Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin.
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.
Department of Physiology, Division of Biological Sciences, Federal University of Paraná (UFPR), Paraná, Brazil.
Priscilla M C
PM
Dos Santos
Fabio P
FP
Freitas
Jeane
J
Mendes
Ana Lucia
AL
Tararthuch
Ricardo
R
Fernandez
D000450:Aldosterone / Q000494:pharmacology* / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology* / Q000502:physiology
D014667:Vasopressins; D000450:Aldosterone; D011189:Potassium Chloride; D006180:Proton-Translocating ATPases; D002118:Calcium