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-center dot(2)-; however, the effects of methylarginines on eNOS-derived O-center dot(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-center dot(2)- production from eNOS under conditions of BH4 depletion. In the absence of BH4, ADMA dose-dependently increased NOS-derived center dot O-2(-) generation, with a maximal increase of 151% at 100 mu M ADMA. L-NMMA also dose-dependently increased NOS-derived center dot O-2(-), but to a lesser extent, demonstrating a 102% increase at 100 mu M L-NMMA. Moreover, the native substrate L-arginine also increased eNOS-derived center dot 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 center dot 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.