Nitric Oxide and Microvessel Permeability In Vivo

描述（由申请人提供）：一氧化氮（NO）是一种重要的信号分子，参与调节血管内皮的许多生理和病理功能。本提案的目的是研究炎症条件下完整微血管中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。要测试的假设是，Ca 2 +/钙调蛋白（CaM）和热休克蛋白90（hsp 90）发挥拮抗作用，小窝蛋白-1调节eNOS活性和微血管通透性通过竞争蛋白质-蛋白质相互作用在完整的微血管，激动剂刺激的NO的生产是至关重要的增加在炎症条件下的渗透性。将使用大鼠肠系膜中单独灌注的完整微静脉微血管进行拟定研究。对于该特定研究，使用单血管灌注技术优于其他整体动物方法的独特优势在于，它允许将NO对血细胞/内皮相互作用和对脉管系统中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产生将在完整微血管中的单个内皮细胞水平上使用荧光成像以时间和空间分辨率定量测量。Ca 2 +/CaM在eNOS活性调节中的作用将通过测量当激动剂诱导的Ca 2+内流在内皮细胞中Ca 2+进入的电化学驱动力改变后被修饰时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hsp 90在形成完整微血管的内皮细胞中内化或遗传表达。在这个建议中，我们结合我们新开发的分子方法与我们以前建立的技术，以研究在完整的微血管中调节eNOS活性的机制。由于急性炎症时微血管壁沿着渗漏部位形成的不均匀性，将在完整微血管的细胞水平上定量评价内皮细胞[Ca 2 +]i和NO产生之间的空间异质性相关性。拟议的研究将提供新的信息，桥接使用整个动物，器官或血管床的研究和纯化蛋白质或培养中的内皮细胞的研究。

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.