CELLULAR MECHANISM OF ADENOSINE'S CARDIAC ACTIONS

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Adenosine (Ado) has electrophysiological effects relevant to the genesis and therapy of a number of cardiac arrhythmias. This proposal is a direct outgrowth of our ongoing research to define the underlying cellular mechanism(s) of Ado actions on cardiac myocytes. Although the main focus of this project is on A1-Ado receptor mediated actions, the cardiac A2-Ado receptor, known to stimulate adenylyl cyclase, will also be investigated. The study to be performed in guinea pig atrial and ventricular myocytes has four objectives aimed to elucidate the electrophysiological signals caused by activation of A1- and A2-Ado receptors and their transmembrane coupling mechanisms during normal and altered cellular responsiveness. The aims are to determine: 1) the coupling mechanism of A1-Ado induced activation of atrial inwardly rectifying K+ current (IKAdo) and isoproterenol (ISO)- stimulated calcium inward current (ICa), 2) the time-course and magnitude of desensitization of A1-Ado mediated-responses and underlying mechanism 3) determine the electrophysiological response to activation of A2-Ado receptors, 4) the effect of Ado on basal ICa (atria) and ISO-stimulated delayed rectifier K+ current (IK, ventricle) and the consequences of these actions for atrial contractility and ventricular action potential duration (APD), respectively. Ado effects on single KAdo-channel activity and on macroscopic currents IKAdo, basal ICa, ISO-stimulated ICa and IK will be measured with the patch-clamp technique in inside-out membrane patches and whole-cells, respectively. To define the A1-Ado receptor coupling mechanism, the effect of the monoclonal antibody-4A (against alpha1) on Ado-mediated effects on membrane currents will be determined. The magnitude and time-course of pertussis toxin-induced ADP-ribosylation of alpha1 will be correlated with inhibition of the actions of Ado and receptor-effector coupling efficiency determined with the irreversible A1- Ado receptor antagonist (M-DITC-XAC). To elucidate potential mechanisms for the altered responsiveness of cells (desensitization) to A1-Ado mediated actions, changes in receptor number and affinity, G-protein subunits, and receptor-effector coupling efficiency will be determined. The hypothesis that A2-agonist. The effect of Ado on basal ICa in atrial cells will be investigated under conditions in which cAMP-mediated effects are abolished by the cAMP-dependent protein kinase inhibitor Rp-cAMPS. Concomitant with ICa measurements, twitch shortening of the myocytes will be monitored with a video-image edge detector. Standard voltage-clamp protocols will be used to determine the action of Ado on ISO-stimulated IK and the extent by which the effect modulates ventricular APD during beta- adrenergic stimulation. This multidisciplinary and detailed approach should provide new insight into the cellular mechanisms underlying the cardiac actions of Ado and further establish the importance of this nucleoside as a modulator of cardiac function.