The endogenous methylarginines asymmetric dimethylarginine (ADMA) and NG-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.
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