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

Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A(1) on cultured rabbit nonpigmented ciliary epithelial cells ( NPE). Cytoplasmic pH and sodium concentrations were measured by digital flu orescence microscopy using BCECF and SBFI respectively. In some experiments , cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A(1) (100 nM) failed to change cytoplasmic pH but it ca used 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 A(1)-treated cells as judged by a 50% increase of o uabain sensitive potassium (Rb-86) uptake. In bafilomycin A(1)-treated cell s, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sens itive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of si milar to 2 mu M. DMA (10 mu M) also prevented the increase of cytoplasmic s odium caused by bafilomycin A(1). Added alone, DMA (10 mu M) failed to chan ge cytoplasmic sodium content but reduced cytoplasmic pH by similar to 0.4 pH units. In cells that first received 10 mu M DMA, the subsequent addition of bafilomycin A(1) (100 nM) caused a further cytoplasmic pH reduction of similar to 0.3 pH units. Taken together, the results suggest H+-ATPase migh t contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A(1), Na-H exchanger activity appears to be sti mulated, so stabilizing cytoplasmic pH but resulting in an increase of cyto plasmic sodium concentration and consequent stimulation of active sodium-po tassium transport.