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Hyperhomocyst(e)inemia is believed to injure endothelial cells in vivo through a number of mechanisms, including the generation of hydrogen peroxide (H 2 O 2 ).Earlier in vitro 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 (p ‫؍‬ 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and nos3 transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H 2 O 2 , Hcy decreases the cell's ability to detoxify H 2 O 2 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 M 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 H 2 O 2 , thus rendering NO more susceptible to oxidative inactivation.Hyperhomocyst(e)inemia is a disease caused by an abnormality in either an enzyme (cystathionine ␤-synthetase or temperature-sensitive methylenetetrahydrofolate reductase) or a cofactor (folate, vitamin B 12 , or vitamin B 6 ) required for homocysteine metabolism. These abnormalities lead to elevations in plasma concentrations of homocyst(e)ine (Hcy) 1 and its precursor methionine as well as a reduction in plasma concentrations of cysteine (1-5). In its most severe form, hyperhomocyst(e)inemia confers a significant risk for thromboembolic complications that are often fatal (6). In contrast, the less severe form of the disease is commonplace and indolent, not presenting with clinical sequelae until the third or fourth decade of life. These individuals manifest atherosclerosis as well as recurrent episodes of acute arterial and venous thrombosis (6) with near normal levels of fasting plasma homocyst(e)ine; following a methionine challenge, however, levels rise significantly compared with normal levels. Many studies demonstrate that hyperhomocyst(e)inemia is an independent risk factor for atherosclerosis in the coronary, cerebral, and peripheral vasculature (7-11), and up to 40% of patients with coronary or cerebrovascular atherosclerosis have hyperhomocyst(e)inemia.The mechanism by which Hcy damages the vessel ...

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Plasma albumin reacts with nitric oxide (NO) to form the bioactive adduct, S-nitroso-albumin (S-NO-albumin). The limited intracellular access of S-NO-albumin suggests the need for a vascular transfer mechanism of NO from a large plasma S-NO-albumin pool to effect biologic function. To study the role of low molecular weight (LMW) thiols in NO transfer in vivo, we administered intravenous S-NOalbumin (1-300 nmol/kg) to rabbits before and after an intravenous infusion of L-cysteine or N-acetyl-L-cysteine. S-NO-albumin produced dose-dependent hypotension that was significantly augmented by prior infusion of either LMW thiol. LMW thiol infusion significantly accelerated the rate of onset and reduced the duration of action of the hypotension induced by S-NO-albumin. The hemodynamic effects of S-NO-albumin after pretreatment with LMW thiols were mimicked by administration of the corresponding LMW S-nitrosothiol. The transfer of NO from albumin to L-cysteine was directly measured in rabbit plasma using a novel technique that couples high performance liquid chromatography to electrochemical detection. These data demonstrate that NO exchange between plasma protein thiolbound NO and available LMW thiol pools (transnitrosa-

GSH peroxidase (Px) catalyzes the reduction of lipid hydroperoxides (LOOH), known metabolic products of platelets and vascular cells. Because interactions between these cells are modulated by nitric oxide (NO) and LOOH inactivate NO, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring S-nitrosothiol, S-nitroso-glutathione (SNO-Glu). Concentrations of SNO-Glu that alone did not inhibit platelet function (subthreshold inhibitory concentrations) were added to plateletrich plasma together with GSH-Px (0.2-20 U/ml); this led to a dose-dependent inhibition of platelet aggregation with an IC50 of 0.6 U/ml GSH-Px. In the presence of subthreshold inhibitory concentrations of SNO-Glu, the LOOH, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, increased platelet aggregation, an effect reversed by GSH-Px. Glutathione and SNO-Glu were equally effective as cosubstrates for GSHPx. Incubation of SNO-Glu with GSH-Px for 1 min led to a 48.5% decrease in the concentration of SNO-Glu. Incubation of SNO-Glu with serum albumin led to the formation of S-nitroso-albumin, an effect enhanced by GSH-Px. These observations suggest that GSH-Px has two functions: reduction of LOOH, thereby preventing inactivation of NO, and metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions. (J. Clin. Invest. 1995. 96:394-400.) Key words: nitric oxide * S-nitrosothiols * endothelium-derived relaxing factor * lipid peroxides glutathione peroxidase