Nitric Oxide and Microvessel Permeability In Vivo

一氧化氮（NO）是一种重要的信号分子，参与调节多种生理和 血管内皮的病理功能。本提案的目的是调查 炎症条件下完整微血管内皮型一氧化氮合酶（eNOS）活性的调节机制 条件及其与微血管通透性的直接关系。有待检验的假设是， Ca ~（2+）/钙调素（CaM）和热休克蛋白90（hsp 90）在细胞膜上对小窝蛋白1有拮抗作用。 通过竞争性蛋白-蛋白相互作用调节eNOS活性和微血管通透性 完整的微血管，激动剂刺激的NO产生是增加渗透性的关键， 炎性条件。将使用大鼠肠系膜中单独灌注的完整微静脉微血管， 进行拟议的研究。单血管灌注技术与其他血管灌注技术相比具有独特的优势， 用于该特定研究的全动物方法的另一个优点是它允许NO对血细胞/内皮细胞的作用 相互作用和对血管系统中血液动力学变化的影响，以区别于其在 微血管通透性的调节。激动剂刺激的NO产生将在以下时间定量测量： 完整微血管中单个内皮细胞水平的时间和空间分辨率， 荧光成像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+内流被改变时NO产生的相应变化， 内皮细胞中Ca 2+进入的电化学驱动力的变化。我们新开发的方法 还允许eNOS的靶向调节蛋白如小窝蛋白-1或hsp 90被内化，或 在内皮细胞中基因表达|形成完整的微血管。在本提案中，我们结合了 新开发的分子方法与我们以前建立的技术，以调查 完整微血管中eNOS活性的调节机制。由于泄漏的不均匀性， 急性炎症时微血管壁沿着部位形成，空间异质性相关 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价， 微血管拟议的研究将提供新的信息，桥梁的研究使用整体 动物、器官或血管床，以及用纯化蛋白质或培养的内皮细胞进行的研究。

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.