Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na(+),K(+)-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na(+),K(+)-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na(+),K(+)-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na(+),K(+)-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D(1) receptor subtype and the sequential activation of phospholipase A(2), arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na(+),K(+)-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

多巴胺（Dopamine, DA）对近端小管细胞中钠钾ATP酶（Na(+),K(+)-ATPase）的抑制作用，与其α、β亚基通过网格蛋白囊泡依赖性通路内吞进入早期及晚期内体的过程增强密切相关。本研究评估了可触发该机制的胞内信号通路，重点探讨了磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI 3-K）的作用——该酶的激活可启动囊泡运输及蛋白质向特定细胞区室的靶向转运。多巴胺可呈时间及剂量依赖性方式激活PI 3-K的活性，而这一效应可被沃曼宁（wortmannin）与LY 294002显著阻断。多巴胺诱导的钠钾ATP酶α亚基内吞作用，同样可被沃曼宁及LY 294002以剂量依赖性方式抑制。通常而言，PI 3-K的激活依赖于与酪氨酸激酶受体的结合，但本研究中，通过磷酸酪氨酸抗体进行的免疫沉淀并未检测到PI 3-K的活性。多巴胺诱导的钠钾ATP酶α亚基内吞作用需要蛋白激酶C（protein kinase C）的参与，且多巴胺激活PI 3-K的能力可被特异性蛋白激酶C抑制剂阻断。PI 3-K的激活通过D₁受体亚型介导，并依次激活磷脂酶A₂、花生四烯酸及蛋白激酶C。研究结果表明，PI 3-K的激活在多巴胺诱导的钠钾ATP酶α亚基内吞的胞吞序列中发挥关键作用，并为蛋白激酶C参与该过程提供了潜在机制。