Tetrahydrobiopterin, but Not l -Arginine, Decreases NO Synthase Uncoupling in Cells Expressing High Levels of Endothelial NO Synthase

Abstract

Abstract-Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R) 6,7, ) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. N -nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH 4 levels were increased as compared with another endothelial cell line, eNOS levels were Ͼ2 orders of magnitude higher. The addition of BH 4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH 4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH 4 , suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH 4 . (Hypertension. 2006;47:87-94.)

Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress

“…Likewise, Bevers et al have recently shown that overexpression of eNOS in microvascular endothelial cells led to enhanced production of both NO and reactive oxygen species. 35 When these cells were treated with H 4 B, NO production dramatically increased and reactive oxygen species production declined. In accord with our present study, Lam et al have recently shown that H 4 B levels are increased in vivo by high shear caused by an aortocaval fistula.…”

Altered Plasma Versus Vascular Biopterins in Human Atherosclerosis Reveal Relationships Between Endothelial Nitric Oxide Synthase Coupling, Endothelial Function, and Inflammation

“…1 Reduced BH 4 availability leads to eNOS uncoupling and the production of superoxide radicals instead of NO. 2,3 Recent evidence suggests that BH 4 -dependent eNOS uncoupling may be an important mechanism that mediates endothelial dysfunction and increased superoxide generation in vascular disease states. 4,5 …”

Hyperoxia-Induced Lung Injury in Gamma-Glutamyl Transferase Deficiency Is Associated with Alterations in Nitrosative and Nitrative Stress

“…In the setting of decreased arginine or tetrahydrobiopterin availability, the eNOS tetramer uncouples into dimers and generates O 2 Ϫ instead of NO. 38,39 In the GGT enu1 mouse lung, it is possible that eNOS plays some role in generating NO, but also participates in the formation of reactive oxygen species through O 2 Ϫ generation. Another study limitation relates to the lack of information about to the interaction between GSNO and cellular targets of nitrosation at the molecular level, 13 the role of glutathione in transnitrosation (the transfer of NO equivalents from one molecule to another) and its impact on caspase activity.…”