The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. Biochem J 2002 Mar 15;362(Pt 3):733-9
Date
03/07/2002Pubmed ID
11879202Pubmed Central ID
PMC1222439DOI
10.1042/0264-6021:3620733Scopus ID
2-s2.0-0037086565 (requires institutional sign-in at Scopus site) 263 CitationsAbstract
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.
Author List
Vásquez-Vivar J, Martásek P, Whitsett J, Joseph J, Kalyanaraman BAuthors
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of WisconsinJeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
ArginineElectron Spin Resonance Spectroscopy
Endothelium, Vascular
Kinetics
NG-Nitroarginine Methyl Ester
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Oxidation-Reduction
Recombinant Proteins
Stereoisomerism
Structure-Activity Relationship
Superoxides