Cardiac A1 and A3 adenosine receptors: Signaling in expression systems and cardiomyocytes

It is well established that adenosine acts on the heart to depress cardiac function (inotropy, chronotropy, dromotropy). These effects are, in part at least, A1 adenosine receptor (AdoR)-mediated through inhibition of adenylyl cyclase. While initially adenosine was proposed to be a mediator in "cardiac preconditioning" by acting solely at A1 AdoRs, more recently A3 AdoRs which also couple to the inhibition of adenylyl cyclase have also been proposed to be involved. Preconditioning is a phenomenon whereby one brief period of ischemia, protects the myocardium against a subsequent, more prolonged ischemic insult. Chick myocytes in culture can be preconditioned, and have been shown to have more than one AdoR subtype coupling to the inhibition of adenylyl cyclase. Chicken A1 and A3 AdoR cDNA were isolated by library screening and shown to be expressed in heart. Both AdoR subtypes were characterized by expression in HEK 293 cells. The agonist radioligand [125I]-ABA bound to both A1 and A3 AdoRs with equal affinities (KD 1--2nM) and was used in competition studies to characterize the affinities of AdoR agonists and antagonists. The agonists tested showed mild selectivity with KiA1/KiA3 values of 5.8, 5.0, and 0.1 for IB-MECA, Cl-IB-MECA and CHA, respectively. The antagonist MRS1191 selectively blocked ligand binding to A3 AdoRs. Conversely, the antagonist WRC0571 selectively blocked ligand binding to A1 AdoRs. Isoproterenol-elevated cAMP levels in HEK-A1 and HEK-A3 were lowered by CHA and Cl-IB-MECA with the same receptor specificities found with ligand binding. AdoR coupling to phospholipase C (PLC) and phospholipase D (PLD) could not be detected. Cultures of 13--14 day embryonic chick ventricular myocytes (>90% pure as assessed by myocyte-specific alpha-actinin immunostaining) were subsequently studied. Binding of [125I]-ABA to AdoRs in myocyte membranes was partitioned into A1 (total binding minus binding in presence of WRC0571) and A3 (total binding minus binding in presence of MRS1191). Myocytes expressed similar concentrations of A 1 and A3 AdoRs. Neither coupling to PLC or PLD could be detected, while both receptors inhibited isoproterenol-elevated cAMP levels. Cl-IB-MECA stimulated PKCepsilon translocation; this translocation was blocked by the A3 selective antagonist MRS1191 but not by the A1 specific antagonist WRC0571.