Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes.
Ca(2+)-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 alpha-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 alpha-subunit.
Lynch
C J
CJ
Department of Cellular and Molecular Physiology and Pharmacology, College of Medicine, Pennsylvania State University, Hershey 17033.
Mader
A C
AC
McCall
K M
KM
Ng
Y C
YC
Hazen
S A
SA
D000818:Animals; D004988:Ethers, Cyclic / Q000494:pharmacology*; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D019319:Okadaic Acid; D010042:Ouabain / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000493:pharmacokinetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D010042:Ouabain; D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium