OKADAIC ACID STIMULATES OUABAIN-SENSITIVE RB-86(-UPTAKE AND PHOSPHORYLATION OF THE NA+())K+-ATPASE ALPHA-SUBUNIT IN RAT HEPATOCYTES/

Ca2+-mobilizing and cAMP-dependent hormones rapidly increase sodium, p otassium-dependent adenosine triphosphatase (Na+/K+-ATPase)-mediated t ransport in rat hepatocytes. To explore the possible role of protein p hosphatases in these responses we used a protein phosphatase inhibitor , okadaic acid. Okadaic acid stimulation of ouabain-sensitive Rb-86(+) -uptake was maximal between two and three minutes and displayed an EC( 50) of 41 +/- 1 nM. Inhibition of Na+/H+ exchange with an amiloride an alog abolished the response to insulin, but had no effect on okadaic a cid-mediated stimulation of Na+/K+-ATPase transport. In hepatocytes me tabolically-radiolabeled with P-32(i), okadaic acid stimulated the inc orporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K+-ATPa se alpha-subunits. In other experiments Na+/K+-ATPase was immunoprecip itated from detergent-solubilized membrane fractions of metabolically- radiolabeled cells with an antisera to purified rat kidney Na+/K+-ATPa se. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K+-A TPase antisera, but not by preimmune serum. Okadaic acid stimulated in corporation of radioactivity into this band by 220 +/- 28%. These find ings provide support for the hypothesis that rapid stimulation of hepa tic Na+/K+-ATPase by hormones may be related to protein kinase/phospha tase-mediated changes in the phosphorylation state of the Na+/K+-ATPas e alpha-subunit.