Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal.
J Cell Physiol. 2011 Sep;226(9):2391-7. doi: 10.1002/jcp.22575.
Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport.
钠钾-ATP 酶（Na(+),K(+)-ATPase）是一种基底外侧转运体，负责肾小管对 Na(+)的重吸收，并为各种溶质和离子的向量转运提供驱动力，也可以作为信号转导物，对与类固醇激素的相互作用做出响应。在纳摩尔浓度下，哇巴因（ouabain）与钠钾-ATP 酶结合，激活信号级联，最终调节包括钠钾-ATP 酶在内的几种膜转运体。本研究评估了哇巴因对钠钾-ATP 酶活性（Na(+)跨上皮通量）和表达的长期影响，研究对象为在培养中具有低（40 代）和高（80 代）传代数的负鼠肾（OK）细胞，已知这些细胞会过表达钠钾-ATP 酶（Silva 等人，2006 年，J Membr Biol 212, 163-175）。通过 Western blot 定量检测 ERK1/2 磷酸化来评估信号级联的激活。通过电生理学技术测定钠钾-ATP 酶活性，通过 Western blot 测定表达水平。哇巴因孵育可诱导 ERK1/2 磷酸化的激活。长期哇巴因孵育仅可诱导具有 80 代传代数的 OK 细胞中 Na(+)跨上皮通量和钠钾-ATP 酶表达的增加。这种增加可被 MEK1/2 和 PI-3K 抑制剂孵育所阻止。总之，由 MEK1/2 和 PI-3K 介导的哇巴因激活的信号级联负责上皮肾细胞中 Na(+)跨上皮通量的长期调节。高传代数的 OK 细胞系被建议构成用于理解哇巴因介导的 Na(+)转运调节的特别有用的模型。
