LRRC8 anion channels, superoxide and RhoA in diabetic erectile dysfunction

项目摘要 勃起功能障碍（艾德）是糖尿病最常见、最严重的并发症之一 （DM）。糖尿病和性功能障碍的结合影响计划生育和整体生活质量， 艾德现在被认为是心血管疾病的早期标志物， 发展到危及生命的状况。高脂血症、高脂血症和炎症都与 与糖尿病和诱导血管活性氧（ROS）的产生。烟酰胺腺嘌呤 二核苷酸磷酸（NADPH）氧化酶（Nox）是血管系统中ROS的主要来源， 诱导阴茎血管收缩的细胞内信号通路。血管平滑肌 细胞（VSMCs），这种信号级联反应始于细胞外超氧化物（O2-·）的产生，由Nox 1在细胞外产生。 质膜来自Nox 1的细胞外O2-·可通过灭活内皮源性 一氧化氮（NO）。但是O2-·也触发RhoA/Rho激酶信号传导的激活，从而引起血管收缩。 阴茎脉管系统在糖尿病中，RhoA/Rho激酶信号传导的激活被夸大，导致ED。 我们已经证明了Nox 1和体积调节蛋白之间的功能和结构相互依赖性。 阴离子通道（VRAC）由富含亮氨酸的重复序列的8（LRRC 8）家族蛋白组成。LRRC 8A是 所有VRAC都需要，并与四种密切相关的LRRC 8亚型（LRRC 8B至E）之一结合， 产生具有独特生物物理特性的通道。我们推测并提供了初步数据， 提供O2-·进入细胞质。我们的初步数据表明，LRRC 8敲除（KO）或通道 抑制增强对照血管中的扩张器功能（或减少收缩），并保护无效小鼠 在体外对抗TNFα和在体内对抗血管紧张素II（AngII）对血管舒张的损害。 这一建议将提供一个结构框架，以了解如何胞外O2-·产生 导致阴茎血管收缩的细胞内信号。我们假设在糖尿病患者中， 功能障碍部分是由于细胞外O2-·从Nox 1通过LRRC 8阴离子通道进入， RhoA/Rho激酶导致血管收缩。三个目标解决这个中心假设：1）测试 在糖尿病中，艾德是由过量的Nox 1和LRRC 8A依赖性的 然后通过LRRC 8A阴离子通道进入VSMC，激活RhoA/Rho激酶， 增加血管张力; 2）测试糖尿病小鼠在血管中缺乏LRRC 8通道的假设， 血管系统不会发生艾德;和3）检验选择性抑制LRRC 8通道的药物 将成为治疗糖尿病艾德性ED的新的治疗剂。这项工作将提供全面的 了解LRRC 8/VRACs、O2-·和RhoA在艾德的病因和发展中的作用的方法 糖尿病男性，并提供了治疗该疾病的新的治疗方法。

PROJECT SUMMARY Erectile dysfunction (ED) is one of the most prevalent and impactful complications of diabetes mellitus (DM). The combination of diabetes and sexual dysfunction affects family planning and overall quality of life, and ED is now considered an early marker for cardiovascular diseases and consequently could predate the development of a life-threatening condition. Hyperglycemia, hyperlipidemia and inflammation are all associated with DM and induce the generation of vascular reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is the main source of ROS in the vasculature and ROS activate intracellular signaling pathways that induce constriction of the penile vasculature. In vascular smooth muscle cells (VSMCs), this signaling cascade begins with extracellular superoxide (O2-•) production by Nox1 at the plasma membrane. Extracellular O2-• from Nox1 can alter vascular tone by inactivating endothelium-derived nitric oxide (NO). But O2-• also triggers activation of RhoA/Rho-kinase signaling to cause constriction of the penile vasculature. In diabetes, activation of RhoA/Rho-kinase signaling is exaggerated leading to ED. We have demonstrated functional and structural inter-dependence between Nox1 and volume-regulated anion channels (VRACs) composed of leucine-rich repeat-containing 8 (LRRC8) family proteins. LRRC8A is required for all VRACs and combines with one of four closely related LRRC8 isoforms (LRRC8B through E) to produce channels with unique biophysical properties. We speculate and provide preliminary data that VRACs provide O2-• access to the cytoplasm. Our preliminary data demonstrate that LRRC8 knockout (KO) or channel inhibition enhances dilator function (or reduces constriction) in control blood vessels and null mice are protected against impairment of vasodilation by TNFα in vitro and angiotensin II (AngII) in vivo. This proposal will provide a structural framework to understand how extracellular O2-• produces intracellular signals that lead to contraction of the penile vasculature. We hypothesize that in diabetes, erectile dysfunction is partly due to the entry of extracellular O2-• from Nox1 via LRRC8 anion channels to activate RhoA/Rho-kinase resulting in vascular contraction. Three aims address this central hypothesis: 1) to test the hypothesis that in diabetes, ED is caused by excess Nox1 and LRRC8A-dependent production of extracellular O2-• which then enters VSMCs via LRRC8A anion channels, activates RhoA/Rho kinase and increases vascular tone; 2) to test the hypothesis that diabetic mice that lack the LRRC8 channel in the vasculature will not develop ED; and 3) to test the hypothesis that drugs that selectively inhibit LRRC8 channels will be novel therapeutic agents for the treatment of ED in diabetes. This work will provide a comprehensive approach to understanding the role of LRRC8/VRACs, O2-• and RhoA in the etiology and development of ED in diabetic males and provide a novel therapeutic approach for treatment of the disorder.