Sweeney G, Niu W, Canfield V A, Levenson R, Klip A
Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8, Canada.
Am J Physiol Cell Physiol. 2001 Dec;281(6):C1797-803. doi: 10.1152/ajpcell.2001.281.6.C1797.
Insulin stimulates K(+) uptake and Na(+) efflux via the Na(+)-K(+) pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic alpha-subunit of the Na(+)-K(+) pump. To analyze specifically the effect of insulin on the alpha(1)-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na(+)-K(+) pump alpha(1)-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of alpha(1)-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of alpha(1)-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface alpha-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of alpha(1) (at serine-18). These results suggest that insulin regulates the Na(+)-K(+) pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of alpha(1)-subunits via a signaling pathway involving PI 3-kinase and PKC.
胰岛素通过肾脏、骨骼肌和大脑中的钠钾泵刺激钾离子摄取和钠离子外流。胰岛素在这些组织中的作用机制有所不同，部分原因是钠钾泵催化α亚基的同工型组成存在差异。为了具体分析胰岛素对该泵α1同工型的影响，我们研究了稳定转染了大鼠钠钾泵α1同工型的人胚肾（HEK）-293细胞，该同工型在其第一个细胞外环上带有血凝素（HA）表位标签。通过将特异性HA抗体与完整细胞结合来定量α1亚基的质膜含量。胰岛素迅速增加了细胞表面α1亚基的数量。这种增加对磷脂酰肌醇（PI）3激酶抑制剂渥曼青霉素和蛋白激酶C（PKC）抑制剂双吲哚马来酰亚胺敏感。此外，胰岛素刺激的表面α亚基增加与仅识别α1非磷酸化形式（丝氨酸18处）的抗体结合增加相关。这些结果表明，胰岛素至少部分地通过涉及PI 3激酶和PKC的信号通路降低丝氨酸磷酸化并增加α1亚基的质膜含量来调节HEK-293细胞中的钠钾泵。
