Purinergic Signaling in Cultured Heart Cells

培养心脏细胞中的嘌呤能信号传导

基本信息

批准号：
6679825
负责人：
BRUCE T LIANG
金额：
$ 36.25万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-03 至 2006-08-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：本研究的总体目标是阐明腺苷A1和A3受体途径中的基本信号传导机制，以定义肌细胞上A1R，A3R和A2AR之间的相互作用，并描述这些潜在重要相互作用的机制。将使用基于转染的鸡心细胞和完整小鼠心脏制剂的药理，功能和细胞方法以及新型心脏模型。具体而言，该研究将1）检验以下假设：A3R通过GA12或GA13信号激活RhoA，从而刺激磷脂酶D（PLD，可能是PLD1），并导致心脏保护并导致心脏保护性，2）确定ADP-孔基化因子（ARF）的功能及其在冥想中的潜在相互作用，该验证的作用3响应了rho的作用。 Rho激酶，PIP2水平和细胞骨架以及直接RhoA-PLD1在介导A3响应中相互作用的直接相互作用，4）特征是A2AR在调节通过A1和A3受体介导的保护效应中的作用，并确定该调节的机械基础，5）研究的机械基础，5）确定了A1和A1是否在A1中，A1和A1 A1和A1 A1和A1 A1和A1 A1和A1 A1和A1均表示，并且A1是A1和A1。 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信号传导和二酰基甘油发射的阳性PKC-KATP通道反馈IOOP也是介导两个受体之间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负突变体以及这些分子的选择性激活剂和抑制剂的cDNA将用于描述完整的肌细胞中的信号级联反应。磷脂酶CBETA2-，β3和β2/beta3-null小鼠与PLD和腺苷受体选择性剂的药理抑制剂结合使用，将用于进一步描述磷脂酶C在介导A1和A3受体的磷脂型介导性心脏保护作用中的信号传导作用。这些研究应提供对腺苷的心脏作用以及基本信号机制的新见解。他们还应该有助于我们对信号通路的理解以及心脏保护和缺血性预处理的机制。