Okadaic acid stimulates ouabain‐sensitive 86Rb+‐uptake and phosphorylation of the Na+/K+‐ATPase α‐subunit in rat hepatocytes

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Abstract
Ca2+‐mobilizing and cAMP‐dependent hormones rapidly increase sodium, potassium‐dependent adenosine triphosphatase (Na+/K+‐ATPase)‐mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain‐sensitive 86Rb+‐uptake was maximal between two and three minutes and displayed an EC50 of 41 ± 1 nM. Inhibition of Na+/H+ exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid‐mediated stimulation of Na+/K+‐ATPase transport. In hepatocytes metabolically‐radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti‐LEAVE peptide antisera, that recognizes Na+/K+‐ATPase α‐subunits. In other experiments Na+/K+‐ATPase was immunoprecipitated from detergent‐solubilized membrane fractions of metabolically‐radiolabeled cells with an antisera to purified rat kidney Na+/K+‐ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti‐Na+/K+‐ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 ± 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K+‐ATPase by hormones may be related to protein kinase/phosphatase‐mediated changes in the phosphorylation state of the Na+/K+‐ATPase α‐subunit.