The main objectives of the present study are to study the process and mechanism of desensitization and sensitization of the cardiac A1 adenosine receptor (A1AR)-mediated physiologic response, to determine whether changes in the levels of A1AR and/or the high-affinity form of the A1AR regulate physiologic responsiveness of the heart to A1AR agonist stimulation, and to determine the molecular mechanism(s) by which activation of A1AR causes the A1AR-mediated contractile response. Monolayer atrial cells cultured from 14 day chick embryo will be used as a model system. Specifically, we will: a) characterize the functional responses elicited by A1AR agonist stimulation; b) carry out biochemical studies of the binding of radioligand antagonist and agonist to the A1AR in membranes of these cultures cells. We will further determine whether 1) A1AR are coupled to the various potential effectors such as the adenylate cyclase, phosphodiesterase, guanylate cyclase or the potassium channel, and we will study the role of these potential effectors in mediating the contractile effects of A1AR agonist; 2) pertussis toxin-sensitive GTP-binding protein(s) is involved in the coupling between A1AR and the various effectors; 3) chronic exposure of cultured atrial cells to A1AR agonist causes attenuation of the A1AR- mediated functional responses and whether such decreased responsiveness is associated with a conversion of the high-affinity A1AR to a low-affinity form, a downregulation of the A1AR, or both; 4) chronic treatment of the culture with an adenosine receptor antagonist or adenosine deaminase induces sensitization of the A1AR-mediated functional response by causing the conversion of low-affinity A1AR to a high-affinity form, an upregulation of the receptor or by both mechanisms; 5) desensitization of A1AR is accompanied by a compensatory increase in the level of stimulatory G protein (Gs) or beta-adrenergic receptors (beta AR) with a concomitant increase in responsiveness to beta-adrenergic stimulation; 6) on the other hand, sensitization of these inhibitory receptors is associated with a compensatory decrease in the level of Gs or beta AR with concomitant decrease in beta-adrenergic responsiveness. These studies should help elucidate the mechanism of sensitization and desensitization of the A1AR system as well as the role of the regulation of A1AR and its high-affinity form in modulating the sensitivity of atrial myocytes to A1AR agonist stimulation. They should also provide insights into the mechanisms responsible for the cellular action of adenosine.
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