Studies on H⁺-ATPase in cultured rabbit nonpigmented ciliary epithelium

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 10μM 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.