Studies on Na,K-ATPase in culture non-pigmented ciliary epithelial cells

Purpose. The sodium pump (Na,K-ATPase) utilizes a large fraction of the ATP generated within the cell. In studies with cultured non-pigmented epithelial cells, we detect a significant decrease of cell ATP content following a period stimulated active sodium transport. In the present study we tested whether ATP depletion might contribute to the apparent down regulation of Na,K-ATPase activity observed in cells following an episode of sodium pump stimulation. Methods. Experiments were conducted with a cell line derived from rabbit non-pigmented ciliary epithelium. The cells were kindly provided by Dr. Coca-Prados (Yale Univ.). Intracellular ATP content was measured using a luciferase assay. Na,K-ATPase activity was measured in digitonin-permeabilized cell monolayers as the difference between ATP hydrolysis in the presence and absence of ouabain. Results. Na,K-ATPase activity was significantly inhibited in cells that had previously been subjected to a brief (10-30 min) period of stimulated active Na-K transport caused by a cold-rewarm maneuver or by exposure to nigericin (5μM). Cell ATP content was also diminished in cells subjected to the cold-rewarm maneuver or nigericin. In some experiments, cells were exposed to glucose-free medium or metabolic inhibitors (rotenone or CCCP) in order to deplete cellular ATP; each of these pretreatment regimens caused significant inhibition of NaJi-ATPase activity measured subsequently under control conditions. Conclusion. Na,K-ATPase activity appears to be inhibited in cells that have been subjected to an episode of ATP depletion. Such a decrease of cytoplasmic ATP can be caused by a period of rapid Na-K transport. We suggest that ATP depletion may contribute the complex Na,K-ATPase response reported earlier in thapsigargin-treated cells (Curr. Eye Res 14, 651-57, 1995).