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 degrees C for 4 h then rewarmed to 37 degrees 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.
Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, KY 40292, USA.
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Kuwahara
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Chin
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Delamere
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002461:Cell Line, Transformed; D002924:Ciliary Body / Q000187:drug effects* / Q000201:enzymology; D003593:Cytoplasm / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D010042:Ouabain / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D012402:Rotenone / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D014475:Uncoupling Agents / Q000494:pharmacology
D014475:Uncoupling Agents; D012402:Rotenone; D015240:Phorbol 12,13-Dibutyrate; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D010042:Ouabain; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium