title: Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide.
abstract: 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) during hydrogen peroxide (H(2)O(2))-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 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide 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 (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), 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 H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Srigiridhar
authors.initials: S
authors.lastname: Kotamraju
authors.forename: Shasi V
authors.initials: SV
authors.lastname: Kalivendi
authors.forename: Anuradha
authors.initials: A
authors.lastname: Dhanasekaran
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D005452:Fluoresceins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007501:Iron / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism*; D010617:Phenanthridines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
chemical_list: D000975:Antioxidants; D002518:Ceramides; D005452:Fluoresceins; D016877:Oxidants; D010617:Phenanthridines; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011990:Receptors, Transferrin; C065013:2',7'-dichlorodihydrofluorescein; D013481:Superoxides; D009569:Nitric Oxide; C058428:hydroethidine; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D007501:Iron; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; D019331:NG-Nitroarginine Methyl Ester