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

J Biol Chem. 2004 Jul 2;279(27):28614-24. doi: 10.1074/jbc.M400977200. Epub 2004 Apr 21.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antioxidants / pharmacology
  • Apoptosis*
  • Blotting, Western
  • Caspase 3
  • Caspases / metabolism
  • Cattle
  • Cells, Cultured
  • Ceramides / metabolism
  • Ceramides / pharmacology*
  • Cytochromes c / metabolism
  • DNA Fragmentation
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / metabolism*
  • Enzyme Activation
  • Fluoresceins / metabolism
  • Glutathione / metabolism
  • Hydrogen Peroxide / pharmacology
  • In Situ Nick-End Labeling
  • Iron / metabolism*
  • Microscopy, Fluorescence
  • Mitochondria / metabolism
  • Models, Chemical
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Neurons / metabolism
  • Nitric Oxide / metabolism*
  • Oxidants / metabolism*
  • Phenanthridines / pharmacology
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Reactive Oxygen Species
  • Receptors, Transferrin / metabolism
  • Signal Transduction
  • Superoxides / metabolism
  • Time Factors


  • Antioxidants
  • Ceramides
  • Fluoresceins
  • Oxidants
  • Phenanthridines
  • Proto-Oncogene Proteins c-bcl-2
  • Reactive Oxygen Species
  • Receptors, Transferrin
  • 2',7'-dichlorodihydrofluorescein
  • Superoxides
  • Nitric Oxide
  • hydroethidine
  • Cytochromes c
  • Hydrogen Peroxide
  • Iron
  • Caspase 3
  • Caspases
  • Glutathione
  • NG-Nitroarginine Methyl Ester