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, O-2(radical anion) ; however, the effects of methylarginines on this O-2(radical anion) generation are poorly understood. Therefore, we measured the dose-dependent effects of ADMA and L-NMMA on the rate and amount of O-2(radical anion) 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 muM) inhibited O-2(radical anion) generation by similar to 60% from a rate of 56 to 23 nmol/mg/min, whereas L-NMMA ( 0.1 - 100 muM) had no effect. L-Arginine markedly decreased the observed O-2(radical anion), adduct formation; however, O-2. generation from the enzyme still occurs at a low rate ( 12.1 nmol/mg/min). This O-2(radical anion) leak is NOS-derived as it is not seen in the absence of calcium and calmodulin and demonstrates that O-2(radical anion) generation from NOS occurs even when normal substrate/cofactor levels are present. Under conditions of BH4 depletion, ADMA had no effect on O-2(radical anion), whereas L-NMMA increased O-2(radical anion) production almost 3-fold. This O-2(radical anion) generation was > 90% inhibited by imidazole, indicating that it occurred at the heme center. Thus, methylarginines can profoundly shift the balance of NO and O-2(radical anion) generation from nNOS. These observations have important implications with regard to the therapeutic use of methylarginine-NOS inhibitors in the treatment of disease.