Epithelial Sodium Channels in Vasopressin and Oxytocin Synthesizing Magnocellular

描述(申请人提供)：盐敏感者的血压对盐的摄入量异常敏感(1)。无论是否患有高血压，盐敏感都会增加死亡风险。此外，盐敏感的人很可能随着年龄的增长而患上高血压(2)。由于盐敏感性在美国很常见，这是一个重大的公共卫生问题。目前的干预方法只能对抗盐敏感型高血压的外周效应，结果往往不令人满意。因此，需要治疗这种疾病的病因的其他疗法。虽然盐敏感的机制还不清楚，但越来越多的证据表明，它至少部分是由大脑中上皮性钠离子通道(ENaCs)的异常调节引起的。在大鼠脑内，包括加压素(VP)和催产素(OT)在内的所有三个ENaC亚基的信使和蛋白质都存在于合成下丘脑视上核(SON)和室旁核(PVN)的大细胞(MNC)(3)。此外，已知的ENaC表达改变的靶点是单核细胞中存在的盐皮质激素受体(MR)。VP和OT从神经垂体释放到全身循环中。VP的分泌在高渗透压、低血容量和低血压时增加，并产生抗利尿和升压作用。血浆OT因高钠血症而增加(5)，并诱导钠尿(6，7)。因为，脑室内注射ENaC阻滞剂显著降低了盐敏感型高血压动物模型的高血压(8，9)，这些发现有力地表明，单核细胞中的ENaC在盐敏感型高血压的发展中起着重要作用。然而，ENaCs的作用及其在大脑中的调节还没有被很好地理解。因此，这项拟议研究的总体目标是表征ENaCs在跨国公司中的功能意义。我们最近的研究表明，ENaC是一种钠离子泄漏电流调节膜电位，并影响MNC诱发的频率动作电位(18)。这表明，调节ENaC活性是根据生理需要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，从而改变VP和OT MNC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的异常调节参与了盐敏感型高血压的发展。为了解决这一假设，我们将使用全细胞膜片钳技术，结合免疫细胞化学、半定量RT-PCR和免疫印迹技术来确定：1)饮食盐摄入如何影响MNC的ENaC活性和神经元活性；2)盐摄入量对MNC中ENaC活性和神经元活性的调节作用；3)MNC中ENaC的激活机制。这一拟议项目的结果将提供有关中枢ENaC抑制作为心血管疾病治疗的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).