Purinergic Signaling in Cultured Heart Cells

描述(申请人提供)：本研究的总体目标是阐明腺苷A1和A3受体通路中的基本信号机制，确定A1R、A3R和A2AR在心肌细胞上的相互作用，并描述这些潜在重要相互作用的机制。将使用药理、功能和细胞方法，以及基于转基因鸡心肌细胞和完整小鼠心脏制剂的新型心脏模型。具体地说，这项研究将1)测试A3R信号通过GA12或Ga13激活RhoA，进而刺激磷脂酶D(PLD，很可能是PLD1)并导致心脏保护的假设；2)确定ADP核糖化因子(ARF)的功能及其与RhoA在完整心肌细胞中介导这些A3反应的潜在相互作用；3)测试Rho激酶、PIP2水平和细胞骨架以及RhoA-PLD1直接相互作用在调节A3反应中的作用；4)表征A2AR在调节通过A1和A3受体介导的保护效应中的作用，并确定这种调节发生的机制5)研究A1和A3受体协同作用的机制，6)确定A1R的激活是否增强了A3R途径中的信号，或者协同作用是否源于激活的A3R对A1R信号的促进作用；7)验证了磷脂酰肌醇衍生的二酰甘油(来自A1R偶联磷脂酶C)及其随后激活的PKC在增强A3R-RhoA-PLD信号中起重要作用的假说，以及二甘油启动的正PKC-KATP通道反馈IOOP也是介导两种受体之间协同作用的重要机制。编码各种信号分子的结构性活性和显性负性突变体以及这些分子上的选择性激活剂和抑制物的cDNA将被用来描述完整心肌细胞中的信号级联。磷脂酶CBeta2-、Beta3和Beta2/Beta3缺失小鼠，结合PLD的药理抑制剂和腺苷受体选择性药物，将进一步阐明磷脂酶C在介导A1和A3受体的心脏保护作用中的信号作用。这些研究将为腺苷的心脏作用以及基本信号机制提供新的见解(S)。它们也应该有助于我们理解信号通路和发生心肌保护和缺血预适应的机制。

DESCRIPTION (provided by applicant): The overall objectives of the present study are to elucidate basic signaling mechanisms in the adenosine A1 and A3 receptor pathways, to define the interaction between A1R, A3R and A2AR on the myocyte, and to delineate the mechanisms underlying these potentially important interactions. Pharmacological, functional and cellular approaches as well as novel cardiac models based on transfected chick cardiac cells and intact mouse heart preparations will be used. Specifically, the study will 1) test the hypothesis that the A3R signals via Ga12 or Ga13 to activate RhoA, which in turn stimulates phospholipase D (PLD, likely PLD1) and causes cardioprotection, 2) determine the function of ADP-ribosylation factor (ARF) and its potential interaction with RhoA in mediating these A3 responses in intact cardiac myocytes, 3) test the role of Rho kinase, PIP2 level and cytoskeleton as well as that of a direct RhoA-PLD1 interaction in mediating the A3 responses, 4) characterize the role of A2AR in modulating the protective effects mediated via A1 and A3 receptors and determine the mechanism by which this modulation occurs, 5) investigate the mechanistic basis of the synergistic interaction between the A1 and A3 receptors, 6) determine whether activation of the A1R enhances the signaling in the A3R pathway or whether the synergism arises from a facilitating effect of activated A3R on the A1R signaling, 7) test the hypotheses that the phosphatidylinositol-derived diacylglycerol (from the A1R coupled phospholipase C) and its subsequent activation of PKC play an important role in enhancing the A3R -RhoA-PLD signaling and that a diacylglycerol-initiated positive PKC-KATP channel feedback Ioop is also an important mechanism in mediating the synergism between the two receptors. The cDNAs encoding constitutively active and dominant negative mutants of the various signaling molecules as well as selective activators and inhibitors at these molecules will be used in delineating the signaling cascades in the intact myocyte. Phospholipase CBeta2-, Beta3 and Beta2/Beta3-null mice, in conjunction with pharmacological inhibitors of PLD and adenosine receptor-selective agents, will be used to further delineate the signaling role of phospholipase C in mediating the cardioprotective effect of A1 and A3 receptors. The studies should provide novel insights into the cardiac actions of adenosine as well as the basic signaling mechanism(s). They should also contribute to our understanding of the signaling pathway and the mechanism by which cardioprotection and ischemic preconditioning occur.