Regulation of eNOS-derived superoxide by endogenous methylarginines

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and N^G-monomethyl-L-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH₄) depletion eNOS also generates ^•O_2-; however, the effects of methylarginines on eNOS-derived ^•O_2- generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on ^•O_2- production from eNOS under conditions of BH ₄ depletion. In the absence of BH₄, ADMA dose-dependently increased NOS-derived ^•O_2- generation, with a maximal increase of 151% at 100 μM ADMA. L-NMMA also dose-dependently increased NOS-derived ^•O_2-, but to a lesser extent, demonstrating a 102% increase at 100 μM L-NMMA. Moreover, the native substrate L-arginine also increased eNOS-derived ^•O _2-, exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either L-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for L-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and ^•O_2- generation from eNOS. These observations have important implications with regard to the therapeutic use of L-arginine and the methylarginine-NOS inhibitors in the treatment of disease.