[Frontiers in Bioscience S3, 1263-1272, June 1, 2011]

Tetrahydrobiopterin attenuates superoxide-induced reduction in nitric oxide

Mark C. Bowers1, Laura A. Hargrove1, Katherine A. Kelly1, Guoyao Wu1,2, Cynthia J. Meininger1

1Cardiovascular Research Institute and Department of Systems Biology and Translational Medicine, Texas A and M Health Science Center, Temple, TX 76504, 2Department of Animal Science, Texas A&M University, College Station, TX 77843


1. Abstract
2. Introduction
3. Materials and methods
3.1. Oxidation of low-density lipoprotein
3.2. Endothelial cell culture
3.3. Superoxide anion production
3.4. BH4 analysis
3.5. Western blotting
3.6. Nitric oxide production
3.7. Statistical analysis
4. Results
5. Discussion


NADPH oxidase, a source of superoxide anion (.O2-), can be stimulated by oxidized low-density lipoprotein (OxLDL). We examined whether tetrahydrobiopterin (BH4) could reduce OxLDL-induced .O2- production by NADPH oxidase, increasing nitric oxide (NO) synthesis. Endothelial cells incubated with OxLDL produced more .O2- (35-67%) than untreated cells, with the highest increase 1 hour after OxLDL addition. The elevated .O2- production correlated with the translocation of the p47phox subunit of NADPH oxidase from the cytosol to the membrane. Cells exhibited a marked decrease in both BH4 (83%) and NO (54%) in the same hour following exposure to OxLDL. An NADPH oxidase inhibitor, apocynin, or antioxidant, N-acetyl-L-cysteine, substantially attenuated the reduction in both BH4 and NO. The .O2- production was increased when cells were pretreated with an inhibitor of BH4 synthesis and decreased following pretreatment with a BH4 precursor, suggesting that NADPH oxidase-induced imbalance of endothelial NO and .O2- production can be modulated by BH4 concentrations. BH4 may be critical in combating oxidative stress, restoring proper redox state, and reducing risk for cardiovascular disease including atherosclerosis.