Cl- and regulation of pH by MDCK-C11 cells.
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 microM), 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 microM 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 microM 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 microM ATP was studied in 0 Na(+) Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 microM Bapta. We have shown that H(+)-ATPase present in MDCK C11 cells depends on Cl(-) ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H(+) transport.
Tararthuch
A L
AL
Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil.
Fernandez
R
R
Malnic
G
G
D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002999:Clone Cells; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D018118:Chloride Channels; D005452:Fluoresceins; D005456:Fluorescent Dyes; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D006180:Proton-Translocating ATPases