Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors
AUTOR(ES)
Vásquez-Vivar, Jeannette
FONTE
The National Academy of Sciences
RESUMO
The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of l-arginine, NG-hydroxy-l-arginine, l-thiocitrulline, and l-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous–dioxygen complex and that occupation of the l-arginine binding site does not inhibit this process. However, the concomitant addition of l-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, l-citrulline production is close to maximal. Our data indicate that BH4 fully couples l-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous–dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=21319Documentos Relacionados
- Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase.
- Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.
- Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages
- Direct measurement of nitric oxide generation from nitric oxide synthase
- Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.