; Ricardo Fernandez |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Canadian journal of physiology and pharmacology Volume: 87 ISSN: 1205-7541 ISO Abbreviation: Can. J. Physiol. Pharmacol. Publication Date: 2009 Sep |
Date Detail:
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Created Date: 2009-10-01 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0372712 Medline TA: Can J Physiol Pharmacol Country: Canada |
Other Details:
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Languages: eng Pagination: 653-65 Citation Subset: IM |
Affiliation:
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Department of Physiology, Division of Biological Sciences, Federal University of Paraná (UFPR), Paraná, Brazil. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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