Mechanism of Metabolism of S-Nitrosothiols

描述(申请人提供)：这项建议寻求资金，以研究一氧化氮(NO)和S-亚硝硫醇(RSNO)的基本细胞行为。一氧化氮(NO)是肺和所有其他组织血管功能的重要组成部分。在肺中，NO具有特别重要的作用，因为吸入NO被用来治疗肺动脉高压，而且许多肺部疾病，包括哮喘和囊性纤维化，都认识到NO稳态的变化。此外，在肺灌洗液中也检测到微量浓度的S-亚硝基谷胱甘肽(GSNO)，在哮喘患者中呈下降趋势。一般来说，RSNO被称为NO生物活性的‘载体’，RSNO被用作细胞培养研究中的‘NO供体’。我们在前一个资助时期的研究已经强调了这样一个事实，即NO和RSNO通过明显不同的机制影响细胞功能。特别是，我们描述了RSNO转运到细胞内的机制，并表明这些化合物可以通过NO依赖和NO不依赖的途径影响细胞功能。这导致了这一提议的总体假设，即lmwtRSNO的形成代表了NO信号的分叉。我们将通过以下三个具体目标来解决这一假设。1)我们将进一步研究lmwtRSNO是如何合成并转运到细胞内的，以及谷胱甘肽/NADH/NADPH如何控制RSNO的活性和稳定性。2)检测所选途径对RSNO和NO调节的差异敏感性。3)使用1mwtRSNO和NO浓度范围来定义细胞内蛋白质的修饰，以揭示蛋白质和通路对S亚硝化和S硫基化的差异敏感性。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把NO对细胞的影响与RSNO的直接影响分开，以便更充分地了解这些物种如何影响细胞功能。重要的是，这些研究将指出RSNO的形成不仅仅是作为没有生物活性的‘载体’，而是作为NO信号通路的一个明显的分叉。

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.