Effects of methylarginines on superoxide production from uncoupled eNOS.

Abstract

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 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 BH4 depletion. In the absence of BH4, ADMA dose‐dependently increased NOS‐derived ▸O2‐ generation, with a maximal increase of 151 % at 100 uM. L‐NMMA also dose‐dependently increased NOS‐derived ▸O2‐, but to a lesser extent, demonstrating a 102 % increase at 100 uM L‐NMMA. Moreover, methylarginines also increased eNOS derived ROS from endothelial cells. Measurements of NADPH consumption from eNOS demonstrated that binding of methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies demonstrate that the binding of ADMA and L‐NMMA shifts the eNOS heme to the high‐spin state, 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 and play an important role in endothelial dysfucntion.