CELLULAR MECHANISMS OF MINERALOCORTICOID ACTION

维持细胞外液容量的动态平衡对于 血流动力学稳定性和肾脏钠处理的异常 与心血管疾病和高血压有关。极致 肾脏钠排泄的调节发生在远端肾单位 并受矿质皮质激素醛固酮的调节。从长远来看 这项研究的目标是了解细胞机制 醛固酮调节钠的重吸收。最初的生理事件 在醛固酮的刺激下，钠的重吸收是心尖部的增加 膜通透性增加所致的数量和开放概率 传导性钠通道。已知这些通道由 异三聚体G蛋白定位于通道附近。这项研究提出 以前不活动或静止的通道被激活 醛固酮刺激对G蛋白定位的影响。G蛋白被认为是 通过顺序的羧甲基化作用靶向于膜和 末端氨基酸的断裂。既有酰化又有 羧甲基化与醛固酮和 是刺激钠转运所必需的。这项研究将 通过电泳法对顶膜蛋白进行鉴定 羧甲基化和/或酰化和戊烯基化 醛固酮。抗G蛋白亚基和抗G蛋白亚基抗体的免疫沉淀 将采用钠离子通道复合体来确定 这些翻译后修饰的蛋白质及其与 频道。脱氢反应和甲基化反应抑制剂的研究 将进行以确定这些反应是否需要 膜定位。因为这些蛋白质在转录上可能是 以及翻译后调节的，代谢的蛋白质印迹 标记的蛋白质将被用来评估调节性G- 蛋白质在细胞质或细胞膜中的丰度增加 在醛固酮刺激后。

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.