Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes.
Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes.
复制标题
冈田酸刺激大鼠肝细胞中哇巴因敏感的 86Rb( ) 摄取和 Na /K( )-ATPase α 亚基的磷酸化。
DOI：
10.1016/0014-5793(94)80085-5
复制
发表时间：
1994
期刊：
影响因子：
3.5
通讯作者：
Hazen,SA
中科院分区：
文献类型：
--
作者：
Lynch,CJ;Mader,AC;McCall,KM;Ng,YC;Hazen,SA
钙动员和cAMP依赖的激素迅速增加钠，钾依赖的三磷酸腺苷酶(Na+/K+-ATPase)介导的大鼠肝细胞转运。为了探索蛋白磷酸酶在这些反应中的可能作用，我们使用了蛋白磷酸酶抑制剂冈田酸。冈田酸对哇巴因敏感的~(86)Rb~+摄取的刺激在2~3分钟达到最大，EC50为41±1 nM。用阿米洛利类似物抑制Na+/H+交换可取消对胰岛素的反应，但不影响冈田酸对Na+/K+-ATPase转运的刺激。在~(32)PI标记的肝细胞中，冈田酸刺激放射性进入几个95 kDa的多肽，其中一个与识别Na~+/K~+-ATP酶α亚基的抗叶多肽抗血清反应。在其他实验中，用纯化的大鼠肾脏Na+/K+-ATPase抗血清从代谢标记细胞经洗涤剂溶解的膜部分免疫沉淀Na+/K+-ATPase。用抗Na+/K+-ATPase抗血清免疫沉淀95 kDa的磷蛋白，而不用前免疫血清。冈田酸使该带的放射性参入率增加了220±28%。这些结果支持激素对肝脏Na+/K+-ATPase的快速刺激可能与蛋白激酶/磷酸酶介导的Na+/K+-ATPaseα亚基磷酸化状态的改变有关的假说。
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-sensitive86Rb+-uptake was maximal between two and three minutes and displayed an EC50of 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 with32Pi, 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.