The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-L-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH4) depletion eNOS also generates •O2-; however, the effects of methylarginines on eNOS-derived •O2- generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on •O2- production from eNOS under conditions of BH 4 depletion. In the absence of BH4, ADMA dose-dependently increased NOS-derived •O2- generation, with a maximal increase of 151% at 100 μM ADMA. L-NMMA also dose-dependently increased NOS-derived •O2-, 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 •O2- 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.
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