Mechanism of Metabolism of S-Nitrosothiols

描述（由申请人提供）：该提案寻求资金，以检查一氧化氮（NO）和S-亚硝基硫醇（RSNO）的基本细胞行为。一氧化氮（NO）是肺和所有其他组织中血管功能的重要组成部分。在肺中，NO具有特别的重要性，因为吸入的NO被用作肺动脉高压的治疗，并且在包括哮喘和囊性纤维化的许多肺部疾病中已经认识到NO稳态的改变。此外，S-亚硝基谷胱甘肽（GSNO）已在肺灌洗中检测到微摩尔浓度，并在哮喘中降低。一般来说，RSNO已被称为NO生物活性的“载体”，RSNO已被用于细胞培养研究中作为“NO供体”。我们在上一个资助期的研究强调了NO和RSNO通过明显不同的机制影响细胞功能的事实。特别是，我们表征了RSNO可以被转运到细胞中的机制，并表明这些化合物可以通过NO依赖性和NO非依赖性途径影响细胞功能。这导致了该提议的总体假设，即lmwtRSNO的形成代表了NO信号传导的分支。我们将通过以下三个具体目标来解决这个假设。1)我们将进一步表征lmwtRSNO是如何合成和转运到细胞中的，以及谷胱甘肽/NADH/NADPH如何控制RSNO的活性和稳定性。2)3）使用一系列lmwtRSNO和NO浓度来定义细胞内蛋白质的修饰，以揭示蛋白质和途径对S-亚硝化和S-硫醇化的不同敏感性。我们将使用肺细胞特异性细胞培养模型、专门的化学发光检测方法和蛋白质组学技术来实现这些目标。这些研究的成功完成将使NO的细胞效应与RSNO的直接效应分开，从而更全面地了解这些物质如何影响细胞功能。重要的是，这些研究将指出RSNO形成的作用不仅仅是作为NO生物活性的“载体”，而是作为NO信号通路中的一个独特的分支。

DESCRIPTION (provided by applicant): This proposal seeks funds to examine the fundamental cellular behavior of nitric oxide (NO) and S- nitrosothiols (RSNO). Nitric oxide (NO) is an essential component of vascular function in pulmonary and all other tissues. In the lung, NO has particular importance as inhaled NO is used as a therapy for pulmonary hypertension, and alterations in NO homeostasis have been appreciated in many pulmonary diseases including asthma and cystic fibrosis. In addition S-nitrosoglutathione (GSNO) has been detected in micro molar concentration in lung lavage and is decreased in asthma. In general RSNO have been invoked as 'carriers' of NO bioactivity and RSNO have been used in cell culture studies as 'NO-donors'. Our studies in the previous funding period have highlighted the fact that NO and RSNO affect cellular functions by clearly different mechanisms. In particular we characterized the mechanisms by which RSNO can be transported into cells and have shown that these compounds can affect cellular functions via both NO-dependent and NO-independent pathways. This has led to the overall hypothesis of this proposal, that lmwtRSNO formation represents a bifurcation in NO signaling. We will address this hypothesis by the following three Specific Aims. 1) We will further characterize how lmwtRSNO are synthesized and transported into cells and how the glutathione/NADH/NADPH can control the activity and stability of RSNO. 2) Examine the differential sensitivity of selected pathways to modulation by RSNO and NO. 3) Define modifications of intracellular proteins using a range of lmwtRSNO and NO concentrations to uncover the differential sensitivity of proteins and pathways to S-nitrosation and S-thiolation. We will accomplish these aims using lung-cell specific cell culture models, specialized chemiluminescence detection methodologies and proteomic techniques. Successful completion of these studies will disentangle the cellular effects of NO from the direct effects of RSNO to more fully understand how these species affect cellular function. Importantly, these studies will point to a role of RSNO formation not simply as 'carriers' of NO bioactivity but as a distinct bifurcation in NO signaling pathways.