T-type Calcium Channels and Aldosterone Secretion

说明（由申请人提供）：生理上醛固酮作用于肾小管，促进盐和水的保留。它的产生调节血管容量，从而调节血压。对于个体的盐状态，醛固酮的不适当分泌是致病性的，通过推进疾病过程如心脏纤维化、肾硬化和动脉硬化而诱导心脏、肾脏和脉管系统的结构和功能改变。虽然醛固酮诱导的心血管和肾脏损害与配体激活的核盐皮质激素受体（MR）的激活有关，但醛固酮的致病作用与MR无关，如血管反应性受损，现已得到广泛认可。因此，有一个强大的理论基础，探索补充MR封锁，直接靶向醛固酮的生产。肾上腺肾小球（ZG）细胞产生的醛固酮是钙依赖性的，循环中的血管紧张素II是主要的醛固酮促分泌素。然而，由于肾上腺的ZG层高度血管化，每个ZG细胞与内皮细胞相邻，因此ZG细胞也是局部产生的腔外分泌的内皮素（ET-1）调节的靶。在这个建议中，我们测试新的假设，醛固酮的生产是由全身和旁分泌激素刺激，转换静止的ZG细胞成一个电兴奋的，用于敏感的生产醛固酮低浓度的生理促分泌素。本研究利用膜片钳电生理学、分子诱变和放射免疫分析等技术，对ZG细胞膜兴奋性的离子基础进行了研究。目的1：建立ZG细胞膜兴奋性的离子基础。具体而言，我们将：（1.1）定义ZG细胞中再生Vm尖峰的基本性质;（1.2）鉴定Cav3.2通道和IKCa在起搏的起始和/或传播中的作用;（1.3）表征由Ang II和/或ET-1引起的再生Vm尖峰的控制;（1.4）量化再生Vm尖峰对净钙进入的影响。目的2：探讨PKC介导Cav3.2通道调控的分子基础。PKD及其对电兴奋性的贡献和由Ang II和/或内皮素引起的醛固酮产生的刺激。具体而言，我们将：（1.1）确定Cav3.2通道蛋白上介导PKC β刺激全细胞和单通道活性的关键残基。PKD;（1.2）测定PKC β 2在PKD中的作用。PKD介导的通道对Ang Ⅱ和/或ET-1引起的通道激活和电兴奋性的调节; PKD调节抗性通道进入肾上腺皮质球状细胞以干扰Ang II和/或ET-1诱导的电兴奋性，并评估由Ang II和/或ET-1引起的醛固酮产生的调节。很可能，ZG细胞的电兴奋性的认识将彻底改变治疗策略，以有效地抑制醛固酮的产生。 公共卫生相关性：在适当的生产醛固酮诱导结构和功能的改变，在心脏，肾脏和脉管系统通过推进疾病的过程，如心脏纤维化，肾硬化和动脉硬化。然而，由于并非所有的心血管和肾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直接靶向调节醛固酮产生的MR阻断剂的补充疗法。在本申请中，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，这彻底改变了对这种内分泌细胞的看法，并且本身改变了醛固酮产生可以被靶向用于通过新的药物疗法抑制的方式。

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.