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）之间的交流对于血压调节和阻力血管的血管舒缩反应是必要的。肌内皮连接（MEJ）是抵抗血管系统所特有的，被认为是一个高度组织的细胞信号传导微域，可以调节内皮细胞和SMCs之间的串扰。最近，在MEJ上发现了小泡，提供了在这个结合点上发生独特信号事件的间接证据。此外，许多报告表明，小泡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，并被区隔化以促进高度定位的细胞信号传导。我们的初步结果证实，eNOS定位于MEJ，并在体外选择性地磷酸化MEJ。为了确定eNOS的潜在激活因子，我们对MEJ富集的蛋白质进行了蛋白质组学分析，发现了烟酰胺磷核糖基转移酶（NAMPT）。重要的是，NAMPT是最近发现的一种脂肪因子，可以刺激eNOS磷酸化、一氧化氮释放和内皮依赖性血管舒张。NAMPT激活eNOS的机制尚不清楚，但有人认为胰岛素受体- 2（也极化到MEJ）起中介作用。因此，本提案的目的是确定NAMPT调节MEJ中一氧化氮释放的机制，特别侧重于了解MEJ中的局部一氧化氮信号。1.)确定eNOS、IR-2和NAMPT在MEJ的局部表达和翻译后修饰，2.)了解NAMPT在MEJ刺激eNOS信号传导的机制，3)生成NAMPT/EC特异性IR-2敲除动物，以确定体内血压和血管运动调节机制。该研究计划还有一个额外的好处，即提供了一个研究框架，可以在此基础上对MEJ区隔化的NAMPT/eNOS信号进行药理学研究，其长期目标是预防或逆转心血管相关疾病。

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.