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.
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