Adenosine receptor is known to relax coronary artery. Pharmacologic characterization of the coronary adenosine receptor follows the physiological evidence in support of the role of adenosine in regulating coronary blood flow. The molecular mechanism and complete characterization of the intracellular messenger system(s) activated by the adenosine agonist-receptor interaction have not been fully established. Apart from involvement of various second messengers, the activation of adenosine receptor(s) causes release of coronary endothelium-derived factors (EDRF, EDHF, NO) which can hyperpolarize the coronary artery, possibly through the coupling to G-proteins. The overall objective of this proposal is to investigate the role of K+ conductance and membrane potential in porcine coronary artery smooth muscle and endothelium in relation to its well-known effect through adenosine receptor(s). The applicant proposes to investigate: 1) adenosine receptor-induced modulation of membrane potential in porcine coronary artery with and without endothelium and establish the order of potency of various adenosine agonists to define the receptor-subtype; 2) a correlation in the changes in membrane potential, cytosolic calcium ([Ca2+]), and vascular tone induced by adenosine agonists through simultaneous measurement of these parameters and the involvement of second messengers (cAMP, cGMP) in signaling of adenosine receptor in coronary artery; 3) replicate the Specific Aim 1 in porcine coronary artery smooth muscle and endothelial cells in culture using electrophysiological technique under more strictly controlled in vitro conditions; 4) effect of adenosine receptor activation on Na+/K+ ATPase, K+ATP, and Ca2+ channels on vascular smooth muscle and endothelial cells in culture using whole cell patch-clamp technique; 5) role of G-protein in the activation of adenosine receptor through modulation of membrane potential; and 6) effect of adenosine receptor activation on nitric oxide synthase (NOS) expression and NO production to establish the order of potency of adenosine agonists and to define adenosine receptor-subtype in porcine coronary artery rings and cultured cells through the following methods: NO production in porcine coronary artery rings and cultured endothelial cells in the presence or absence of adenosine agonists and antagonists; NOS activity using [14C]-arginine through the conversion of L-arginine to citrulline and NADPH diaphorase histochemistry; and reverse transcription/PCR (RT-PCR), Northern and Western Blotting for iNOS and cNOS expression/message. The outcome of these studies will further understanding of the role of endothelium in adenosine receptor-stimulated vascular smooth muscle hyperpolarization/relaxation factors and may be of major importance in understanding cardiovascular dysfunctions. PEER REVIEWERS' COMMENTS The resume and other initial sections of the summary statement are the authoritative representation of the final outcome of group discussion. The comments attached were prepared by the reviewers assigned to this application. The application was discussed and scored by all reviewers present. Thus, the commentaries may not necessarily reflect the position of the authors at the close of group discussion nor the final majority opinion of the group.
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