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.
Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden.
R M
RM
Andersson
S X
SX
Cheng
A
A
Aperia
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001665:Binding Sites; D019556:COS Cells / Q000187:drug effects / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003847:Deoxyglucose / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects*; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010042:Ouabain / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D010726:Phosphodiesterase Inhibitors; D005576:Colforsin; D010042:Ouabain; D000968:Antimycin A; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D015056:1-Methyl-3-isobutylxanthine