Nitric oxide regulation of atrioventricular node excitability

The role of nitric oxide in the autonomical regulation of atrioventricular (AV) spontaneous action potentials and L-type calcium current (I(Ca-L)) in isolated single AV nodal cells from rabbit heart was examined by using the whole cell parch clamp technique, immunohistochemical staining and single cell reverse transcription polymerase chain reaction analysis. The nitric oxide donor 3-morpholino-sydnonimine (SIN-1) (0.1 mmol/L) suppressed the beta-agonist isoproterenol- (1 μmol/L) stimulated increase in I(Ca-L) and decreased the frequency and amplitude of spontaneous action potentials. In cells in which I(Ca-L) had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 μmol/L), SIN-1 had no additive effect. Intracellular dialysis with the nitric oxide synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh- but not SIN-1-induced I(Ca-L) attenuation. However, intracellular dialysis with methylene blue (20 μmol/L), which inhibits nitric oxide-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN-1 on I(Ca-L). In these cells, neither L-NMMA nor methylene blue affected the CCh-activated potassium current (I(K(ACh))). Internal dialysis with cGMP (10 μmol/L) significantly inhibited isoproterenol-stimulated I(Ca-L) without affecting I(K(ACh)). In AV nodal cells internally perfused with either a non-hydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit I(Ca-L) but still activated I(K(ACh)). CCh-induced I(Ca-L) attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (20 μmol/L) but not by milrinone (5 μmol/L), which only inhibits the cGMP-inhibited phosphodiesterase isozyme (PDE3). Immunohistochemical staining identified the presence of the endothelial constitutive nitric oxide synthase (NOS3) in both single AV node cells in vitro and in cryostat sections of AV node tissue in situ. These results demonstrate that endogenous nitric oxide is involved in the muscarinic cholinergic attenuation of I(Ca-L) in AV nodal cells; the mechanism likely involves the cGMP-stimulated phosphodiesterase.