Abstract 1427: Regulation of eNOS derived Superoxide by Endogenous Methylarginines
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 L-arginine or tetrahydrobiopterin (BH4) depletion eNOS also generates superoxide, however, the effects of methylarginines on eNOS derived superoxide generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose dependent effects of ADMA and L-NMMA on superoxide production from eNOS under conditions of BH4 depletion. In the absence of BH4, ADMA dose dependently increased NOS derived superoxide generation, with a 43 % increase at 1.0 μM, a 125 % increase at 10 μM, and a 151 % increase at 100 μM ADMA. L-NMMA dose dependently increased NOS derived superoxide generation similar to that observed with ADMA, with a 18 % increase at 1.0 μM, an 80 % increase at 10 μM, and a 102 % increase at 100 μM L-NMMA. Experiments were also carried out to determine whether the native substrate L-arginine had any effects of NOS derived superoxide. Surprisingly, L-arg dose dependently increased eNOS derived superoxide with a 26 % increase at 1.0 μM, a 116 % increase at 10 μM, and a 152 % increase at 100 μM L-arginine. In support of these findings, measurements of NADPH consumption rate from eNOS demonstrated that binding of either substrate or methylarginines increased the rate from a basal value of 54.7 nmols/mg/min to 86.0 nmols/mg/min for ADMA; 81.3 nmols/mg/min for L-NMMA and 89.2 nmols/mg/min for L-arg. Measurements of the heme spin state of eNOS demonstrated that binding of ADMA, L-NMMA and L-arg resulted in an increase in the heme reduction potential and enhanced electron transfer from the reductase to the oxygenase site, thus augmenting superoxide generation. 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.