The cardiac actions of adenosine and adenosine 5'-triphosphate (ATP) are mediated by purinergic receptors present on cardiac myocytes. These receptors are classified into receptors for ATP (P2 purinergic) and receptors for adenosine (P1 purinergic), which is further subdivided into the adenosine A1 and A2 receptors. The adenosine A2 receptor, coupled to stimulation of adenylyl cyclase activity and myocyte contractility on ventricular myocytes represents a novel stimulatory purinergic receptor of potential physiologic and pathophysiologic significance in the heart. While cardiac adenosine A1 receptors have been characterized, very little is known about the adenosine A2 receptor and its subtypes (A2a and A2b), or the interaction between the A1 and A2 receptors. Further, since ATP exerts a pronounced positive inotropic effect and is readily hydrolyzed to adenosine, questions arise regarding whether activation of the adenosine A2 receptor contributes to the positive inotropic effect of ATP. The overall objective of the present proposal is to study the function and regulation of subtypes of adenosine A2 receptor and the interaction between subtypes of purinergic receptors. Specifically, using ventricular myocytes cultured from 14-day chick embryos as a model which express both A1 and A2 adenosine receptors, the following hypotheses will be tested. 1) The A2 subtypes exhibits differential regulation in response to adenosine agonists compared to the A1 subtype. 2) activation of the A2 subtype is capable of opposing the A1 subtype- mediated effect on the basal contractile amplitude. 3) stimulation of the A2 subtype contributes to the positive inotropic effect of ATP by a mechanism that involves an ATP-induced, adenosine A2 receptor-mediated stimulation of adenylyl cyclase activity and cyclic AMP accumulation. 4) activation of the A2 subtype is capable of opposing the A1 subtype- mediated antagonism of isoproterenol-stimulated increase in myocyte contractility, that is, the A1 subtype-mediated anti-adrenergic response to adenosine. A1-selective agonist and antagonist, selective desensitization of the adenosine A2 receptor pathway and blocking with an A2-selective antagonist will be used to determine the potential contractile functions of the A2 subtype as outlined in 2) through 4). Atrial myocytes cultured from 14-day embryos express the A1 but not the A2 subtype and exhibit a decrease in basal contractile amplitude in response to adenosine as well as a diminished positive inotropic response to ATP. The atrial myocyte therefore represents a useful cardiac model to test the relative importance of the A2a and A2b subtypes in mediating the various contractile functions listed in 2) thr 4). The cDNA encoding either the rate A2a or A2b receptor (inserted in the plasmid pcDNA 1, driven by CMV promoter) will be transfected individually into cultured atrial myocytes; the contractile effects of adenosine and ATP in myocytes expressing selectively the A2a or the A2b subtypes will be compared. These studies should provide significant insights into the function and regulation of subtypes of adenosine A2 receptor and into the mechanisms of cardiac action of ATP and adenosine in the heart.
Showing the most recent 10 out of 41 publications
