Mechanisms of Intracellular NAMPT-regulated GSNOR in Vessel Wall

摘要 阻力动脉张力的调节涉及血管平滑肌和血管张力之间的信息传递。 内皮细胞，它被一个错综复杂但尚未完全定义的细胞信号过程严格控制。 最近，我们发现S-亚硝化/脱亚硝化，一种一氧化氮的添加或去除 来自半胱氨酸-硫醇侧链的基团，是连接蛋白43的重要翻译后修饰 缝隙连接(GJ)蛋白的这种修饰与阻力动脉张力的控制有关。 连接蛋白43亚硝化的调节似乎在肌内皮细胞连接(Mej)处占优势， 阻力动脉内皮细胞和平滑肌细胞接触的点。在Mej酒店， 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) 协同调节GJS的渗透性。ENOS活性的调控机制一直很好 然而，调控GSNOR活性的分子机制仍然知之甚少。至 鉴定MEJ上能够调节GSNOR活性的浓缩蛋白，我们最近在体外进行了一项 MEJ蛋白质组筛选。从这个分析中，我们发现烟酰胺的丰富表达 磷酸核糖转移酶(NAMPT)，烟酰胺腺嘌呤二核苷酸(NAD)中的限速酶 生物合成途径。细胞内NAMPT在MEJ的定位蛋白表达提示 这对于调节NAD水平至关重要，NAD水平已知调节GSNOR活动，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，我们探索了其中的关键要素 细胞内NAMPT可以调节GSNOR活性和阻力动脉张力。基座 在这些观察的基础上，我们形成了一个中心假设，即血管阻力，因此，全身血液 压力控制通过NAMPT调节的局部GSNOR机制进行调节。我们将对此进行测试 使用三个特定目标的假设：目标1将测试NAMPT是否调节GSNOR活性和 在体外异细胞通讯中，AIM 2将确定NAMPT是否在耐药调节中起关键作用 动脉张力，AIM 3将阐明细胞类型特异性地调节内皮或血管内皮细胞NAMPT表达 在阻力动脉中，平滑肌改变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 确定NAMPT和GSNOR的表达和/或活性异常是否有助于 包括高血压在内的心血管疾病。

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.