Mediation by nitric oxide of the indirect effects of adenosine on calcium current in rabbit heart pacemaker cells

Adenosine (ADO) is a potent negative chronotropic agent in the mammalian myocardium. We have used single myocytes from rabbit sino-atrial node (SAN) to examine whether nitric oxide (NO) is a significant mediator of the effects of ADO on the pacemaker activity, or the underlying Ca2+ and K+ currents. 2. SAN pacemaker cells were isolated from rabbit hearts by enzymatic dispersion, and Ca2+ and K+ currents were recorded by the nystatin-perforated patch voltage clamp method. ADO was applied in the presence of the beta-adrenoceptor agonist, isopremaline (Iso) to mimic the adrenergic tone which the SAN is subjected to in vivo. 3. Control experiments confirmed that isolated SAN cells responded to ADO (10-100 microM) with the expected (i) small increase in background inwardly rectifying K+ current, IK-ADOi and (ii) pronounced decrease in L-type Ca2+ current, ICa-L. These effects were mimicked by a selective A1 purinoceptor agonist, N6-cyclopentyladenosine (CPA, 10 microM); and were inhibited following bath application of the antagonist, DPCPX (10 microM), which selectively blocks A1 purinoceptors. DMPX (10 microM), a blocker of A2 purinoceptor, had no effect on the actions of ADO. 4. A nitric oxide synthase inhibitor, L-NMMA (100 microM), abolished the inhibitory effect of ADO on ICa-L but did not alter activation of IK-ADO. After L-NMMA washoff, it was possible to obtain the normal response (inhibition) of ICa-L to ADO in the same cell. 5. To evaluate whether the observed effect of nitric oxide (NO) on ICa-L was mediated by an increase in guanylyl cyclase (GC) activity and cyclic GMP formation, the guanylyl cyclase inhibitor, LY 83583 (40 microM) was applied prior to ADO. Under these conditions, the inhibitory effect of ADO on ICa-L was abolished, but the activation of IK-ADO was still observed. 6. In combination, these findings strongly suggest that in mammalian primary pacemaker tissue which is under adrenergic tone, the effects of ADO on ICa-L are mediated by NO.