Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells

Objective: To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current ( I Ca) in atrioventricular (AV) nodal myocytes. Methods: The whole-cell patch-clamp technique was used to record I Ca from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Results: Isoprenaline, 0.1 μM, increased peak I Ca from 0.58±0.08 to 1.23±0.1 nA, and this increase was reversibly inhibited by adenosine, 10 μM (83±6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 μM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on I Ca. Methylene blue (10 μM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide ( · O2−), did not prevent, but increased, the inhibiting action of adenosine (49.5±6.6%, P <0.01). Methylene blue (50 μM) caused a reduction of I Ca, with further inhibition when combined with adenosine. A · O2−-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on I Ca. Inhibition of I Ca by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 μM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, l-NAME (0.5 mM). Conclusions: The inhibitory effect of adenosine on I Ca in rabbit AV nodal myocytes can be mediated by two mechanisms—stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with · O2− production.