Epithelial Sodium Channels in Vasopressin and Oxytocin Synthesizing Magnocellular

描述（由申请人提供）：盐敏感个体的血压对盐摄入量异常敏感（1）。无论一个人是否患有高血压，盐敏感性都会增加死亡风险。此外，对盐敏感的人随着年龄的增长可能会患上高血压（2）。因为盐敏感在美国很常见，这是一个重大公共卫生问题。目前的介入治疗方法只能对抗盐敏感性高血压的外周效应，结果往往不令人满意。因此，需要治疗疾病原因的其他疗法。虽然盐敏感性的机制还不清楚，但越来越多的证据表明，它至少部分是由大脑中上皮Na+通道（ENaCs）的异常调节引起的。所有三种ENaC亚基的信使和蛋白质均在大鼠脑中得到证实，包括在下丘脑视上核（SON）和室旁核（PVN）中合成加压素（VP）和催产素（OT）的大细胞（MNC）中（3）。此外，一个已知的ENaC表达改变的靶点，盐皮质激素受体（MR），存在于MNC中（3）。VP和OT从神经垂体释放到全身循环中。VP的分泌在高渗、低血容量和低血压时增加，并产生抗利尿和升压作用（4）。血浆OT在高钠血症时升高（5），并诱导尿钠排泄（6，7）。由于脑室内输注ENaC阻滞剂显著减弱了盐敏感性高血压动物模型中的高血压（8，9），这些发现强烈表明MNC中的ENaC在盐敏感性高血压的发展中起重要作用。然而，ENaCs的作用及其在大脑中的调节还不清楚。因此，本研究的总体目标是描述ENaCs在跨国公司中的功能意义。我们最近的研究表明，ENaC是一种Na+漏电流，可调节膜电位并影响MNC中诱发的频率动作电位（18）。这意味着ENaC活性的调节是根据生理需求调节激素分泌的有力手段。根据我的初步研究结果，我假设饮食中的Na+摄入量影响ENaCs的活动，改变VP和OT MNCs的动作电位模式，最终影响这些激素的分泌。因此，这些神经元中ENaCs的异常调节有助于盐敏感性高血压的发展。为了验证这一假设，我们将采用全细胞膜片钳技术结合免疫细胞化学、半定量RT-PCR和免疫印迹来确定：1）膳食盐摄入如何影响MNCs中ENaC活性和神经元活性; 2）盐皮质激素醛固酮和VP对MNCs中ENaC表达的调节作用; 3）MNCs中ENaC的激活机制。该拟议项目的结果将提供关于中枢ENaC抑制作为心血管疾病治疗潜在新靶点的关键信息（10）。

DESCRIPTION (provided by applicant): Salt-sensitive individuals have blood pressure that is unusually sensitive to salt intake (1). Salt-sensitivity increases the risk of death whether or nota person has high blood pressure. Furthermore, salt-sensitive persons are likely to develop high blood pressure as they age (2). Because salt sensitivity is common in the U.S., it is of significan public health concern. Current interventional approaches counter only the peripheral effects of salt sensitive hypertension, and the results are often unsatisfactory. Therefore, additional therapies that treat the cause of the disorder are needed. Although the mechanism of salt sensitivity is not well understood, a growing body of evidence suggests that it is caused by, at least partly, an abnormal regulation of the epithelial Na+ channels (ENaCs) in the brain. Both messengers and proteins for all three ENaC subunits were demonstrated in the rat brain including in vasopressin (VP) and oxytocin (OT) synthesizing magnocellular cells (MNCs) in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei (3). In addition, a known target for altered ENaC expression, the mineralocorticoid receptor (MR), is present in MNCs (3). VP and OT are released from the neurohypophysis into the general circulation. The secretion of VP increases in response to hyperosmolality, hypovolemia, and hypotension, and produces antidiuretic and pressor effects (4). Plasma OT increases in response to hypernatremia (5) and induces natriuresis (6, 7). Because, intracerebroventricular infusion of the ENaC blockers significantly attenuated the hypertension in animal models with salt-sensitive hypertension (8, 9), these findings strongly suggest that ENaC in MNCs play a significant role in the development of salt-sensitive hypertension. However, the role of ENaCs and their regulation in the brain is not well understood. Therefore, the overall objective of this proposed research is to characterize the functional significance of ENaCs in MNCs. Our recent study demonstrated that ENaC is a Na+-leak current modulating membrane potential and affecting the frequency action potentials evoked in MNCs (18). This implies that modulation of ENaC activity is a powerful means to modulate hormone secretion according to physiological demands. Based on results from my preliminary study, I hypothesize that dietary Na+ intake affects ENaCs activity that alters the patterns of action potentials in VP and OT MNCs which ultimately affect the secretion of these hormones. Therefore, abnormal regulation of ENaCs in these neurons contributes to the development of salt-sensitive hypertension. To address this hypothesis, we will employ the whole-cell patch clamp technique combined with immunocytochemistry, semi-quantitative RT-PCR and immunoblotting to determine: 1) how dietary salt intake affects both ENaC activity and the neuronal activity in MNCs; 2) the regulatory roles of the mineralocorticoid aldosterone and VP on ENaC expression in MNCs; and 3) the activation mechanisms of ENaCs in MNCs. Results from this proposed project will provide critical information concerning central ENaC inhibition as a potential new target in the treatment of cardiovascular disease (10).