Khundmiri Syed J, Metzler Melissa A, Ameen Mohamed, Amin Vishal, Rane Madhavi J, Delamere Nicholas A
Department of Medicine, University of Louisville, Louisville, Kentucky 40202, USA.
Am J Physiol Cell Physiol. 2006 Dec;291(6):C1247-57. doi: 10.1152/ajpcell.00593.2005. Epub 2006 Jun 28.
Cardiotonic glycosides, like ouabain, inhibit Na(+)-K(+)-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser(473) phosphorylation, as evidenced by an increase in phospho-Akt Ser(473) band density. Ouabain-stimulated Akt Ser(473) phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser(473) phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser(473) phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in (86)Rb uptake but did not significantly alter Na(+)-K(+)-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na(+)-K(+)-ATPase-mediated ion transport.
强心苷，如哇巴因，可抑制钠钾ATP酶。最近的证据表明，低摩尔浓度的哇巴因会改变细胞生长。本研究旨在探讨哇巴因对负鼠肾（OK）近端小管细胞中Akt磷酸化及细胞增殖速率的影响。暴露于10 nM哇巴因的细胞显示Akt Ser(473)磷酸化增加，磷酸化Akt Ser(473)条带密度增加证明了这一点。用磷脂酰肌醇3激酶（PI3K）抑制剂（LY294002和渥曼青霉素）、磷脂酶C抑制剂（依地福新）和Akt抑制剂预处理可抑制哇巴因刺激的Akt Ser(473)磷酸化。此外，细胞外钙减少（EGTA）或细胞内钙用BAPTA-AM缓冲时，哇巴因介导的Akt Ser(473)磷酸化受到抑制。钙库释放抑制剂（TMB-8）和通过储存-操纵性钙通道进入的钙抑制剂（SKF96365）也抑制哇巴因介导的Akt Ser(473)磷酸化。在装载fura-2 AM的细胞中，10 nM哇巴因增加了储存性钙内流（CCE）。10 nM哇巴因对对照细胞的基线细胞质钙浓度没有显著影响。然而，用10 nM哇巴因处理导致ATP介导的钙库释放显著增加。24小时后，10 nM哇巴因增加了细胞增殖速率。Akt抑制剂、BAPTA-AM、SKF96365和环匹阿尼酸抑制了10 nM哇巴因引起的细胞增殖速率增加。10 nM哇巴因导致（86）Rb摄取可检测到增加，但对粗膜中钠钾ATP酶（哇巴因敏感的对硝基苯磷酸酶）活性或细胞钠含量没有显著影响。综上所述，结果表明，低浓度哇巴因可引起CCE和Akt磷酸化，从而增加细胞增殖速率，而不抑制钠钾ATP酶介导的离子转运。
