Nitric Oxide and Microvessel Permeability In Vivo

一氧化氮(NO)是一种重要的信号分子，参与了许多生理和病理过程的调节。 血管内皮的病理功能。这项提案的目的是调查 炎症状态下完整微血管内皮型一氧化氮合酶活性的调节机制 条件及其与微血管通透性的直接关系。需要检验的假设是 钙调素(CaM)和热休克蛋白90(HSP90)对小窝蛋白-1具有拮抗作用。 内皮型一氧化氮合酶活性和微血管通透性通过竞争性蛋白质相互作用调节 完整的微血管和激动剂刺激的NO产生是增加通透性的关键 炎症状态。单独灌流的大鼠肠系膜上完整的微血管将用于 执行建议的研究。单支血管灌注技术与其他技术相比的独特优势 这项特殊研究的整体动物方法是，它允许对血细胞/内皮细胞没有影响 相互作用和血管系统血流动力学的变化与其在血管中的作用 微血管通透性的调节。激动剂刺激的NO产量将在 应用时间和空间分辨率研究完整微血管中单个内皮细胞的水平 荧光成像。钙/钙调素在eNOS活性调节中的作用将通过 测定激动剂诱导的钙内流改变时NO产生的相应变化 内皮细胞内钙离子内流的电化学驱动力的变化。我们新开发的方法 还允许eNOS的靶向调控蛋白，如小窝蛋白-1或HSP90内化或 基因表达于内皮细胞|S，形成完整的微血管。在这项提案中，我们结合了我们的 新开发的分子方法和我们以前建立的技术来研究 调节完整微血管中eNOS活性的机制。因为漏水的不均匀性质 急性炎症期间沿微血管壁部位的形成与空间异质性的相关性 内皮细胞[Ca~(2+)]i和NO产生之间的关系将在细胞水平上进行定量评估 微血管。拟议的研究将提供新的信息，利用整体 动物、器官或血管床以及培养中纯化蛋白质或内皮细胞的研究。

Nitric oxide (NO) is an important signaling molecule involved in the regulation of many physiological and pathological functions of the vascular endothelium. The objectives of this proposal are to investigate the mechanisms regulating endothelial NO synthase (eNOS) activity in intact microvessels under inflammatory conditions and their direct relationship with microvessel permeability. The hypothesis to be tested is that Ca2+/calmodulin (CaM) and heat shock protein 90 (hsp90) play antagonistic roles to caveolin-1 in the regulation of eNOS activity and microvessel permeability through competing protein-protein interactions in intact microvessels, and agonist-stimulated NO production is critical for increases in permeability under inflammatory conditions. Individually perfused intact venular microvessels in rat mesentery will be used to perform the proposed studies. The unique advantage of using single vessel perfusion technique over other whole animal approaches for this specific study is that it allows the NO effect on blood cell/endothelium interactions and on the changes in hemodynamics in the vasculature to be distinguished from its role in the regulation of microvessel permeability. Agonist-stimulated NO production will be quantitatively measured at levels of individual endothelial cells in intact microvessels with temporal and spatial resolution using fluorescence imaging. The role of Ca2+/CaM in the regulation of eNOS activity will be investigated by measuring corresponding changes in NO production when agonist-induced Ca2+ influx is modified following changes in electrochemical driving force for Ca2+ entry in endothelial cells. Our newly developed methods also allow a targeted regulatory protein for eNOS such as caveolin-1 or hsp90 to be internalized or genetically expressed in endothelial cel|s that form intact microvessels. In this proposal we combined our newly developed molecular approaches with our previously established techniques to investigate the mechanisms regulating eNOS activity in intact microvessels. Because of the nonuniformity nature of leaky site formation along the microvessel walls during acute inflammation, the spatial heterogeneity correlation between endothelial [Ca2+]i and NO production will be quantitatively evaluated at cellular levels in intact microvessels. The proposed research will provide new information that bridges the studies using whole animal, organ, or vascular beds and studies with purified proteins or endothelial cells in culture.