Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide. J Biol Chem 2004 Jul 02;279(27):28614-24
Date
04/23/2004Pubmed ID
15102832DOI
10.1074/jbc.M400977200Scopus ID
2-s2.0-3142543756 (requires institutional sign-in at Scopus site) 93 CitationsAbstract
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.
Author List
Matsunaga T, Kotamraju S, Kalivendi SV, Dhanasekaran A, Joseph J, Kalyanaraman BAuthor
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAntioxidants
Apoptosis
Blotting, Western
Caspase 3
Caspases
Cattle
Cells, Cultured
Ceramides
Cytochromes c
DNA Fragmentation
Dose-Response Relationship, Drug
Endothelium, Vascular
Enzyme Activation
Fluoresceins
Glutathione
Hydrogen Peroxide
In Situ Nick-End Labeling
Iron
Microscopy, Fluorescence
Mitochondria
Models, Chemical
NG-Nitroarginine Methyl Ester
Neurons
Nitric Oxide
Oxidants
Phenanthridines
Proto-Oncogene Proteins c-bcl-2
Reactive Oxygen Species
Receptors, Transferrin
Signal Transduction
Superoxides
Time Factors