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

Abstract

Objective: To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes.

Methods: The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion.

Results: Isoprenaline, 0.1 microM, increased peak ICa from 0.58 +/- to 1.23 +/- 0.1 nA, and this increase was reversibly inhibited by adenosine, 10 microM (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 microM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa-Methylene blue (10 microM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (.02-), did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01). Methylene blue (50 microM) caused a reduction of ICa, 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 ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 microM) 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 ICa 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.