NITRIC OXIDE AND PULMONARY ARTERY ENDOTHELIAL CELL FUNCTION

肺血管内皮细胞暴露于一氧化氮(NO)。 来自外源环境来源和来自内源细胞 消息来源。大量细胞对NO的反应。可能的范围从 与cGMP升高到细胞停滞相关的信号事件和 细胞毒性。这些反应取决于NO的浓度。和 暴露的时间长短。一般来说，NO的一过性激活。合成酶 (NO)导致信号反应，而延长NO。生产 在诱导性一氧化氮合酶(炎症)或长期接触一氧化氮之后。在……里面 环境与细胞的戏剧性变化有关 功能和潜在的细胞死亡。不是的。是一种氧化还原活性分子， 在有氧条件下，可以与蛋白质巯基反应并修饰， 酶铁/硫络合物、蛋白质、血红素铁等活性物质 组。文献中的最新证据和我们自己的初步数据 提示NO可以修饰蛋白质的巯基，特别是那些低分子的 PKA与许多蛋白质的活性部位有关，包括 脱氢酶。在这个项目中，我们将通过以下方式确定机制 当然不是。脱氢酶中活性中心的硫基修饰 以甘油醛-3-磷酸脱氢酶为原型 酵素。GAPDH在这方面特别重要，因为 酶是内皮细胞中细胞存活的关键，因为这些细胞 几乎完全依赖于糖酵解来产生ATP。在其他 细胞类型，NO以外的介质对GAPDH的氧化抑制。是 与细胞死亡有关，并涉及活性部位的氧化 巯基。最近，其他人使用分离的酶制剂所做的工作 将GAPDH确定为NO的潜在目标。修改可能是在 同样的巯基。修改的确切性质或是否 在完整的细胞中是否会发生修饰尚不清楚。此外，这个角色 细胞抗氧化系统，如谷胱甘肽氧化还原循环， 谷氧还蛋白和硫氧还蛋白对NO诱导程度的调节 受伤情况尚不清楚。我们的假设是否定的。抑制谷氨酸脱氢酶的活性 通过与分离酶相似的机制形成完整的细胞 而且抑制的程度是由氧化还原调节的 单元的状态。我们的方法将是确定通过哪些机制 不是的。抑制GAPDH，研究抑制的可逆性和 研究细胞氧化还原机制在调节这些方面的作用 事件。实验将使用分离的酶系统和 完整的肺动脉内皮细胞。

Pulmonary vascular endothelial cells are exposed to nitric oxide (NO.) from both exogenous environmental sources and from endogenous cellular sources. Numerous cellular responses to NO. are possible ranging from signalling events related to elevation of cGMP to cytostasis and cytotoxicity. These responses depend on both the concentration of NO. and the length of exposure. In general, transient activation of NO. synthase (NOS) leads to signalling responses while prolonged NO. production following induction of NOS (inflammation) or prolonged exposure to NO. in the environment is associated with dramatic alterations in cellular function and potentially cell death. NO. is a redox active molecule which under aerobic conditions can react with and modify protein sulfhydryls, enzyme iron/sulfur complexes, protein heme iron, and other reactive groups. Recent evidence in the literature and our own preliminary data suggests that NO modifies protein sulfhydryls, particularly those with low pKa's which are associated with the active site of many proteins including dehydrogenases. In this project, we will determine the mechanisms through which NO. modifies active site sulfhydryls in dehydrogenases with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the prototypical enzyme. GAPDH is particularly important in this regard because this enzyme is key for cell viability in endothelial cells since these cells are depend almost exclusively on glycolysis for ATP generation. In other cells types, oxidative inhibition of GAPDH by mediators other than NO. is associated with cell death and involves oxidation of the active site sulfhydryl. Recent work by others using isolated enzyme preparations has established GAPDH as a potential target for NO. modification presumably at the same sulfhydryl. The exact nature of the modification or whether modifications can occur in intact cells is not known. Moreover, the role of cellular antioxidant systems such as the glutathione redox cycle, glutaredoxin, and thioredoxin, in regulating the extent of NO.-induce injury is not known. Our hypothesis is that NO. inhibits GAPDH activity in intact cells via mechanisms similar to those that occur in isolated enzyme preparations and that the extent of inhibition is regulated by the redox status of the cell. Our approach will be to determine mechanisms by which NO. inhibits GAPDH, investigate the reversibility of inhibition and investigate the role of cellular redox mechanisms in regulating these events. Experiments will be carried out using isolated enzyme systems and intact pulmonary artery endothelial cells.