Forskolin–induced down–regulation of Na+,K+–ATPase activity is not associated with internalization of the enzyme

Activation by protein kinase A by forskolin phosphorylates and inactivates Na+,K(+)-ATPase in COS-7 cells (Cheng et al. 1997b). In this study we show, using [3H]ouabain binding, that forskolin-induced inhibition of Na+,K(+)-ATPase activity is not because of internalization of the enzyme. The effect of forskolin on Na+,K(+)-ATPase activity was examined by two independent methods, ouabain-sensitive 86Rb+ uptake in intact cells and ATP hydrolysis in microsomal preparations from cells. The change in number of functional pumps on cell surface before and after protein kinase A activation was assessed by [3H]ouabain binding measured under equilibrium conditions. Cells, which had been ATP-depleted by antimycin A and 2-deoxyglucose treatment, served as a positive control for the internalization of Na+,K(+)-ATPase. Activation of protein kinase A with forskolin in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine, inhibited Na+,K(+)-ATPase activity, but this treatment had no effect on specific ouabain binding. No change in ouabain binding was found following activation of protein kinase C by phorbol ester or diacyl glycerol analogue treatment in cells. These data suggest that protein kinase A phosphorylation and inhibition of Na+,K(+)-ATPase activity does not lead to any internalization of the enzyme in COS-7 cells.