Project 5: ROS, Glutathione and Vascular Response to Diesel Exhaust

暴露于与交通有关的空气污染，如柴油机废气（DE），与 多种细胞类型中活性氧（ROS）的水平。这些活性氧包括超氧阴离子 自由基、过氧化氢、脂质过氧化物和羟基自由基。ROS可对细胞具有有害作用， 但也已知它们参与正常的跨膜信号传导和代谢控制， 心血管系统例如，两种有效的血管收缩剂，血管紧张素II和内皮素-1， 增加平滑肌细胞NADPH氧化酶产生的ROS，从而导致 超氧化物和过氧化氢的细胞内浓度。超氧化物可与一氧化氮（NO）结合 形成过氧亚硝酸盐，其可引起细胞损伤，但也限制可用于血管舒张的NO的量。 以这种方式产生的ROS也可以氧化四氢生物蝶呤，这是NO的必要辅因子 合成酶（NOS），导致解偶联的NOS和生产更多的超氧化物和过氧亚硝酸盐。 以这种方式通过超氧化物消耗NO可导致血管收缩。因此，ROS能够影响 心血管性能和血管反应性。谷胱甘肽（GSH）是一种含量丰富的非蛋白巯基 它是活性氧的有效清除剂，包括过氧化氢，脂质过氧化物，重要的是， 过氧化氢（ONOO-）。GSH是一种三肽硫醇，在大多数细胞中以毫摩尔浓度存在。第一和 谷胱甘肽合成中的限速步骤是由谷氨酸半胱氨酸连接酶（GCL）进行的， 由两个亚基组成，催化亚基（GCLC）和修饰或调节亚基（GCLM）。 重要的是，GCLC和GCLM中的单核苷酸多态性（SNP）已被证明是 在心肌梗塞中以及控制人类血管反应性中是重要的。在这个项目中，我们将 通过比较的方法研究GCL这种效果的原因。我们将1）使用鼠标 GCL表达和活性的差异模型，以研究其在DE诱导的血管变化中的作用。 反应性2），使用来自这些小鼠和人类的培养内皮细胞来研究潜在的 负责GSH和DE诱导的内皮NO产生调节的生化事件;和3） 表征在对DE的血管反应中具有变异性的遗传定义的近交系小鼠， 并确定单核苷酸多态性（SNP）和数量性状基因座，与这些相关， 老鼠的变化使用这些工具，我们将确定GSH合成在DE诱导的变化中的作用， 血管反应性，研究潜在的病理生理机制，并绘制和识别遗传 DE诱导的血管反应性变化的决定因素。这些候选基因将被称为 项目1和2，将进一步评价它们作为DE诱导的血管病变中的潜在遗传因素 人类的异常

Exposure to traffic-related air pollution such as diesel exhaust (DE) is associated with an increase in the level of reactive oxygen species (ROS) in multiple cell types. These ROS include the superoxide anion radical, hydrogen peroxide, lipid peroxides and hydroxyl radical. ROS can have deleterious effects on cells, but they are also known to be involved in normal transmembrane signaling and metabolic control in the cardiovascular system. For instance, two potent vasoconstrictors, angiotensin II and endothelin-1, can increase production of ROS by smooth muscle cell NADPH oxidase, thus causing an increase in the intracellular concentrations of superoxide and hydrogen peroxide. Superoxide can bind to nitric oxide (NO) to form peroxynitrite, which can cause cellular injury, but also limit the amount of NO available for vasorelaxation. ROS generated in this way can also oxidize tetrahydrobiopterin, a necessary cofactor for NO synthase (NOS), resulting in uncoupling of NOS and production of more superoxide and peroxynitrite. Consumption of NO by superoxide in this way can result in vasoconstriction. ROS are thus able to influence cardiovascular performance and vascular reactivity. Glutathione (GSH) is an abundant non-protein thiol which is a potent scavenger of ROS, including hydrogen peroxide, lipid peroxides, and importantly, peroxitrite (ONOO-). GSH is a tripeptide thiol present in millimolar concentrations in most cells. The first and rate-limiting step in GSH synthesis is carried out by the enzyme glutamate cysteine ligase (GCL), which is composed of two subunits, a catalytic subunit (GCLC) and a modifier or regulatory subunit (GCLM). Importantly, single nucleotide polymorphisms (SNPs) in both GCLC and GCLM have been shown to be important in myocardial infarction, as well as controlling vascular reactivity in humans. In this project, we will investigate the reasons for this effect of GCL by using a comparative approach. We will 1) use mouse models of differential GCL expression and activity to investigate its role in DE-induced changes in vascular reactivity 2), use cultured endothelial cells from these mice and from humans to investigate the underlying biochemical events responsible for GSH and DE-induced modulation of endothelial NO production; and 3) characterize genetically defined inbred strains of mice that have variability in their vascular responses to DE, and identify single nucleotide polymorphisms (SNPs) and quantitative trait loci that are associated with these changes in mice. Using these tools we will define the role of GSH synthesis in DE-induced changes in vascular reactivity, investigate the underlying pathophysiological mechanisms, and map and identify genetic determinants of DE-induced changes in vascular reactivity. These candidate genes will then be referred to Projects 1 and 2 where they will be further evaluated as potential genetic factors in DE-induced vascular abnormalities in humans.