Mechanisms of intracellular NAMPT-regulated GSNOR in vessel wall

描述（由申请人提供）：摘要阻力动脉张力的调节涉及血管平滑肌和内皮之间的通信，其由复杂但尚未完全定义的细胞信号传导过程严格控制。最近，我们发现S-亚硝基化/脱亚硝基化，从半胱氨酸-巯基侧链添加或去除一氧化氮基团，作为连接蛋白43间隙连接（GJ）蛋白的重要翻译后修饰，并且这种修饰与阻力动脉张力的控制相关。连接蛋白43亚硝酰化的调节似乎在肌内皮连接处（MEJ）占主导地位，该点是内皮细胞和平滑肌细胞在阻力动脉中接触的地方。在MEJ处，内皮型一氧化氮合酶（eNOS）和脱硝酶S-亚硝基谷胱甘肽还原酶（GSNOR）协同工作以调节GJ的渗透性。调控eNOS活性的机制已被很好地表征，然而调控GSNOR活性的分子机制仍然知之甚少。为了鉴定能够调节GSNOR活性的MEJ富集蛋白，我们最近进行了体外MEJ蛋白质组学筛选。从这个分析中，我们发现富集表达的烟酰胺磷酸核糖转移酶（NAMPT），在烟酰胺腺嘌呤二核苷酸（NAD）生物合成途径的限速酶。在MEJ的细胞内NAMPT的定位蛋白表达向我们表明，这是至关重要的NAD水平的调节，已知的调节GSNOR活性，从而可能控制在血管壁中的异细胞通讯。在我们的初步研究中，我们通过显示细胞内NAMPT可以调节GSNOR活性和阻力动脉张力来探索这一概念的关键要素。基于这些观察结果，我们提出了一个中心假设，即血管阻力和全身血压控制是通过局部NAMPT调节的GSNOR机制介导的。我们将使用三个特定的目标来测试这一假设：AIM 1将测试NAMPT是否在体外调节GSNOR活性和异细胞通讯，AIM 2将确定NAMPT是否在阻力动脉张力的调节中至关重要，AIM 3将阐明内皮或平滑肌中NAMPT表达的细胞类型特异性调节如何改变阻力动脉中对血管收缩剂或血管舒张剂的反应。我们的结果将 影响了我们对这些酶在血压控制中的理解，并提供了一个框架，以确定NAMPT和GSNOR的表达和/或活性功能障碍是否有助于心血管疾病，包括高血压。

DESCRIPTION (provided by applicant): Abstract The regulation of resistance arterial tone involves communication between vascular smooth muscle and endothelium, which is tightly controlled by an intricate, but yet to be fully defined, cell signaling processes. Recently, we made the discovery that S-nitrosylation/denitrosylation, the addition or removal of a nitric oxide group from a cysteine-thiol side chain, serves as an important post-translational modification on connexin 43 gap junction (GJ) proteins, and that this modification is associated with control of resistance arterial tone. Regulation of connexin 43 nitrosylation appeared to be predominant at the myoendothelial junction (MEJ), the point where endothelial cells and smooth muscle cells make contact in resistance arteries. At the MEJ, endothelial nitric oxide synthase (eNOS), and the denitrosylase S-nitrosoglutathione reductase (GSNOR), work in concert to modulate the permeability of GJs. The mechanisms regulating eNOS activity have been well characterized, however the molecular mechanisms regulating GSNOR activity remain poorly understood. To identify enriched proteins at the MEJ capable of regulating GSNOR activity, we recently performed an in vitro MEJ proteomic screen. From this analysis, we found enriched expression of nicotinamide phoshoribosyltransferase (NAMPT), a rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthesis pathway. The localized protein expression of intracellular NAMPT at the MEJ suggested to us that it is critical for the regulation of NAD levels which are known to modulate GSNOR activity and thus might control heterocellular communication in the vessel wall. In our pilot studies, we explored key elements of this concept by showing that intracellular NAMPT can regulate GSNOR activity and resistance arterial tone. Based on these observations we formulated the central hypothesis that vascular resistance and thus, systemic blood pressure control is mediated through a localized NAMPT-regulated GSNOR mechanism. We will test this hypothesis using three specific aims: AIM 1 will test whether NAMPT regulates GSNOR activity and heterocellular communication in vitro, AIM 2 will determine if NAMPT is critical in the regulation of resistance arterial tone, AIM 3 will elucidate how cell-type specific modulation of NAMPT expression in endothelium or smooth muscle modifies the responses to vasoconstrictors or vasodilators in resistance arteries. Our results will impact our understanding of these enzymes in blood pressure control and provide a framework to determine whether dysfunctions in the expression and/or activity of NAMPT and GSNOR contribute to cardiovascular diseases including hypertension.