Cl- and regulation of pH by MDCK-C11 cells

Abstract

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.

Regulation of intracellular pH by Ca2+- activated proton channel

“…Using the pH-sensitive fluorescent probe 2′7′bis(carboxyethyl) -5(6)-carboxyfluorescein (BCECF) pHi was measured [18]. Macrophages were loaded with the probe by incubation with the acetoxymethyl ester form of BCECF (5 μM in RPMI 1640) for 30 min at 37°C.…”

“…The fluorescence intensity ratio (Fpeak/Fiso) was calibrated to pHi using the standard K + -nigericin technique [18].…”