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.
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