CONTROL OF MICROCIRCULATORY EXCHANGE FUNCTION

描述（由申请方提供）：炎症过程的特征为微血管对大分子的渗透性（高渗透性）增加。 血管壁和血细胞之间的信号相互作用提供了一种独特的方式来沟通，协调和整合体内组织环境变化的适当生理反应。 在体内微循环的复杂细胞环境中，有关渗透性相关信号传导机制的信息很少。 这些重要的相互作用在体内的知识是了解微血管运输的综合调节和血管疾病的功能改变的基础。 特别是，一氧化氮（NO）在微血管通透性控制中的作用仍然存在争议。 我们提出在体内测试三个主要假设：1）eNOS相关的信号级联调节微血管转运。 2)eNOS从膜到其它细胞区室的分子运动（易位、运输）是内皮介导的调节机制中功能上和差异上重要的步骤。 3)eNOS信号传导机制调节微血管对缺血-再灌注（I-R）的通透性反应。 为了检验这些假设，我们将：A）确定在eNOS -/-小鼠和它们的野生型eNOS +/+对照中体内eNOS活性和高通透性之间是否存在因果关系; B）测试在用纯血管扩张剂（ACh）和引起高通透性的非血管扩张剂（PAF）刺激后eNOS从膜到胞质溶胶的易位; C）确定在eNOS +/+和eNOS -/-小鼠中是否存在微血管渗透性反应的差异以及在基线下和I-R后eNOS的亚细胞定位。 此外，我们将确定是否eNOS磷酸化的变化发生在响应ACh，PAF和I-R，并评估PKB和MAP激酶在这些过程中的意义。 免疫沉淀，蛋白质印迹，活体和共聚焦显微镜，以及NO测量和计算机辅助数字图像分析的方法将被应用于评估在体内微血管通透性的调节。

DESCRIPTION (provided by applicant): Inflammatory processes are characterized by an increase in microvascular permeability (hypermeability) to macromolecules. Signaling interactions between vascular wall and blood cells provide a unique way of communicating, coordinating, and integrating an appropriate physiologic response to the changing tissue environment in vivo. There is a paucity of information on permeability-related signaling mechanisms in the complex cellular environment of the in vivo microcirculation. Knowledge of these important interactions in vivo is fundamental to understand the integrated regulation of microvascular transport and its functional alterations in vascular disease. In particular, the role of nitric oxide (NO) in the control of microvascular permeability remains controversial. We propose to test in vivo three major hypotheses: 1) an eNOS-assoctated signaling cascade regulates microvascular transport. 2) molecular movement (translocation, trafficking) of eNOS from membrane to other cellular compartments is a functionally and differentially important step in the endothelium-mediated regulatory mechanisms. 3) eNOS signaling mechanisms regulate microvascular permeability responses to ischemia-reperfusion (I-R). To test these hypotheses, we will A) determine whether or not a cause-effect relationship exists between eNOS activity and hyperpermeability in vivo in eNOS -/- mice and their wild-type eNOS +/+ control; B) test translocation of eNOS from membrane to cytosol after stimulation with a pure vasodilator (ACh) and a non-vasodilator that causes hyperpermeability (PAF); C) determine whether or not differences in microvascular permeability responses exist in eNOS +/+ and eNOS -/- mice and the subcellular location of eNOS under baseline and after I-R. In addition, we will determine whether or not changes in eNOS phosphorylation occur in response ACh, PAF and to I-R, and assess the significance of PKB and MAP kinases in these processes. Methods of immunoprecipitation, western blotting, intravital and confocal microscopy, as well as NO measurements and computer-assisted digital image analysis will be applied to evaluate the regulation of microvascular permeability in vivo.