Mechanism of Coronary Endothelium-mediated Vasodilation

描述（来自应用程序的逐字记录）：我们实验室的最新研究 和其他人已经表明，环腺苷二磷酸核糖（cADPR） 在冠状动脉平滑肌细胞中产生和水解， 这种核苷酸作为第二信使刺激Ca 24从 肌浆网（SR）。本提案将审查以下假设： 内源性cADPR诱导的Ca 2+释放有助于[Ca 2 +]的控制， 血管平滑肌细胞从小冠状动脉和NO减少 细胞内cADPR的产生，从而降低[Ca 2 +]， 导致血管舒张。我们将首先研究cADPR在 利用SR Ca 2+调节Ryanodine受体/Ca 2+释放通道活性 通道重建和脂质双层钳技术。那就 进一步确定cADPR介导的兰尼碱受体激活的作用， 在冠状动脉平滑肌细胞内[Ca 2 +]的控制中 以及该信号通路对NO抑制作用的贡献 [Ca ~（2+）]i的影响。自从cADPR 据报道，通过Ca 2+诱导的Ca 2+释放（CICR），我们将 解决cADPR介导的NO效应是否与抑制有关 或兰尼碱受体的信号我们还将研究NO对 通过反相HPLC分析cADPR的产生或代谢 ADP-核糖环化酶和cADPR水解酶活性及其作用机制探讨 NO通过这种途径调节冠状动脉平滑肌中这些酶的活性， 肌肉. cGMP和亚硝基化介导的二聚化的参与 ADP-核糖基环化酶在NO作用下的作用将被确定。使用 视频显微镜分离加压小冠状动脉，我们将 确定cADPR，CICR和ryanodine受体在发展中的作用， 冠状动脉张力和介导血管扩张反应NO。最后，我们 将探索cADPR结合或激活ryanodine的精确机制 受体并使用放射性配体结合从SR释放Ca 2+，或 ADP核糖基化测定。这些研究将定义一种新的信号机制 调节冠状动脉阻力动脉[Ca 2 +]i和血管反应， 增加我们对调节血管扩张剂的细胞机制的理解 效果不。

DESCRIPTION (Verbatim from the application): Recent studies from our laboratory and by others have indicated that cyclic adenosine diphosphate-ribose (cADPR) is produced and hydrolyzed in coronary arterial smooth muscle cells, and that this nucleotide serves as a second messenger to stimulate Ca24 release from the sarcoplasmic reticulum (SR). This proposal will examine the hypothesis that endogenous cADPR-induced Ca2+ release contributes to the control of [Ca2+], in vascular smooth muscle cells from small coronary arteries and that NO decreases intracellular cADPR production and consequently lowers [Ca2+], thereby resulting in vasodilation. We will first examine the role of cADPR in the regulation of ryanodine receptor/Ca2+ release channel activity using SR Ca2+ channel reconstitution and lipid bilayer clamp techniques. Then, we will further determine the role of cADPR-mediated activation of ryanodine receptors in the control of intracellular [Ca2+] in coronary arterial smooth muscle cells and the contribution of this signaling pathway to the inhibitory effect of NO on [Ca2+]i using single cell fluorescence microscopic spectrometry. Since cADPR has been reported to act through Ca2+-induced Ca2+ release (CICR), we will address whether the cADPR-mediated effects of NO are associated with inhibition of CICR or ryanodine receptors. We will also examine the effects of NO on the production or metabolism of cADPR by reverse phase-HPLC analysis of ADP-ribosylcyclase and cADPR hydrolase activities and to explore the mechanisms by which NO modulates these enzyme activities in coronary arterial smooth muscle. The involvement of cGMP and nitrosylation-mediated dimerization of ADP-ribosylcyclase in the effect of NO will be determined. Using videomicroscopy of isolated pressurized small coronary arteries, we will determine the role of cADPR, CICR and ryanodine receptors in the development of coronary tension and in mediating the vasodilator response to NO. Finally, we will explore the precise mechanisms by which cADPR binds or activates ryanodine receptors and produces Ca2+ release from the SR using radioligand binding or ADP ribosylation assays. These studies will define a new signaling mechanism regulating [Ca2+]i and vasomotor response in coronary resistance arteries and increase our understanding of the cellular mechanism mediating the vasodilator effect of NO.