NITRIC OXIDE AND PULMONARY ARTERY ENDOTHELIAL CELL FUNCTION

肺血管内皮细胞暴露于一氧化氮（NO.） 从外源性环境来源和内源性细胞来源 源许多细胞对NO的反应是可能的， 与cGMP升高至细胞停滞相关的信号传导事件， 细胞毒这些反应取决于NO的浓度， 暴露的时间长度。一般来说，NO合酶的瞬时激活 (NOS)导致信号反应，而延长NO。 在诱导NOS（炎症）或长期暴露于NO后， 这种环境与细胞的急剧变化有关， 功能和潜在的细胞死亡。NO是氧化还原活性分子， 在有氧条件下可以与蛋白质巯基反应并修饰蛋白质巯基， 酶铁/硫复合物，蛋白质血红素铁，和其他反应 组文献中的最新证据和我们自己的初步数据 表明NO修饰蛋白质巯基，特别是那些低巯基的蛋白质。 与许多蛋白质的活性位点相关的pKa，包括 - 是的在本项目中，我们将通过以下方式确定机制： 其中NO.修饰酶中的活性位点巯基， 甘油醛-3-磷酸脱氢酶（GAPDH）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活力的关键，因为这些细胞 几乎完全依赖于糖酵解产生ATP。换句 细胞类型，通过NO以外的介质氧化抑制GAPDH， 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的工作， 确定GAPDH为NO修饰的潜在靶点，推测为 相同的巯基。修改的确切性质或是否 在完整细胞中可能发生的修饰是未知的。此外，作用 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在调节NO. -诱导 伤害是未知的。我们的假设是NO抑制GAPDH活性， 通过类似于在分离酶中发生的机制， 制备和抑制的程度是由氧化还原调节 细胞的状态。我们的方法将是确定机制， 号抑制GAPDH，研究抑制的可逆性， 研究细胞氧化还原机制在调节这些过程中的作用， 事件实验将使用分离的酶系统进行， 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.