Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca2+ current in human atrial myocytes

1. The whole-cell patch-clamp technique was used to examine the participation of nitric oxide synthase (NOS) and soluble guanylyl cyclase in the muscarinic regulation of the L-type Ca2+ current (I-Ca) in freshly isolated human atrial myocytes. 2. Acetylcholine (ACh, 1 mu M) decreased basal I-Ca by 39.1 +/- 5.5% (n = 8) under control conditions, and by 38.0 +/- 6.1.% (n = 6) in the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 mu M), a potent guanylyl cyclase inhibitor, and N-G-monomethyl-L-arginine (L-NMMA, 1 mM), a competitive NOS inhibitor. L-NMMA alone had no effect on I-Ca, whilst ODQ increased I-Ca in 50% of the cells. 3. The accentuated antagonism of ACh on I-Ca, i.e. its ability to antagonize the stimulatory effect of beta-adrenergic agonists and, by extension, of other cAMP-elevating agents, was examined after the current was stimulated by either the beta-adrenergic agonist isoprenaline (Iso) or serotonin (5-HT). ACh (100 nM or 1 mu M) completely blocked the stimulatory effects of 10 nM Iso or 10 nM 5-HT on I-Ca. 4. Extracellular application of Methylene Blue (MBlue, 10 mu M), a guanylyl cyclase inhibitor, antagonized the inhibitory effect of 1 mu M ACh on Iso- or 5-HT-stimulated I-Ca. However, this effect was overcome by a 100-fold higher ACh concentration and was not mimicked by an intracellular application of MBlue. 5. Inhibition of NOS and soluble guanylyl cyclase activities by addition of ODQ (10 mu M) and L-NMMA (1 mM) to both extracellular and intracellular solutions, or by a 2 h pre-incubation of the cells with these inhibitors, modified neither the Iso (10 nM) response nor the inhibitory effect of ACh (100 nM or 1 mu M) on Iso-stimulated I-Ca. 6. Extracellular application of the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) at 100 nM produced a stimulatory effect on I-Ca in control conditions. This stimulatory effect was abolished by intracellular MBlue (20 mu M) or by intracellular and extracellular application of ODQ (10 mu M) in combination with L-NMMA (1 mM). 7. We conclude that the NO-cGMP pathway does not contribute significantly to the muscarinic regulation of I-Ca in human atrial myocytes.