Epithelial Sodium Channels in the Supraoptic Vasopressin and Oxytocin Neurons

描述（由申请人提供）：神经垂体激素加压素（VP）和催产素（OT）在下丘脑室旁核（PVN）和视上核（SON）内的大细胞神经元（MNCs）中合成，并根据生理需求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，并产生抗利尿和加压作用（Sladek 2000）。除了众所周知的OT在分娩和哺乳期的作用外，血浆OT在高钠血症的反应中增加（Huang et al., 1995）并诱导尿钠（Conrad et al., 1986; Huang et al., 1994）。非电压依赖性、酰胺敏感的上皮Na+通道（ENaCs）存在于肾脏中，已知有助于Na+和水的稳态（Benos等，1995）。在人类中，大多数已知的高血压遗传原因是由于ENaC本身或其调节缺陷导致肾Na+重吸收异常增加（Dahlberg et al., 2007; Lifton 1996; Mune et al., 1995; Shimkets et al., 1994； Zhou等，2007）。有趣的是，所有三个ENaC亚基（1、2和3）的信使和蛋白质都已在大鼠大脑的心血管调节中心得到证实，包括SON和PVN中的MNCs （Amin et al., 2005）。脑室内注射ENaC阻滞剂（阿米洛利类似物苯并米）可显著减轻盐依赖性高血压动物模型中的高血压（Gomez-Sanchez and Gomez-Sanchez 1995; Nishimura et al., 1998）。此外，已知ENaC表达改变的靶点，即矿化皮质激素受体（MR）存在于跨国公司中（Amin et al., 2005）。这些发现表明，中枢ENaC抑制可能是治疗心血管疾病的潜在新靶点（Teiwes和Toto 2007）。尽管有这些发现，ENaCs的功能意义以及它们在跨国公司中由MRs调控是完全未知的。因此，本研究项目的总体目标是表征ENaCs在跨国公司中的功能意义。我们假设ENaCs影响VP和OT神经元的放电模式，最终影响这些激素的分泌，并且ENaCs在这些神经元中的异常表达/调节至少部分地导致了盐敏感个体VP和/或OT的异常分泌。为了解决这一假设，我们将采用全细胞膜片钳技术结合单细胞RT-PCR和免疫细胞化学来确定：1)enact介导的电流在VP和OT神经元中的存在和电生理特征；2)在盐敏感大鼠动物模型中是否观察到ENaCs在VP和OT神经元中的异常表达/调控。上皮性钠通道存在于肾脏中，在人类高血压的发展中起重要作用，在下丘脑的脑加压素（VP）和催产素（OT）神经元的心血管调节中心也被发现。虽然脑ENaC可能是治疗心血管疾病的潜在新靶点，但ENaC在VP和OT神经元中的功能意义尚不清楚。本提案的研究将阐明这一关键机制，并将提高我们管理高血压的能力。

DESCRIPTION (provided by applicant): The neurohypophysial hormones vasopressin (VP) and oxytocin (OT) are synthesized in the magnocellular neurons (MNCs) located within the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus, and released from the neurohypophysis into the general circulation in response to physiological demands. The secretion of VP increases in response to hyperosmolality, hypovolemia, and hypotension, and produces antidiuretic and pressor effects (Sladek 2000). In addition to the well known effects of OT during parturition and lactation, plasma OT increases in response to hypernatremia (Huang et al., 1995) and induces natriuresis (Conrad et al., 1986; Huang et al., 1994). The non-voltage-dependent, amiloride-sensitive Epithelial Na+ channels (ENaCs) are present in kidney and are known to contribute to Na+ and water homeostasis (Benos et al., 1995). In humans, most of the known genetic causes of hypertension are due to defects in ENaC itself or its regulation, which results in abnormal increases in renal Na+ reabsorption (Dahlberg et al., 2007; Lifton 1996; Mune et al., 1995; Shimkets et al., 1994; Zhou et al., 2007). Interestingly, both messengers and proteins for all three ENaC subunits (1, 2, and 3) have been demonstrated in the cardiovascular regulatory centers of the rat brain including the MNCs in the SON and PVN (Amin et al., 2005). Intracerebroventricular injections of the ENaC blocker, the amiloride analogue benzamil, significantly attenuated the hypertension in animal models with salt-dependent forms of hypertension (Gomez-Sanchez and Gomez-Sanchez 1995; Nishimura et al., 1998). In addition, a known target for altered ENaC expression, the mineralocorticoid receptor (MR), is present in MNCs (Amin et al., 2005). These findings suggest central ENaC inhibition may be a potential new target in the treatment of cardiovascular disease (Teiwes and Toto 2007). Despite these findings, the functional significance of ENaCs and their regulation by MRs in MNCs is completely unknown. Therefore, the overall objective of this research project is to characterize the functional significance of ENaCs in MNCs. We hypothesize that ENaCs affect the firing patterns of VP and OT neurons that ultimately affect the secretion of these hormones, and abnormal expression/regulation of ENaCs in these neurons contributes, at least partly, to abnormal secretion of VP and/or OT in salt-sensitive individuals. To address this hypothesis, we will employ whole-cell patch clamp technique combined with single-cell RT-PCR and immunocytochemisry to determine: 1) the presence and electrophysiological characteristics of ENaC-mediated current in VP and OT neurons; 2) whether abnormal expression/regulation of ENaCs in VP and OT neurons is observed in an animal model of the salt-sensitive rat. PUBLIC HEALTH RELEVANCE Epithelial sodium channels which present in kidney and which play an important role in development of hypertension in human, have been also found in the cardiovascular regulatory centers of the brain vasopressin (VP) and oxytocin (OT) neurons in the hypothalamus. While the brain ENaC may be a potential new target in the treatment of cardiovascular disease, the functional significance of ENaCs in VP and OT neurons is unknown. The research in this proposal will elucidate this critical mechanism, and will increase our ability to manage hypertension.