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.
The aim of the present study was to analyze the effects of CaSR stimulation on the activity of the vacuolar H+‐ATPase in a cellular model of proximal tubule, OKP cells. H+‐ATPase activity was accessed by a biochemical method. Intracellular pH (pHi) was monitored with BCECF and changes in [Ca2+]i was determined using Fluo‐4 AM. A significant increase of H+‐ATPase activity was observed when the CaSR was stimulated with agonists such as 300μM Gd3+ and 200μM neomycin. This activity was also stimulated in a dose‐dependent fashion when Ca2+ concentration was increased between 10‐4mM and 2mM. Neomycin induced a significant alkalinization of pHi (∆pHi 0.57±0.04) that was significantly reduced in the presence of 10‐8M concanamycin. Gd3+ and neomycin produced a sustained rise of cytosolic ionized calcium (4.77±0.47 and 12.79±0.86%, respectively, p<0.0001 vs basal), an effect that disappeared when extracellular calcium was removed in the presence of 0.1μM thapsigargin. Inhibition of phospholipase C activity with U73122 (5x10‐8M) reduced the increase in [Ca2+]i induced by neomycin. These data allow us to conclude that CaSR stimulation of OKP cells induces an increase in vacuolar H+‐ATPase activity. This effect involves an increase in intracellular calcium, resulting from the combination of Ca2+ release from intracellular stores and the influx of Ca2+ from the extracellular medium, an effect that requires PLC activity. Grant Funding Source: Supported by CAPES (Brazil)
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