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Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca 2+ current in human atrial myocytes

Abstract : 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 (ICa) in freshly isolated human atrial myocytes. Acetylcholine (ACh, 1 μM) decreased basal ICa 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 μM), a potent guanylyl cyclase inhibitor, and NG-monomethyl-L-arginine (L-NMMA, 1 mM), a competitive NOS inhibitor. L-NMMA alone had no effect on ICa, whilst ODQ increased ICa in 50 % of the cells. The accentuated antagonism of ACh on ICa, i.e. its ability to antagonize the stimulatory effect of β-adrenergic agonists and, by extension, of other cAMP-elevating agents, was examined after the current was stimulated by either the β-adrenergic agonist isoprenaline (Iso) or serotonin (5-HT). ACh (100 nM or 1 μM) completely blocked the stimulatory effects of 10 nM Iso or 10 nM 5-HT on ICa. Extracellular application of Methylene Blue (MBlue, 10 μM), a guanylyl cyclase inhibitor, antagonized the inhibitory effect of 1 μM ACh on Iso- or 5-HT-stimulated ICa. However, this effect was overcome by a 100-fold higher ACh concentration and was not mimicked by an intracellular application of MBlue. Inhibition of NOS and soluble guanylyl cyclase activities by addition of ODQ (10 μM) 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 μM) on Iso-stimulated ICa. Extracellular application of the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) at 100 nM produced a stimulatory effect on ICa in control conditions. This stimulatory effect was abolished by intracellular MBlue (20 μM) or by intracellular and extracellular application of ODQ (10 μM) in combination with L-NMMA (1 mM). We conclude that the NO-cGMP pathway does not contribute significantly to the muscarinic regulation of ICa in human atrial myocytes.
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Grégoire Vandecasteele, Thomas Eschenhagen, Rodolphe Fischmeister. Role of the NO-cGMP pathway in the muscarinic regulation of the L-type Ca 2+ current in human atrial myocytes. The Journal of Physiology, Wiley, 1998, 506 (3), pp.653-663. ⟨10.1111/j.1469-7793.1998.653bv.x⟩. ⟨hal-03616987⟩

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