Document Detail

Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin.
MedLine Citation:
PMID:  19794516     Owner:  NLM     Status:  In-Process    
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.
Priscilla M C Dos Santos; Fabio P Freitas; Jeane Mendes; Ana Lucia Tararthuch; Ricardo Fernandez
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Canadian journal of physiology and pharmacology     Volume:  87     ISSN:  1205-7541     ISO Abbreviation:  Can. J. Physiol. Pharmacol.     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-10-01     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0372712     Medline TA:  Can J Physiol Pharmacol     Country:  Canada    
Other Details:
Languages:  eng     Pagination:  653-65     Citation Subset:  IM    
Department of Physiology, Division of Biological Sciences, Federal University of Paraná (UFPR), Paraná, Brazil.
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