NITRIC OXIDE EFFECTS ON PULMONARY ENDOTHELIAL CELLS

一氧化氮对肺内皮细胞的影响

• 批准号: 2232406
• 负责人: A RICHARD WHORTON
• 金额: $ 18.24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2232406
• 关键词: ADP ribosylation; cytokine; electrofocusing; enzyme activity; enzyme inhibitors; glutathione; glyceraldehyde 3 phosphate dehydrogenase; immunoprecipitation; laboratory rabbit; nitric oxide; northern blottings; stoichiometry; thiols; thioredoxin; vascular endothelium

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 cytostatis 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 pK/a'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 lung endothelial cells since these cells are dependent 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. However 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.

肺血管内皮细胞暴露于一氧化氮（NO）， 外源性环境来源和内源性细胞来源。 许多细胞对NO的反应是可能的， 与cGMP升高至细胞抑制和细胞毒性相关的事件。 这些 响应取决于NO的浓度和 exposure. 通常，NO合酶（NOS）的瞬时激活导致 信号转导反应，而诱导后延长NO产生， NOS（炎症）或长期暴露于环境中的NO， 与细胞功能的巨大变化有关， 细胞死亡 NO是一种氧化还原活性分子， 可与蛋白质巯基、酶铁/硫反应并修饰 复合物、蛋白质血红素铁和其他反应基团。 最近的证据 在文献和我们自己的初步数据表明，NO修改 蛋白质巯基，特别是那些具有低pK/a的巯基， 与许多蛋白质的活性位点相关，包括蛋白酶。 在这个项目中，我们将确定的机制，通过不 用甘油醛-3-修饰酶中的活性位点巯基 磷酸脱氢酶（GAPDH）作为原型酶。 GAPDH是 在这方面特别重要，因为这种酶是细胞生长的关键。 由于这些细胞几乎依赖于 专门靠糖酵解生成ATP。 在其他细胞类型中， NO以外的介质对GAPDH的氧化抑制与 与细胞死亡有关，并涉及活性部位巯基的氧化。 最近其他人使用分离的酶制剂的工作已经建立了 GAPDH作为NO修饰的潜在靶点，推测在相同的时间点， 巯基。 然而，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诱导的 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， NO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。