T-type Calcium Channels and Aldosterone Secretion

描述(申请人提供)：从生理上讲，醛固酮作用于肾小管，促进盐分和水分的保留。它的产生调节血管容量，从而调节血压。不适当的醛固酮分泌对个体的盐分状态是致病的，通过促进心脏纤维化、肾硬化症和动脉硬化等疾病过程，导致心脏、肾脏和血管系统的结构和功能变化。虽然醛固酮引起的心血管和肾脏损伤与配体激活的核盐皮质激素受体(MR)激活有关，但现在公认的是，醛固酮的致病作用不依赖于MR，如血管反应性障碍。因此，探索直接针对醛固酮生产的MR BLOKKAK的补充疗法是有充分理由的。肾上腺球状带(ZG)细胞产生的醛固酮是钙依赖的，循环中的Ang II是主要的醛固酮促分泌剂。然而，由于肾上腺的ZG层是高度血管化的，每个ZG细胞都与内皮细胞相邻，因此ZG细胞也是局部产生的白蛋白分泌的内皮素(ET-1)调节的靶点。在这个提议中，我们测试了一个新的假设，即醛固酮的产生是由全身和旁分泌激素刺激的，这些激素将静止的ZG细胞转化为电兴奋的细胞，从而使醛固酮的产生对低浓度的生理促分泌剂敏感。利用膜片钳电生理学、分子诱变和放射免疫分析等技术，我们对以下几个方面进行了研究：目的1：建立ZG细胞膜兴奋性的离子基础。具体地说，我们将：(1.1)定义ZG细胞中再生VM峰的基本特性；(1.2)确定Cav3.2通道和IKCa在起搏的启动和/或传播中的作用；(1.3)表征由Ang II和/或ET-1引起的再生VM峰的控制；(1.4)量化再生VM峰对净钙内流的影响。目的：研究PKC？？PKD调节Cav3.2通道的分子基础及其对血管紧张素转换酶II和/或内皮素刺激血管紧张素Ⅱ和/或内皮素刺激的醛固酮生成的作用。具体地说，我们将：(1.1)确定介导PKC？？PKD刺激全细胞和单通道活动的Cav3.2通道蛋白上的关键残基；(1.2)确定PKC？？PKD介导的通道调节对Ang II和/或ORET-1诱导的通道激活和电兴奋性的贡献；(1.3)将PKC？？PKD调节抗性通道引入肾上腺肾小球细胞，以扰乱Ang II和/或ET-1诱导的电兴奋性，并评价其对Ang II和/或ET-1诱导的醛固酮产生的调节。对ZG细胞电兴奋性的认识很可能将彻底改变治疗策略，有效地抑制醛固酮的产生。 与公共卫生相关：适当生产醛固酮可通过促进心脏纤维化、肾硬化症和动脉硬化等疾病进程，导致心脏、肾脏和血管系统的结构和功能变化。然而，由于并不是所有的心血管和肾脏损伤都与醛固酮核受体(MR)的激活有关，因此有充分的理由探索补充MR阻断的直接针对醛固酮产生的调节的治疗方法。在这一应用中，我们探索了调节醛固酮产生的重要机制，并提供了数据表明，肾上腺的醛固酮产生细胞是电兴奋的，这彻底改变了对内分泌细胞的看法，并本身改变了通过新的药物治疗靶向抑制醛固酮产生的方式。

DESCRIPTION (provided by applicant): Physiologically aldosterone acts at the renal tubule to promote salt and water retention. Its production regulates vascular volume and hence blood pressure. Inappropriate secretion of aldosterone for the salt status of an individual is pathogenic, inducing structural and functional alterations in the heart, kidneys, and vasculature by advancing disease processes such as cardiac fibrosis, nephrosclerosis and arteriosclerosis. Although aldosterone induced cardiovascular and renal damage has been associated with the activation of the ligand-activated nuclear mineralocorticoid receptor (MR), pathogenic contributions of aldosterone that are independent of the MR, such as impairment in vascular reactivity, are now well-acknowledged . Therefore, there is a strong rationale for exploring therapies that are complementary to MR blockade that directly target the production of aldosterone. Aldosterone production from adrenal zona glomerulosa (ZG) cells is Ca-dependent and circulating Ang II is the predominant aldosterone secretagogue. Nevertheless, because the ZG layer of the adrenal gland is highly vascularized, every ZG cells is adjacent to an endothelial cell, and thus ZG cells are also targets for regulation by locally produced abluminally secreted endothelin (ET-1). In this proposal we test the novel hypothesis that aldosterone production is stimulated by systemic and paracrine hormones that convert the quiescent ZG cell into an electrically excitable one that serves to sensitize the production of aldosterone to low concentrations of physiological secretagogues. Using techniques of patch-clamp electrophysiology, molecular mutagenesis and radioimmunoassay we evaluate the following specific aims: Aim 1: Establish the ionic basis for ZG cell membrane excitability. Specifically we will: (1.1) Define the basic properties of regenerative Vm spiking in ZG cells; (1.2) Identify the role of Cav3.2 channels and IKCa in the initiation and/or propagation of pacemaking; (1.3) Characterize the control of regenerative Vm spiking elicited by Ang II and/or ET-1; (1.4) Quantify the impact of regenerative Vm spiking on net calcium entry. Aim 2: Determine the molecular basis for the regulation of Cav3.2 channels by PKC??PKD and its contribution to electrical excitability and the stimulation of aldosterone production elicited by Ang II and/or endothelin. Specifically we will: (1.1) Identify critical residues on the Cav3.2 channel protein that mediate stimulation of whole-cell and single channel activity by PKC??PKD; (1.2) Determine the contribution of PKC??PKD-mediated channel regulation to channel activation and electrical excitability elicited by Ang II and/orET-1; (1.3) Introduce PKC??PKD regulation resistant channels into adrenal zona glomerulosa cells to perturb Ang II and or ET-1 induced electrical excitability and evaluate the regulation of aldosterone production elicited by Ang II and/or ET-1. It is likely that the recognition of the electrical excitability of the ZG cell will revolutionize therapeutic strategies to efficiently inhibit the production of aldosterone. PUBLIC HEALTH RELEVANCE: In appropriate production of aldosterone induces structural and functional alterations in the heart, kidneys and vasculature by advancing disease processes such as cardiac fibrosis, nephrosclerosis and arteriosclerosis. However, because not all cardiovascular and renal damage has been associated with the activation of the aldosterone nuclear receptor (MR) there is a strong rationale for exploring therapies that are complementary to MR blockade that target directly the regulation of aldosterone production. In this application we explore important mechanisms that regulate the production of aldosterone and provide data showing that the aldosterone producing cell of the adrenal gland is electrically excitable which revolutionizes the view of this endocrine cell and in itself changes the way in which aldosterone production could be targeted for inhibition by new drug therapies.