ALDOSTERONE INDUCES EARLY ACTIVATION AND LATE ACCUMULATION OF NA-K-ATPASE AT SURFACE OF A6 CELLS

In A6 cells cultured on filters, aldosterone (10(-6) M) induces an early increase in the initial rate of ouabain binding to intact monolayers (in K-free buffer) that parallels an early increase in Na transport. This effect is independent of apical Na influx and precedes an increase in the synthesis of Na-K-adenosinetriphosphatase (ATPase) subunits. In the present study we observed in addition a late aldosterone-induced increase in the rate of ouabain binding (2.5 times from 3 to 120 h aldosterone). The role of Na-K-ATPase accumulation and/or translocation to the cell surface was tested by Western blotting, saturation ouabain binding, and cell-surface labeling using sulfosuccinimidobiotin or enzyme-mediated radioiodination. Only cell-surface beta(1)-subunit was detected by blotting with streptavidin or autoradiography, because the alpha(1)-subunit was not efficiently labeled. Three hours after hormone addition, none of the three parameters had increased significantly, whereas after 20 and 120 h similar increases (similar to 1.6 and 2.3 times, respectively) were detected by all three methods. In addition, it was shown that increasing intracellular Na with amphotericin B or a K-free preincubation also stimulated the rate of ouabain binding without increasing the surface labeling of P l-subunits. Taken together, these results suggest that a short aldosterone treatment or an increase in intracellular Na leads to an increase in the rate of ouabain binding that is due to an in situ activation of cell-surface Na-K-ATPase molecules. In contrast, the late increase in the rate of ouabain binding parallels an increase in the number of pumps.