Mechanism of Coronary Endothelium-mediated Vasodilation

描述(来自申请表的逐字)：我们实验室的最新研究 另有研究表明，环腺苷二磷酸核糖(CADPR) 在冠状动脉平滑肌细胞中产生和降解，并且 这种核苷酸作为第二信使刺激CA24从细胞中释放 肌浆网(SR)。这项提议将检验这样一个假设 内源性cADPR诱导的Ca~(2+)释放参与[Ca~(2+)]_i的控制 小冠状动脉来源的血管平滑肌细胞和NO的减少 细胞内cADPR的产生，从而降低[Ca2+]，从而 导致血管扩张。我们将首先研究cadpr在 肌质网钙离子对兰尼定受体/钙释放通道活性的调节 通道重建和脂质双层钳制技术。那么，我们会 进一步确定cADPR介导的ryanodine受体激活的作用 冠状动脉平滑肌细胞内钙离子的调控 以及该信号通路在NO抑制效应中的作用 用单细胞荧光显微光谱法测定细胞内[Ca~(2+)]i。由于cadpr 已经被报道通过钙离子诱导的钙释放(CICR)起作用，我们将 探讨cADPR介导的NO效应是否与抑制有关 CICR或Ryanodine受体。我们还将研究一氧化氮对人体健康的影响 反相高效液相色谱法分析cADPR的生产或代谢 ADP-核糖环化酶和cADPR水解酶活性及其机制探讨 NO通过其调节冠脉平滑肌中这些酶的活性 肌肉。CGMP和亚硝化介导的血管内皮细胞二聚 将测定ADP-核糖环化酶对NO的影响。vbl.使用 分离的加压小冠状动脉的视频显微镜检查，我们将 确定cADPR、CICR和Ryanodine受体在卵巢癌发生发展中的作用 冠脉张力和介导血管扩张剂对NO的反应。最后，我们 将探索cADPR结合或激活ryanodine的精确机制 受体，并通过放射性配体结合或从SR产生钙释放 ADP核糖化检测。这些研究将定义一种新的信号机制 调节冠脉阻力血管的[Ca~(2+)]i和血管舒缩反应 加深对介导血管扩张剂的细胞机制的了解 NO的影响。

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.