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 partic ipation of nitric oxide synthase (NOS) and soluble guanylyl cyclase in the muscarinic regulation of the L-type Ca2+ current (I-Ca) in freshl y isolated human atrial myocytes. 2. Acetylcholine (ACh, 1 mu M) decre ased 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 inhibit or, and N-G-monomethyl-L-arginine (L-NMMA, 1 mM), a competitive NOS in hibitor. 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 exami ned after the current was stimulated by either the beta-adrenergic ago nist isoprenaline (Iso) or serotonin (5-HT). ACh (100 nM or 1 mu M) co mpletely 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 ov ercome by a 100-fold higher ACh concentration and was not mimicked by an intracellular application of MBlue. 5. Inhibition of NOS and solubl e 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 th e Iso (10 nM) response nor the inhibitory effect of ACh (100 nM or 1 m u M) on Iso-stimulated I-Ca. 6. Extracellular application of the NO do nor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) at 100 nM produced a s timulatory effect on I-Ca in control conditions. This stimulatory effe ct was abolished by intracellular MBlue (20 mu M) or by intracellular and extracellular application of ODQ (10 mu M) in combination with L-N MMA (1 mM). 7. We conclude that the NO-cGMP pathway does not contribut e significantly to the muscarinic regulation of I-Ca in human atrial m yocytes.