CONTROL OF MICROCIRCULATORY EXCHANGE FUNCTION

描述（由申请人提供）：炎症过程的特征是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沟通、协调和整合体内组织环境变化的适当生理反应的方式。在体内微循环的复杂细胞环境中，关于通透性相关的信号机制的信息缺乏。了解这些重要的体内相互作用是理解微血管运输的综合调控及其在血管疾病中的功能改变的基础。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。我们提出在体内验证三个主要假设：1)与enos相关的信号级联调节微血管运输。2) eNOS从细胞膜到其他细胞间室的分子运动（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一步。3) eNOS信号机制调控微血管通透性对缺血-再灌注（I-R）的响应。为了验证这些假设，我们将A)确定eNOS -/-小鼠及其野生型eNOS +/+对照体内eNOS活性与高通透性之间是否存在因果关系；B)用纯血管扩张剂（ACh）和非血管扩张剂刺激引起高渗透性（PAF）后，测试eNOS从膜到细胞质的易位；C)确定eNOS +/+和eNOS -/-小鼠微血管通透性反应是否存在差异，以及基线下和I-R后eNOS的亚细胞位置。此外，我们将确定eNOS磷酸化是否在响应ACh、PAF和I-R时发生变化，并评估PKB和MAP激酶在这些过程中的意义。

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.