The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study

Abstract

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 N^G-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.