Mechanisms of NAMPT-stimulated nitric oxide release at the myoendothelial junctio

描述(由申请人提供)：NAMPT刺激的肌内皮连接处一氧化氮释放的机制内皮细胞(ECs)和平滑肌细胞(SMCs)之间的通信对于血压调节和阻力血管系统的血管运动反应是必不可少的。肌内皮细胞连接(Mej)是抵抗血管系统所特有的，被认为是一个高度组织化的细胞信号微域，可以调节ECs和SMC之间的串扰。最近，在MEJ发现了小凹，提供了间接证据，表明在这个结合点发生了独特的信号事件。此外，大量的报道表明，小窝调节内皮型一氧化氮合酶(ENOS)的运输和活性。这些发现使我们假设eNOS合成酶可能极化到MEJ，并被划分为促进高度定位的细胞信号。我们的初步结果证实，eNOS定位于MEJ，并在体外选择性地在MEJ处被磷酸化。为了确定eNOS的潜在激活物，我们对在MEJ富含的蛋白质进行了蛋白质组学分析，发现了烟酰胺磷酸核糖转移酶(NAMPT)。重要的是，NAMPT是一种新近鉴定的脂肪因子，它可以刺激eNOS的磷酸化、一氧化氮的释放和内皮依赖性的血管扩张。NAMPT激活eNOS的机制尚不清楚，但已有研究表明，也极化到MEJ的胰岛素受体-2发挥了中介作用。因此，这项建议的目的是确定NAMPT调节的MEJ一氧化氮释放的机制，特别是侧重于了解MEJ的局部一氧化氮信号。定义eNOS、IR-2和NAMPT在MEJ和翻译后修饰的本地化表达，2)了解NAMPT刺激MEJ的eNOS信号的机制；3)建立NAMPT/EC特异性IR-2基因敲除动物，以确定体内血压和血管运动调节机制。这项研究计划的另一个好处是提供了一个研究框架，可以在此框架上对MEJ的分区NAMPT/eNOS信号进行药理学研究，长期目标是预防和/或逆转心血管相关疾病。 公共卫生相关性：在抵抗中，血管内皮细胞和血管平滑肌细胞部分通过肌内皮细胞连接(MEJ)连接。这项建议的重点是内皮型一氧化氮合酶(ENOS)和烟酰胺磷酸核糖转移酶(NAMPT)在MEJ的区划和功能。我们将试图阐明NAMPT和eNOS之间的相互作用，以及这可能对血压和血管运动的调节产生的潜在影响。

DESCRIPTION (provided by applicant): Mechanisms of NAMPT-stimulated nitric oxide release at the myoendothelial junction Communication between endothelial cells (ECs) and smooth muscle cells (SMCs) is imperative for blood pressure regulation and vasomotor responses in the resistance vasculature. The myoendothelial junction (MEJ) is unique to the resistance vasculature and is hypothesized to be a highly organized cell-signaling microdomain that can regulate crosstalk between ECs and SMCs. Recently, caveolae were identified at the MEJ, providing circumstantial evidence that unique signaling events at this juncture. Additionally, numerous reports have demonstrated that caveolae regulate endothelial nitric oxide synthase (eNOS) transport and activity. These discoveries led us to hypothesize that eNOS synthase may polarize to the MEJ and is compartmentalized to promote highly localized cell signaling. Our preliminary results confirm that eNOS localizes to the MEJ and is selectively phosphorylated at the MEJ in vitro. To identify potential activators of eNOS, we performed a proteomic analysis of proteins that were enriched at the MEJ and found nicotinamide phoshoribosyltransferase (NAMPT). Importantly, NAMPT is a recently characterized adipokine that can stimulate eNOS phosphorylation, nitric oxide release, and endothelium dependent vasodialation. The mechanisms by which NAMPT can activate eNOS remain unclear, however it has been suggested that the insulin receptor- 2, which also polarizes to the MEJ, plays an intermediary role. Therefore, the aim of this proposal is to identify mechanisms of NAMPT regulated nitric oxide release at the MEJ, with a particular focus on understanding localized nitric oxide signaling at the MEJ Specifically, the goals of this proposal are to; 1.) define localized expression of eNOS, IR-2, and NAMPT at the MEJ and post-translational modifications, 2.) understand the mechanism of NAMPT stimulated eNOS signaling at the MEJ, 3) generate a NAMPT/EC specific IR-2 knockout animal to determine in vivo mechanisms of blood pressure and vasomotion regulation. This research plan has the additional benefit of providing an investigational framework upon which pharmacological studies of compartmentalized NAMPT/eNOS signaling at the MEJ may be performed, with the long term aim of preventing and or reversing cardiovascular related diseases. PUBLIC HEALTH RELEVANCE: Within the resistance vasculature endothelial cells and vascular smooth muscle cells are coupled in part by myoendothelial junctions (MEJ). The focus of this proposal entails the compartmentalization and function of endothelial nitric oxide synthase (eNOS) and nicotinamide phoshoribosyltransferase (NAMPT) at the MEJ. We will attempt to elucidate the interaction between NAMPT and eNOS as well as the potential implications this may have on the regulation of blood pressure and vasomotion.