The interaction between H + extrusion via H + -ATPase and Cl -conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH 4 Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na + Ringer and 0.032 ± 0.003 in the absence of Na + (0 Na + ). With 0 Na + plus the Cl -channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl -channels, increased dpHi/dt in 0 Na + to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl -dependence of H + -ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K + was tested in our cells. In Na + Ringer, dpHi/dt was increased, but no effect was detected in 0 Na + Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl -channels on dpHi/ dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na + Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H + -ATPase present in MDCK C11 cells depends on Clions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H + transport.

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.

It is well known that the aminoglycoside antibiotic gentamicin is capable of causing damage to kidney cells. Given the known involvement of Ca2+ in the nephrotoxic action of gentamicin, the purpose of this study was to establish a relationship between the concentration of intracellular Ca2+ ([Ca2+]i) and cellular cytotoxicity using MDCK-C11 cells, a clone that has several properties that resemble those of intercalated cells of the distal nephron. Changes in [Ca2+]i was determined using fluorescence microscopy. Cell viability was evaluated by the neutral red method, and cell cytotoxicity by the MTT method. The [Ca2+]i gradually increased when cells were exposed to 0.1 mM gentamicin for 10, 20, and 30 min. The presence of extracellular Ca2+ was found to be necessary to stimulate the increase in [Ca2+]i induced by gentamicin, since this stimulus disappeared by using 1.8 mM EGTA (a Ca2+ chelator). Morphological changes were observed with scanning electron microscopy in epithelial cells exposed to the antibiotic. Furthermore, with the MTT method, a decrease in metabolic activity induced by gentamicin was observed, which indicates a cytotoxic effect. In conclusion, gentamicin was able to alter [Ca2+]i, change the morphology of MDCK-C11 cells, and promote cytotoxicity.

The interaction between H + extrusion via H + -ATPase and Cl -conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH 4 Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na + Ringer and 0.032 ± 0.003 in the absence of Na + (0 Na + ). With 0 Na + plus the Cl -channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl -channels, increased dpHi/dt in 0 Na + to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl -dependence of H + -ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K + was tested in our cells. In Na + Ringer, dpHi/dt was increased, but no effect was detected in 0 Na + Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl -channels on dpHi/ dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na + Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H + -ATPase present in MDCK C11 cells depends on Clions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H + transport.

CaSR stimulation induces an increase in the vacuolar H-ATPase activity of OKP cells, an effect that involves an increase in [Ca] and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.