Effects of N-Monomethyl-L-arginine on Ca Current and Nitric-Oxide Synthase in Rat Ventricular Myocytes

The effects of N-monomethyl-L-arginine (L-NMMA), a nitricoxide synthase (NOS) inhibitor, on the L-type Ca current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 mM) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 mM) or milrinone (10 mM), a cGMP-inhibited cAMPphosphodiesterase (PDE), L-NMMA (10 and 100 mM) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 mM) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 mM), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 mM). 8-Bromo-cGMP (200 mM) inhibited 100 mM L-NMMAinduced ICa increase in the simultaneous application of forskolin (1 mM). Acetylcholine (ACh; 1 and 3 mM) inhibited 1 mM forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 mM). In the continuing presence of both L-NMMA (100 mM) and forskolin (1 mM), ACh (6 mM) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS. The conversion of L-arginine to L-citrulline plus nitric oxide (NO) is produced by constitutive NO synthase (cNOS), or after cytokine stimulation, by inducible NOS (Moncada et al., 1991; Nathan and Xie, 1994). cNOS in saline-treated rats and inducible NOS in rats after pretreatment with endotoxin or cytokine are found in ventricular tissue slices as well as in isolated cardiac myocytes (Schultz et al., 1992). Furthermore, two isoforms of cNOS have been cloned from rat vascular endothelium (ecNOS) and brain (Nathan and Xie, 1994). The ecNOS protein expression also occurs in rat cardiac myocytes (Balligand et al., 1995). Administration of the NOS inhibitor N-monomethyl-Larginine (L-NMMA) causes increases in mean blood pressure and systemic vascular resistance suppresses cardiac output in anesthetized dogs pretreated with either saline or endotoxin (Klabunde and Ritger, 1991). Perfusion with L-methylL-arginine in an isolated rat heart inhibits cardiac contractility in isoproterenol-stimulated hearts and this inhibitory action is accompanied by a decrease in both myocardial cGMP and cAMP concentrations (Klabunde et al., 1992). In contrast, N-nitro-L-arginine, one of the NOS inhibitors, potentiates positive inotropic action of isoproterenol on electrically stimulated rat cardiac myocytes but does not significantly alter basal contractility (Balligand et al., 1993). In addition, Balligand et al. (1995) reported that L-NMMA attenuated the inhibitory effects of carbamylcholine on the increase in contractility induced by isoproterenol. Nawrath et al. (1995), however, demonstrated that L-NMMA failed to influence the muscarinic effect on the force of contraction or frequency in rat and guinea pig hearts. Therefore, contradictory opinions remain as to how NOS inhibitors influence myocardial contractility in the b-adrenergically stimulated condition with or without the activation of muscarinic recep-