Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH4). Thus alterations in endothelial BH4 levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH4, and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH4 diminished superoxide release from eNOS, with efficiency BH4>6-methyl-BH4>5-methyl-BH4. In contrast, partially oxidized BH4 analogues, 7,8-dihydrobiopterin (7,8-BH2) and sepiapterin had no effect. Neither l-arginine nor NG-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH4 and l-arginine stimulated ˙NO production at maximal rates of 148nmol/min per mg of protein. These results indicate that BH4 acts as a ‘redox switch’, decreasing superoxide release and enhancing ˙NO formation. This role was verified by adding 7,8-BH2 or sepiapterin to fully active eNOS. Both 7,8-BH2 and sepiapterin enhanced superoxide release while inhibiting ˙NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH4 metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.
Abbreviations used: 7,8-BH2, 7,8-dihydrobiopterin; BH4, 5,6,7,8-tetrahydrobiopterin; 5Me-BH4, 5-methyl-BH4; 6Me-BH4, 6-methyl-BH4; DTPA, diethylenetriaminepenta-acetic acid; EMPO, 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide; eNOS, endothelial nitric oxide synthase; l-NAME, NG-nitro-l-arginine methyl ester; l-NMMA, NG-monomethyl-l-arginine.