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

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 mu M, increased peak I-Ca from 0.58 +/- 0.08 to 1.23 +/- 0.1 nA, and this Increase was reversibly inhibited by adenosine, 10 mu M (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 mu M). an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect af adenosine on I-Ca. Methylene blue (10 mu M), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (O-.(2)-),did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01), Methylene blue (50 mu M) caused a reduction of I-Ca, with further inhibition when combined with adenosine. A.O(2)(-)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 mu M) 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 O-.(2)- production.