Endogenous methylarginines modulate superoxide as well as nitric oxide generation from neuronal nitric-oxide synthase: Differences in the effects of monomethyl- and dimethylarginines in the presence and absence of tetrahydrobiopterin

The endogenous methylarginines asymmetric dimethylarginine (ADMA) and N^G-monomethyl-L-arginine (L-NMMA) regulate nitric oxide (NO) production from neuronal NO synthase (nNOS). Under conditions of L-arginine or tetrahydrobiopterin (BH₄) depletion, nNOS also generates superoxide, O₂^•- however, the effects of methylarginines on this O₂^•- generation are poorly understood. Therefore, we measured the dose-dependent effects of ADMA and L-NMMA on the rate and amount of O₂^•- production from nNOS under conditions of L-arginine and/or BH₄ depletion, using electron paramagnetic resonance spin trapping. In the absence of L-arginine, ADMA (1 μM) inhibited O₂^•- generation by ∼60% from a rate of 56 to 23 nmol/mg/ min, whereas L-NMMA (0.1-100 μM) had no effect. L-Arginine markedly decreased the observed O₂^•- adduct formation; however, O₂^•- generation from the enzyme still occurs at a low rate (12.1 nmol/mg/min). This O₂^•- leak is NOS-derived as it is not seen in the absence of calcium and calmodulin and demonstrates that O₂^•- generation from NOS occurs even when normal substrate/ cofactor levels are present. Under conditions of BH₄ depletion, ADMA had no effect on O₂^•-, whereas L-NMMA increased O₂^•- production almost 3-fold. This O₂^•- generation was >90% inhibited by imidazole, indicating that it occurred at the heme center. Thus, methylarginines can profoundly shift the balance of NO and O₂ ^•- generation from nNOS. These observations have important implications with regard to the therapeutic use of methylarginine-NOS inhibitors in the treatment of disease.