Mechanisms of intracellular NAMPT-regulated GSNOR in vessel wall

描述(申请人提供)：阻力动脉张力的调节涉及血管平滑肌和内皮之间的通讯，这是由一个复杂但尚未完全确定的细胞信号过程严格控制的。最近，我们发现，在半胱氨酸-硫醇侧链上添加或去掉一个一氧化氮基团的S-亚硝化/去硝化是连接蛋白43缝隙连接蛋白(GJ)蛋白的一种重要的翻译后修饰，这种修饰与阻力动脉张力的控制有关。连接蛋白43亚硝化的调节似乎主要发生在肌内皮细胞连接处(Mej)，这是阻力动脉中内皮细胞和平滑肌细胞接触的地方。在MEJ，内皮型一氧化氮合酶(ENOS)和脱硝基酶S-亚硝基谷胱甘肽还原酶(GSNOR)协同作用，调节GJS的通透性。调控eNOS活性的机制已被很好地描述，但调控GSNOR活性的分子机制仍不清楚。为了确定在MEJ处能够调节GSNOR活性的丰富蛋白质，我们最近进行了体外MEJ蛋白质组筛选。通过这一分析，我们发现烟酰胺磷酸核糖转移酶(NAMPT)的表达丰富，NAMPT是烟酰胺腺嘌呤二核苷酸(NAD)生物合成途径中的限速酶。细胞内NAMPT在MEJ的定位蛋白表达向我们表明，它对于调节NAD水平至关重要，NAD水平是调节GSNOR活性的已知因素，因此可能控制管壁中的异质细胞通讯。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，我们提出了中心假设，即血管阻力和全身血压控制是通过NAMPT调节的局部GSNOR机制调节的。我们将通过三个特定的目标来验证这一假说：目的1将测试NAMPT是否在体外调节GSNOR活性和异质细胞通讯，AIM 2将确定NAMPT是否在阻力动脉张力的调节中起关键作用，AIM 3将阐明细胞类型特异性调节血管内皮或平滑肌中NAMPT表达如何改变阻力动脉对血管收缩药或血管扩张剂的反应。我们的结果将 影响我们对这些酶在血压控制中的理解，并提供一个框架来确定NAMPT和GSNOR表达和/或活性异常是否导致包括高血压在内的心血管疾病。

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.