Partial inhibition of Na,K-ATPase activity in cultured rabbit non-pigmented ciliary epithelium following an episode of cytoplasmic ATP depletion

Ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured in digitonin-permeabilized monolayers of cultured cells derived from rabbit non-pigmented ciliary epithelium. Diminished Na,K-ATPase activity was observed in cells that had been pre-treated 10 min with the protein kinase C activator, PDBu, as well as in cells that had been cooled to 4°C for 4 h then rewarmed to 37°C for 30 min (cool-rewarm manoeuvre). In the intact cells, ouabain binding was not decreased either by PDBu treatment or the cool-rewarm manoeuvre. However, both PDBu and the cool-rewarm manoeuvre increased the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake measured in intact cells. Cell ATP content was diminished in PDBu-treated cells and cells subjected to the cool-rewarm manoeuvre. We suggest that an episode of ATP depletion might initiate a mechanism which causes lasting, partial inhibition of Na,K-ATPase activity. In keeping with this suggestion, diminished Na,K-ATPase activity was observed in cells that had been pre-treated 20 min with the metabolic inhibitors CCCP or rotenone and in cells pre-treated 2.5 h in dextrose-free medium. This study illustrates that Na,K-ATPase activity measured in the permeabilized cell is a complex parameter which is not necessarily a reliable indicator of sodium pump responses in the intact cell.