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J. Biol. Chem., Vol. 279, Issue 27, 28614-28624, July 2, 2004

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Ceramide-induced Intracellular Oxidant Formation, Iron Signaling, and Apoptosis in Endothelial Cells

PROTECTIVE ROLE OF ENDOGENOUS NITRIC OXIDE^*

Toshiyuki Matsunaga, Srigiridhar Kotamraju, Shasi V. Kalivendi, Anuradha Dhanasekaran, Joy Joseph, and B. Kalyanaraman

From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

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-, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (^·NO) during hydrogen peroxide (H₂O₂)-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C₂-ceramide (C₂-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C₂-cer (5–20 µM) to BAECs enhanced ^·NO generation. However, at higher concentrations of C₂-cer (20 µM), ^·NO generation did not increase proportionately. C₂-cer (20–50 µM) also resulted in H₂O₂-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of ⁵⁵Fe, 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 C₂-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C₂-cer (10 µM) but was greatly suppressed at higher concentrations (>10 µM). Intracellular scavengers of H₂O₂, cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C₂-cer- and/or L-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C₂-cer-induced H₂O₂ and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that ^·NO exerts an antioxidative and cytoprotective role.

Received for publication, January 28, 2004 , and in revised form, March 31, 2004.

^* This work was supported by National Institutes of Health Grants HL073056-01 and 1PO1HL68769-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-4035; Fax: 414-456-6512; E-mail: balarama{at}mcw.edu.

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