PEROXYNITRITE MEDIATED MECHANISMS IN CEREBRAL ISCHEMIA

这个项目的长期目标是增进我们对 与中风有关的机制。中风是导致脑部疾病的主要原因 美国的疾病，涉及血管的异常调节 在缺血和随后的再灌流期间的音调。众所周知，免费 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/再灌流过程中产生。过亚硝酸根(ONOO-)是 一种高活性的氧化剂，由NO和O2-反应形成。 来自我们自己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因子。在建议的 实验中，我们将使用新分离的大鼠细胞和血管 Willis动脉的脑环。细胞成像和定量 将使用视频显微镜来定义单个细胞的收缩反应 细胞和小动脉通向ONOO。离子通道是主要的决定因素 血管紧张性及其对静息膜电位的影响 血管内皮细胞和血管平滑肌细胞。我们的初步数据 提示ONOO-激活血管内皮细胞的阳离子电流 细胞和钙激活钾电流在脑血管平滑肌中的作用 肌肉细胞。膜片钳技术将被用来(1)识别 由ONOO激活的内皮细胞离子通道-以及 负责膜去极化，以及(2)定义 血管平滑肌细胞的ONOO-On K+通道。O2和NO的通量 将相互变化和滴定，以定义潜在的 对ONOO的电和收缩反应的化学基础。我们 将测试通道活动和细胞收缩的调制是否 由ONOO-是硫醇依赖的，涉及谷胱甘肽。通过汇聚在一起 自由基化学、离子通道电生理学与全血管 收缩反应，这些研究有望揭示重要的 关于NO、O2-和ONOO影响的机制信息 膜电位、细胞-离子信号和血管紧张性 脑血管系统。这些信息将为我们提供对 调节大脑中的血管动态平衡，有助于识别 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.