Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A1 on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A1 (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A1-treated cells as judged by a 50% increase of ouabain sensitive potassium (86Rb) uptake. In bafilomycin A1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A1. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A1 (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
The Journal of Membrane Biology – Springer Journals
Published: Jan 1, 2000
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