To examine the role of nitric oxide (.NO) in vascular endothelial cell injury, cultured porcine pulmonary artery endothelial cells (PAEC) were treated with H2O2 (100-500 microM) for 30 min in the presence or absence of the .NO donors (+/-)S-nitroso-N-acetylpenicillamine (SNAP) or diethylamine nitric oxide (DEANO). H2O2 caused dose-dependent PAEC cytotoxicity detected 2 h after H2O2 treatment as the release of lactate dehydrogenase. SNAP (100 microM) and DEANO (100 microM) attenuated H2O2-induced cytotoxicity if present during H2O2 treatment. In contrast, restricting treatment with .NO donors to periods before (30 min) or after (2 h) incubation with H2O2 did not prevent PAEC injury. Furthermore, the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (1 mM) sensitized PAEC to H2O2-induced injury. SNAP also attenuated H2O2-induced PAEC lipid peroxidation even if restricted to periods before or after exposure to H2O2. Thus, although .NO effectively attenuated H2O2-mediated PAEC lipid peroxidation and cytotoxicity, these effects were clearly dissociated, suggesting that the antiperoxidative effects of .NO are not sufficient to account for its cytoprotective properties.

Altering the fatty acid composition of cultured porcine pulmonary artery endothelial cells (PAEC) modulates their susceptibility to oxidant stress. This study demonstrates that supplementing PAEC with oleic acid (18:1 omega 9), but not gamma-linolenic acid (18:3 omega 6), provided dose-dependent protection from hydrogen peroxide (H2O2)-induced cytotoxicity. It was hypothesized that 18:1 reduced PAEC susceptibility to oxidant stress by altering H2O2 metabolism. To test this hypothesis, confluent PAEC monolayers were treated with 100-200 microM H2O2 or control conditions 24 h after supplementation with 0.1 mM 18:1, 18:3, or vehicle for 3 h. Intracellular [H2O2] in control cells (14.4-29.0 pM), estimated from the rate of aminotriazole-mediated inactivation of endogenous catalase activity, increased following treatment with 200 microM H2O2 (19.0-37.3 pM). Supplementation with 18:1 attenuated increases in intracellular [H2O2] only in oxidant-exposed cells, whereas supplementation with 18:3 attenuated intracellular [H2O2] only in control cells. Supplementation with 18:1 or 18:3 tended to reduce or enhance PAEC lipid hydroperoxide content following H2O2 exposure, respectively, but did not alter PAEC reduced glutathione content, the activities of glutathione peroxidase or catalase, or H2O2 uptake and release. Alteration of H2O2 metabolism in cultured PAEC may contribute to the ability of fatty acids to modulate cellular oxidant susceptibility.

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