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 (BH(4)). Thus alterations in endothelial BH(4) levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH(4), 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 BH(4) diminished superoxide release from eNOS, with efficiency BH(4)>6-methyl-BH(4)>5-methyl-BH(4). In contrast, partially oxidized BH(4) analogues, 7,8-dihydrobiopterin (7,8-BH(2)) and sepiapterin had no effect. Neither l-arginine nor N(G)-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH(4) and l-arginine stimulated .NO production at maximal rates of 148 nmol/min per mg of protein. These results indicate that BH(4) acts as a "redox switch", decreasing superoxide release and enhancing .NO formation. This role was verified by adding 7,8-BH(2) or sepiapterin to fully active eNOS. Both 7,8-BH(2) and sepiapterin enhanced superoxide release while inhibiting (.)NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH(4) metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.

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.

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Objective-4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs). Methods and Results-Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH 4 ) is a limiting factor in non supplemented cells. This was verified by quantification of BH 4 by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH 4 by 4-HNE is attributable to specific mechanisms involving protein degradation. Key Words: tetrahydrobiopterin Ⅲ eNOS phosphorylation Ⅲ 2-hydroxyethidium Ⅲ glutathione Ⅲ ascorbate N itric oxide (NO) plays an essential role in preserving vascular function and health. Animal models of genetic eNOS deficiency have shown that interrupted NO supply affects a variety of functions including blood vessel remodeling, 1,2 permeability, 3 blood flow, 4 endothelial adhesiveness, and blood pressure. 4 Alteration of one or more of these parameters is considered a powerful predictor for the development of cardiovascular disease. Conclusions-4-HNE by altering BHProduction of NO from eNOS is regulated by a complex process dependent on optimal L-arginine and tetrahydrobiopterin (BH 4 ) supply. 5 At the cellular level, eNOS activity is regulated by interaction with different proteins such as hsp90, caveolin-1 (scaffolding peptide), and G ␣12 . 6,7 Additionally, posttranslational eNOS modifications by covalent attachment of lipids (myristoylation, palmitoylation) or phosphate groups (serine 1177, serine 635, threonine 495) 8,9 have been shown to effectively modulate NO production in endothelial cells without changes in eNOS protein levels. However, under certain conditions NO production coincides with upregulation of eNOS expression. 10,11 There has been an increasing interest in defining the relative role of each of these modifications in the mechanisms altering NO production in hypercholesterolemic patients. Several risk factors for atherothrombosis including hypercholesterolemia, hypertension, and diabetes also promote vascular oxidant stress. Thus, it has been suggested that reactive oxygen species are important in the pathophysiogical process leading to decreased NO and acute plaque activation in the atherothrombotic mechanism.Supplementation with BH 4 improves vascular relaxation in hypercholesterolemic animal models and hu...