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

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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.

Keywords: COS-7; K+-ATPase; Na+; PKA; cAMP; forskolin; internalization; ouabain binding; phosphorylation

Document Type: Original Article


Affiliations: Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden

Publication date: September 1, 1998

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