Regulation of the epithelial Na+ channel by Ras and Sgk

描述（由申请人提供）：钠依赖性液体的正确控制 远端肾单位的重吸收对于血压稳态至关重要。 最重要的全身调节剂醛固酮的细胞机制 Na 的任意重吸收的影响，人们知之甚少。长期目标 新任命的调查员的这个（修订后的）第一个提案是他们的 阐明。这项研究与许多器官系统的生理学相关 包括泌尿系统、心血管系统和呼吸系统，以及 高血压和其他与液体有关的疾病的病理生理学 不平衡。阿米洛利敏感的上皮钠离子通道 (ENaC) 的活性 限制了 Na 的重吸收。醛固酮影响基因表达，然后 增加 ENaC 的活性。然而，编码 ENaC 的基因并不 自己最初诱发的。因此，醛固酮增加 将信息转导至 ENaC 的中间信号蛋白。基因 传统上很难编码醛固酮诱导的转录本 识别。利用现代技术，两个醛固酮诱导的转录本，相关 对于信号转导，最近已确定：血清和 糖皮质激素调节激酶 (Sgk) 和小 G 蛋白 K-RasA （K-rasA）。这些转录物的诱导是醛固酮的主要作用 上皮细胞，并转化为 Sgk 和 K-RasA 蛋白水平的增加。 这些蛋白质与 Na 重吸收和 ENaC 的关系仍然很差 明白了。当前提案的具体目标将直接决定 醛固酮诱导的 Sgk 和 K-RasA 信号传导的潜在新作用 调节上皮细胞中的 ENaC 活性。我推测醛固酮激活 KRasA和Sgk通过信号传导汇聚稳定ENaC中 打开状态并增加管腔膜中 ENaC 的数量。的效果 ENaC 上的 Sgk 和 K-RasA 信号传导将在 A6 细胞模型中进行研究 使用全面且新颖的实验方法对远端肾单位上皮进行研究。 蛋白质成分水平和活性的生化评估 Sgk 和 K-RasA 信号通路对醛固酮的反应将是其中之一 最终测量。另一个是 ENaC 的电生理测量 活性、动力学和数量。特定分子的影响 Sgk 和 K-RasA 信号转导的药理调节剂 过程将用于以系统的方式描绘细胞 醛固酮增加天然钠重吸收的作用机制 上皮细胞。

DESCRIPTION (provided by applicant): Proper control of Na+-dependent fluid reabsorption at the distal nephron is critical to blood pressure homeostasis. The cellular mechanisms of aldosterone, the most important systemic modulator of discretionarily Na+ reabsorption, are poorly understood. The long-term goal of this (revised) first proposal of a newly appointed investigator is their elucidation. This research is relevant to the physiology of many organ systems including the urinary, cardiovascular and respiratory systems, as well as to the pathophysiology of hypertension and other diseases associated with fluid imbalance. Activity of the amiloride-sensitive, epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption. Aldosterone affects gene expression and then increases the activity of ENaC. However, the genes encoding ENaC are not themselves initially induced. Thus, aldosterone increases expression of intermediary signaling proteins that transduce information to ENaC. Genes encoding aldosterone-induced transcripts traditionally have been difficult to identify. With modem technology, two aldosterone-induced transcripts, relevant to signal transduction, recently have been identified: serum- and glucocorticoid-regulated kinase (Sgk), and the small G protein, K-RasA (K-rasA). Induction of these transcripts is a primary action of aldosterone in epithelia, and translates into an increase in Sgk and K-RasA protein levels. The relation of these proteins to Na+ reabsorption and ENaC remain poorly understood. The Specific Aims of the current proposal will directly determine the potential novel roles of aldosterone-induced Sgk and K-RasA signaling in regulating ENaC activity in epithelia. I hypothesize that aldosterone-activated KRasA and Sgk through signal transduction convergence stabilize ENaC in the open state and increase number of ENaC in the luminal membrane. The effect of Sgk and K-RasA signaling on ENaC will be investigated in the A6 cell model of distal nephron epithelia using a comprehensive and novel experimental approach. Biochemical assessment of the levels and activities of the protein constituents of Sgk and K-RasA signaling pathways in response to aldosterone will be one end-measurement. The other will be electrophysiological measurement of ENaC activity, kinetics and number. The effects of specific molecular and pharmacological modulators of Sgk and K-RasA signal transduction on these processes will be used to delineate in a systematic manner the cellular mechanisms of aldosterone action to increase Na+ reabsorption in native epithelia.