Homocyst(e)ine Decreases Bioavailable Nitric Oxide by a Mechanism Involving Glutathione Peroxidase

Abstract

Hyperhomocyst(e)inemia is believed to injure endothelial cells <i>in vivo</i> through a number of mechanisms, including the generation of hydrogen peroxide (H₂O₂). Earlier <i>in vitro</i> studies demonstrated that homocyst(e)ine (Hcy) decreases the biological activity of endothelium-derived relaxing factor and that this decrease can be reversed by preventing the generation of hydrogen peroxide. Here we show that Hcy treatment of bovine aortic endothelial cells leads to a dose-dependent decrease in NO _x (<i>p</i> = 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and <i>nos3</i> transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H₂O₂, Hcy decreases the cell's ability to detoxify H₂O₂ by impairing intracellular antioxidant enzymes, specifically the intracellular isoform of glutathione peroxidase (GPx). To test this hypothesis, confluent bovine aortic endothelial cells were treated with a range of concentrations of Hcy, and intracellular GPx activity was determined. Compared with control cells, cells treated with Hcy showed a significant reduction in GPx activity (up to 81% at 250 μm Hcy). In parallel with the decrease in GPx activity, steady-state GPx mRNA levels were also significantly decreased compared with control levels after exposure to Hcy, which appeared not to be a consequence of message destabilization. These data suggest a novel mechanism by which Hcy, in addition to increasing the generation of hydrogen peroxide, may selectively impair the endothelial cell's ability to detoxify H₂O₂, thus rendering NO more susceptible to oxidative inactivation.