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.