Purinergic Signaling in Cultured Heart Cells

描述（由申请方提供）：本研究的总体目的是阐明腺苷A1和A3受体途径中的基本信号传导机制，确定肌细胞上A1 R、A3 R和A2 AR之间的相互作用，并描述这些潜在重要相互作用的机制。将使用药理学、功能和细胞方法以及基于转染的鸡心脏细胞和完整小鼠心脏制备物的新型心脏模型。具体而言，该研究将1）测试以下假设：A3 R通过Ga 12或Ga 13发出信号以激活RhoA，这反过来刺激磷脂酶D（PLD，可能是PLD 1）并引起心脏保护，2）确定ADP-核糖基化因子（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激酶的作用，PIP 2水平和细胞骨架以及RhoA-PLD 1直接相互作用在介导A3反应中的作用，4）表征A2 AR在调节通过A1和A3受体介导的保护作用中的作用，并确定这种调节发生的机制，5）研究A1和A3受体之间协同相互作用的机制基础，6）确定A1 R的活化是否增强A3 R途径中的信号传导，或者协同作用是否由活化的A3 R对A1 R信号传导的促进作用引起，7）检验磷脂酰肌醇衍生的二酰甘油（来自A1 R偶联磷脂酶C）及其随后的PKC激活在增强A3 R-RhoA-PLD信号转导中起重要作用，并且二酰基甘油引发的阳性PKC-KATP通道反馈环也是介导两种受体协同作用的重要机制。编码各种信号分子的组成型活性和显性失活突变体以及这些分子的选择性激活剂和抑制剂的cDNA将用于描绘完整肌细胞中的信号级联。磷脂酶C β 2、β 3和β 2/β 3缺失小鼠，与PLD的药理学抑制剂和腺苷受体选择性药物联合，将用于进一步描述磷脂酶C在介导A1和A3受体的心脏保护作用中的信号传导作用。这些研究将为腺苷的心脏作用及其基本信号传导机制提供新的见解。它们也将有助于我们理解心肌保护和缺血预适应发生的信号通路和机制。

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.