Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.
The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus "clamped" at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.
D000818:Animals; D001693:Biological Transport, Active; D002460:Cell Line; D002477:Cells / Q000201:enzymology / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D007477:Ions; D008985:Monensin / Q000494:pharmacology*; D009761:Nystatin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D007477:Ions; D003840:Deoxycholic Acid; D009761:Nystatin; D010042:Ouabain; D008985:Monensin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium