PEROXYNITRITE MEDIATED MECHANISMS IN CEREBRAL ISCHEMIA

这个项目的长期目标是促进我们对 与中风有关的机制。中风是导致大脑 疾病在美国，并涉及血管调节异常， 在缺血和随后的再灌注期间的张力。众所周知， 自由基和活性氧化剂，包括一氧化氮（NO）和超氧化物 (O2-)在缺血/再灌注期间产生。过氧亚硝酸盐（ONOO-） 由NO和O2-反应形成的高活性氧化剂。 我们实验室和其他实验室的初步证据表明 ONOO-可能是血管张力的关键调节剂。拟议 实验中，我们将使用新鲜分离的细胞和大鼠血管 大脑威利斯动脉环。细胞成像和定量 视频显微镜将被用来定义收缩反应的单一 细胞和小动脉到ONOO。离子通道是细胞内 血管张力通过其对静息膜电位的影响， 内皮细胞和血管平滑肌细胞。我们的初步数据 表明ONOO-激活血管内皮细胞中阳离子电流 抑制脑血管平滑肌细胞钙激活钾电流 肌肉细胞膜片钳技术将用于（1）识别 内皮细胞离子通道被ONOO激活， 负责膜去极化，和（2）定义的影响， ONOO-对血管平滑肌细胞K+通道的影响。O2和NO通量 将是不同的，并相互滴定，以确定潜在的 化学基础的电和收缩反应ONOO。我们 将测试通道活动和细胞收缩的调节是否 由ONOO-是硫醇依赖性的，涉及谷胱甘肽。通过汇集 自由基化学、离子通道电生理学和全血管 收缩反应，这些研究有望揭示重要的 关于NO、O2-和ONOO对 膜电位，细胞离子信号和血管张力内 脑血管系统。这些信息将提供新的见解， 调节大脑中的血管稳态，可以帮助识别 减少中风影响的新措施。

The long-term goal of this project is to advance our understanding of the mechanisms that are involved in stroke. Stroke is a leading cause of brain disease in the United States, and involves abnormal regulation of vascular tone during ischemia and subsequent reperfusion. It is known that free radicals and reactive oxidants, including nitric oxide (NO) and superoxide (O2-), are produced during ischemia/reperfusion. Peroxynitrite (ONOO-) is a highly reactive oxidant formed by the reaction of NO and O2-. Preliminary evidence from our own laboratory and from others indicates that ONOO- may be a key modulator of vascular tone. In the proposed experiments, we will use freshly isolated cells and vessels from the rat brain Circle of Willis arteries. Cell-imaging amd quantitative videomicroscopy will be used to define the contraction responses of single cells and small arteries to ONOO. Ion channels are major determinants of vascular tone through their influence on resting membrane potential in endothelial and vascular smooth muscle cells. Our preliminary data indicate that ONOO-activates a cation current in vascular endothelial cells and inhibits calcium-activated K+ current in cerebrovascular smooth muscle cells. The patch-clamp technique will be used to (1) identify the endothelial cell ion channels that are activated by ONOO- and that are responsible for membrane depolarization, and (2) define the effect of ONOO- on K+ channels in vascular smooth muscle cells. Fluxes of O2 and NO will be varied and titrated against each other to define the underlying chemical basis for the electrical and contractile responses to ONOO. We will test whether the modulation of channel activity and cell contraction by ONOO- is thiol-dependent and involves glutathione. By bringing together free radical chemistry, ion channel electrophysiology and whole-vessel contractile responses, these studies are expected to reveal important mechanistic information regarding the effects of NO, O2- and ONOO on membrane potential, cellular-ionic signaling and vascular tone within the cerebral vasculature. This information will provide new insight into the regulation of vascular homeostasis in the brain and could help identify new measures to reduce the impact of stroke.