Nitric Oxide and Microvessel Permeability In Vivo

说明书(申请人提供)：一氧化氮(NO)是一种重要的信号分子，参与调节血管内皮细胞的许多生理和病理功能。本研究旨在探讨炎症状态下完整微血管内皮型一氧化氮合酶(ENOS)活性的调节机制及其与微血管通透性的直接关系。需要检验的假设是，钙/钙调蛋白(CaM)和热休克蛋白90(HSP90)通过在完整的微血管中竞争蛋白-蛋白质相互作用而拮抗小窝蛋白-1调节eNOS活性和微血管通透性的作用，而激动剂刺激的NO产生是炎症条件下通透性增加的关键。我们将使用单独灌流的大鼠肠系膜完整的微血管来进行拟议的研究。在这项特定的研究中，使用单血管灌流技术相对于其他全动物方法的独特优势在于，它允许将对血细胞/内皮细胞相互作用和对血管系统血流动力学变化的影响与其在微血管通透性调节中的作用区分开来。激动剂刺激的NO的产生将在完整微血管中单个内皮细胞的水平上以时间和空间分辨率使用荧光成像进行定量测量。钙离子/钙调素在调节eNOS活性中的作用将通过检测激动剂诱导的钙离子内流在改变内皮细胞内钙离子进入的电化学驱动力改变时NO产生的相应变化来研究。我们新开发的方法还允许eNOS的靶向调节蛋白，如小窝蛋白-1或HSP90，内化或在形成完整微血管的内皮细胞中遗传表达。在这项建议中，我们结合了我们新开发的分子方法和我们以前建立的技术来研究完整微血管中eNOS活性的调节机制。由于急性炎症时微血管壁上渗漏部位形成的非均质性，在完整微血管中内皮细胞[Ca~(2+)]i和NO产生的空间异质性将在细胞水平上被定量评估。这项拟议的研究将提供新的信息，将使用整个动物、器官或血管床的研究与使用培养中的纯化蛋白或内皮细胞的研究联系起来。

DESCRIPTION (provided by applicant): 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 cells 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.