The influence of thapsigargin on Na,K-ATPase activity in cultured nonpigmented ciliary epithelial cells.
Experiments were conducted to test the influence of thapsigargin on the NaK-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 microM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.
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Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine 40292, USA.
Kuwahara
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S
Delamere
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NA
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability; D002924:Ciliary Body / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D006868:Hydrolysis; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D013449:Sulfonamides; D013729:Terpenes; D014268:Trifluoperazine; D010042:Ouabain; C017967:W 7; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium; D016257:Fura-2