Khundmiri Syed J, Amin Vishal, Henson Jeff, Lewis John, Ameen Mohamed, Rane Madhavi J, Delamere Nicholas A
Kidney Disease Program, Univ. of Louisville, 570 S Preston St., South POD 102, Louisville, KY 40202, USA.
Am J Physiol Cell Physiol. 2007 Sep;293(3):C1171-80. doi: 10.1152/ajpcell.00535.2006. Epub 2007 Jul 18.
Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na(+)/K(+)-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na(+)/K(+)-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na(+)/K(+)-ATPase-mediated (86)Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser(473). Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser(473), cell proliferation, and stimulation of Na(+)/K(+)-ATPase-mediated (86)Rb uptake in OK cells.
内源性强心苷与质膜钠泵（Na⁺/K⁺-ATP酶）的抑制性结合位点结合。在几种疾病状态下，如原发性高血压和尿毒症，内源性强心苷的血浆水平会升高。低浓度的哇巴因不抑制Na⁺/K⁺-ATP酶，但可诱导细胞增殖。哇巴因介导的反应机制尚不清楚。最近，我们证明在负鼠肾（OK）近端肾小管细胞中，低浓度的哇巴因通过蛋白激酶B（Akt）的磷酸化以钙依赖的方式诱导细胞增殖。在本研究中，我们确定细胞外信号调节激酶（ERK）是哇巴因刺激细胞中调节Akt激活的上游激酶。此外，我们提供证据表明，低浓度的哇巴因以Akt、ERK和Src激酶依赖的方式刺激Na⁺/K⁺-ATP酶介导的⁸⁶Rb摄取。细胞内钙释放、钙内流、酪氨酸激酶和磷脂酶C的阻断可抑制哇巴因介导的ERK磷酸化。磷酸肌醇-3激酶和Akt的药理学抑制未能抑制哇巴因刺激的ERK磷酸化。MEK/ERK抑制剂U0126可抑制哇巴因介导的Akt磷酸化，表明哇巴因介导的Akt磷酸化依赖于ERK。在体外激酶试验中，活性重组ERK使重组Akt在Ser⁴⁷³位点磷酸化。此外，用ERK的上游调节因子组成型活性MEK1进行瞬时转染，可增加Akt磷酸化和激活，而组成型活性Akt的过表达未能刺激ERK磷酸化。低浓度的哇巴因也以ERK依赖的方式促进细胞增殖。这些发现表明，哇巴因刺激的ERK磷酸化是OK细胞中Akt在Ser⁴⁷³位点磷酸化、细胞增殖以及刺激Na⁺/K⁺-ATP酶介导的⁸⁶Rb摄取所必需的。
