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Okadaic acid stimulates ouabain‐sensitive 86 Rb + ‐uptake and phosphorylation of the Na + /K + ‐ATPase α‐subunit in rat hepatocytes
Author(s) -
Lynch Christopher J.,
Mader Annie Cielo L.,
McCall Kenneth M.,
Ng Yuk-Chow,
Hazen Stacy A.
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80085-5
Subject(s) - ouabain , okadaic acid , chemistry , phosphorylation , atpase , protein subunit , phosphatase , radiochemistry , microbiology and biotechnology , enzyme , sodium , biochemistry , biology , organic chemistry , gene
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 86 Rb + ‐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 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 32 P i , 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.