CELLULAR MECHANISMS OF MINERALOCORTICOID ACTION

维持细胞外液容量的稳态对于 血液动力学稳定性和肾钠处理异常 与心血管疾病和高血压有关。 终极 肾脏钠排泄的调节发生在远端肾单位 并由盐皮质激素醛固酮调节。 长期 这项研究的目的是了解细胞机制， 醛固酮调节钠重吸收。 最初的生理事件 在醛固酮刺激钠重吸收中， 膜渗透性由于数量和开放的概率增加， 传导性钠通道 已知这些通道由 位于通道附近的异源三聚体G蛋白。 本研究提出 先前不活跃或静止的通道被激活， 醛固酮刺激G蛋白定位。 G蛋白被认为是 通过连续羧甲基化靶向膜， 末端氨基酸的锂化。 酰化和 羧甲基化与醛固酮的作用有关， 对刺激钠转运至关重要。 本研究将 通过电泳表征顶端膜蛋白， 羧甲基化和/或酰化和异戊二烯化 醛固酮 G蛋白亚单位抗体的免疫沉淀， 钠通道复合物将用于确定 这些后修饰的蛋白质及其与 频道 锂化和甲基化反应抑制剂的研究 将进行，以确定是否需要这些反应， 膜定位 由于这些蛋白质可能在转录水平上是 以及代谢后调节的蛋白质印迹， 标记的蛋白质将进行评估是否调节G- 蛋白质在胞质溶胶或细胞膜中的丰度增加 醛固酮刺激后。

Maintenance of extracellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension. Ultimate regulation of sodium excretion in the kidney occurs in the distal nephron and is modulated by the mineralocorticoid aldosterone. The long term goals of this research are to understand the cellular mechanisms by which aldosterone regulates sodium reabsorption. The initial physiologic event in aldosterone stimulation of sodium reabsorption is an increase in apical membrane permeability due to an increase in number and open probability of conductive sodium channels. These channels are known to be gated by heterotrimeric G proteins localized near the channel. This study proposes that previously inactive or quiescent channels are activated by aldosterone stimulation of G-protein localization. G-proteins are thought to be targeted to membranes by sequential carboxylmethylation and lapidation of terminal amino acids. Both acylation and carboxylmethylation have been implicated in the action of aldosterone and are essential for stimulation of sodium transport. This study will characterize by electrophoresis the apical membrane proteins which are carboxylmethylated and/or acylated and prenylated in response to aldosterone. Immunoprecipitation by antibodies to G-protein subunits and to sodium channel complex will be employed to determine the identify of these post-translationally modified proteins and their relationship to the channel. Studies with inhibitors of lapidation and methylation reactions will be carried out to determine whether these reactions are required for membrane localization. Since these proteins may be transcriptionally as well as post-translationally regulated, Western blotting of metabolically labelled proteins will be performed to assess whether regulatory G- proteins are increased in abundance in either cytosol or membranes following aldosterone stimulation.