Beta arrestin and NO signaling in endothelial cells

描述(由申请人提供)：调节eNOS活动的一个重要因素是它的本地化。已有研究表明，质膜是eNOS在内皮细胞中产生NO的最有效部位。此外，许多研究表明，HSP90在增加eNOS介导的NO生成中起着重要作用。然而，eNOS-HSP90相互作用的分子机制尚不完全清楚。阻滞素是广泛存在的G蛋白偶联受体的多功能接头蛋白。最近研究表明，β-arrestin 2与HSP90结合。然而，β-拦阻蛋白在调节HSP90-eNOS相互作用中的作用尚未得到解决。我们的初步数据表明，β-arrestin 1或2、HSP90和eNOS是由钙激活剂形成的三元复合体，并被钙抑制剂减弱。过表达β-arrestin或β-arrestin 2会导致较高的NO产生和较低的eNOS产生的超氧化物，而双敲除β-arrestin 1和2会抑制NO的产生并增加eNOS产生的超氧化物。这些数据导致我们提出假设，即β-arrestin通过介导HSP90的膜转位促进eNOS-HSP90复合体在膜上的形成。氧化应激参与了许多心血管疾病的发病过程。我们的初步数据表明，氧化应激降低了β-arrestin的表达。在我们的肺动脉高压分流模型中，它的表达减少。我们先前已经证明，在这些羔羊中，eNOS是解偶联的，eNOS证明HSPO相互作用减少，超氧化物生成增加。我们推测，β-arrestin表达的减少有助于肺动脉高压中eNOS的解偶联。已有研究表明，过表达CuZnSOD可减轻血管内皮细胞损伤，但其确切机制尚不清楚。我们的初步数据显示，SOD1的过表达增加了β-arrestini的表达，增强了eNOS-HSP90的相互作用，增加了NO的产生，并增加了β-arrestini的表达。我们假设，通过β-arrestin过表达SOD1可以恢复eNOS的解偶联。相关性(见说明)；我们的研究旨在确定eNOS酶调节的信号机制，该酶产生一氧化氮，一氧化氮是血管内稳态的基本重要分子。特别是，我们试图确定当eNOS产生NO而不是超氧化物时，负责维持eNOS处于最佳“偶联”状态的细胞因素，后者是与高血压等疾病相关的内皮功能障碍的关键介质。糖尿病、动脉粥样硬化等。

DESCRIPTION (provided by applicant): An important factor in regulating eNOS activity is its localization. It has been demonstrated that the plasma membrane is the most efficient location for eNOS to produce NO in endothelial cells. Further, many studies have shown that HSP90 plays an important role in increasing eNOS mediated NO generation. However the molecular mechanisms by which eNOS-HSP90 interactions are stimulated are not fully understood. Beta arrestins are ubiquitous multifunctional adaptor proteins of G-protein coupled receptors (GPCR). Beta arrestin 2 has recently been shown to bind to HSP90. However, the role of beta arrestins in regulating HSP90-eNOS interactions has not been resolved. Our preliminary data indicate that a ternary complex of beta arrestin 1 or 2, HSP90 and eNOS is formed by calcium activating agents and is attenuated by calcium inhibiting agents. Overexpression of either beta arrestin or beta arrestin 2 results in higher NO production and lower eNOS-derived superoxide generation while the double knockdown of beta arrestin 1&2 expression attenuates NO production and augments eNOS-derived superoxide generation. These data have led us to form the hypothesis that beta arrestin promotes eNOS-HSP90 complex formation in the membrane by mediating the membrane translocation of HSP90. Oxidative stress mediates the pathogenesis of many cardiovascular diseases. Our preliminary data indicate that oxidative stress decreases the expression of beta arrestin. Its expression is decreased in our shunt model of pulmonary hypertension. We have previously shown that in these lambs, eNOS is uncoupled as evidenced by eNOS HSPO interactions are reduced, superoxide generation is enhanced. We hypothesize that a decrease in beta arrestin expression contributes to eNOS uncoupling in pulmonary hypertension. It has been shown that overexpression of CuZnSOD attenuates endothelial injury but the exact mechanisms of such protection remain unclear. Our preliminary data show that overexpression of SODl increases beta arrestini expression, enhances eNOS-HSP90 interactions and increases NO production and beta arrestini expression is increased in lambs treated with SODL We hypothesize that eNOS uncoupling can be restored by SODl overexpression via beta arrestin. RELEVANCE (See instructions); Our research is aimed at identifying the signaling mechanism of regulation of the enzyme eNOS, which produces nitric oxide, the molecule of fundamental importance for vascular homeostasis. In particular we trying to identify the cellular factor responsible for maintaining eNOS in the optimal "coupled" state when It generates NO and not superoxide, which is a key mediator of endothelial dysfunction associated with diseases like hypertension. diabetes, atherosclerosis etc.