The role of H+-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro'' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na+- and Cl−-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl− channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na+ solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min−1, not significantly different from zero (P > 0.42). For superfusion with Na+0 Ringer, this variation was 0.081 ± 0.015 min−1, P < 0.001 against 0. These slopes were markedly reduced by the Cl− channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na+-free medium is probably due to a vacuolar H+-ATPase, whose activity is stimulated in the presence of Cl−, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl− and on the integrity of the cytoskeleton.
The Journal of Membrane Biology – Springer Journals
Published: Nov 15, 2000
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