Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells - Protective role of endogenous nitric oxide

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide ((NO)-N-.) during hydrogen peroxide (H2O2)-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C-2-ceramide (C-2-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C-2-cer (5-20 muM) to BAECs enhanced (NO)-N-. generation. However, at higher concentrations of C-2-cer (greater than or equal to20 muM), (NO)-N-. generation did not increase proportionately. C-2-cer (20-50 muM) also resulted in H2O2-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of Fe-55, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. Nw-Nitro-L-arginine methyl ester (L-NAME), a nonspecific inhibitor of nitric-oxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C-2-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C-2-cer (less than or equal to10 muM) but was greatly suppressed at higher concentrations (> 10 muM). Intracellular scavengers of H2O2, cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C-2-cer- and/or L-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C-2-cer-induced H2O2 and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that (NO)-N-. exerts an antioxidative and cytoprotective role.