Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin

Abstract

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 (Pi) 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·L–1, with an apparent Kmfor the release of Piof 0.13 mmol·L–1and Vmaxof 22.01 nmol·mg–1·min–1. Incubation of cell monolayers with 10−8 mol·L–1aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10−5 mol·L–1spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10−11 mol·L–1vasopressin, 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.