This competing renewal application is an extension of our earlier work on the understanding of the cellular and molecular mechanisms of the regulation of coronary vascular tone (CVT) by adenosine. Considerable progress (27 peer reviewed articles) towards the understanding on how adenosine interacts with the coronary artery to produce relaxation has been made from this laboratory. Our studies suggest that coronary smooth muscle and endothelium contain more than one adenosine receptor (AR) subtype that may have opposing actions. Our earlier studies demonstrated a role for relaxing (A2A) AR in the regulation of CVT. Also, this laboratory has demonstrated earlier that endothelium may modulate the action of adenosine through the release of NO via stimulation of AZAAR. Additionally, our studies from the past support provided indirect evidence indicating a role for A2B (relaxing) and A1/A3 (contracting) AR in the regulation CVT. However, the relative contributions of each AR subtype (A1, A2A, A2B and A3) and their signaling mechanisms in the regulation of CVT are not well understood. Our central hypothesis of the current proposal is that the modulation of CVT by adenosine is determined by a net effect of AR's that mediate relaxation and contraction in response to adenosine. Binding of adenosine to these receptors (including endothelium) may initiate various signaling mechanisms (e.g. NO, PKC, PLC, p38- MAPK) through the activation of different G proteins leading to the activation of distinct effectors to modulate the CVT through changes in Cai ++. The advent of targeted gene deletion allows more precise examination of adenosine receptor-mediated responses than previously possible through agonist/antagonist studies alone. By combining receptor knockout technology with the traditional receptor-ligand approach, the physiological roles of each adenosine receptor can be elucidated despite the unavailability of selective and potent agonists and antagonists (e.g.A2B). Therefore, we propose three specific aims: (I) to further characterize the relaxing and contracting AR's using isolated mouse heart and aorta from AR KO's and their respective WT's; (II) to identify the signaling pathways for the relaxing (NO) and contracting (PKC, PLC, p3-8MAPK) AR's involved in the action of adenosine in isolated mouse coronary artery smooth muscle and endothelial cells from AR KO's and their respective WT's; and finally, (III) to investigate the functional significance of the signaling pathways coupled to relaxing and contracting AR's identified in aim I employing selective signaling blockers using isolated mouse heart and aorta from KO's and their respective WT's. The identification of the heterogeneity of AR's along with their signaling pathways and their functional significance in the regulation of vascular tone by adenosine may lead to the development of novel pharmacological agents for the treatment of vascular disorders.
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