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 (BH 4) 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 BH 4, ADMA dose-dependently increased NOS-derived (*)O 2 (-) generation, with a maximal increase of 151% at 100 microM ADMA. L-NMMA also dose-dependently increased NOS-derived (*)O 2 (-), but to a lesser extent, demonstrating a 102% increase at 100 microM 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.
Druhan
Lawrence J
LJ
Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
Forbes
Scott P
SP
Pope
Arthur J
AJ
Chen
Chun-An
CA
Zweier
Jay L
JL
Cardounel
Arturo J
AJ
D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism; D006801:Humans; D009249:NADP / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D019323:omega-N-Methylarginine / Q000494:pharmacology*
D013481:Superoxides; D001708:Biopterins; D019323:omega-N-Methylarginine; D006418:Heme; D009249:NADP; C018524:N,N-dimethylarginine; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin