Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

doi:10.1074/jbc.272.17.11369

Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

ROLE OF MITOCHONDRIAL GLUTATHIONE*

From the Instituto Investigaciones Biomédicas, Consejo Superior Investigaciones Científicas and the Liver Unit and Servicio de Bioquímica, Department of Medicine, Hospital Clinic i Provincial, Universidad de Barcelona, Barcelona 08036, Spain

^∥ To whom all correspondence and reprint requests should be addressed:
Liver Unit, Hospital Clinic i Provincial, Villarroel, 170, 08036 Barcelona, Spain.
Fax: 34-3-451-5272.

Abstract

Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C₂-ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 μM and maximal at 1-5 μM C₂, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C₂, C₂-dihydroceramide increased hydrogen peroxide at 1-5 μM. Sphingosine generated hydrogen peroxide at concentrations ≥10 μM, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C₂ was not triggered by mitochondrial permeability transition as C₂ did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C₂; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C₂. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity.

Footnotes

↵^‡ Supported by a postdoctoral contract from the Ministerio de Educación y Ciencia.
↵^§ Supported in part by a grant from Europharma.
↵^¶ Fellow of the FISS.
↵* This study was supported in part by Grant AA09526 from NIAAA, National Institutes of Health; Grants PB92-1110 and PM 95-0185 from Dirección General Política Científica y Técnica; Grant 94-0046/01 from Fondo Investigaciones Sanitarias (FISS), and Europharma. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TNF

tumor necrosis factor

AA

antimycin A

BSO

buthionine-L-sulfoximine

C₂

N-acetylsphingosine

C₆

N-hexanoylsphingosine

C₂DH

dihydro-C₂

DAG

1,2-diacylglycerol

DEM

diethylmaleate

DHR

dihydrorhodamine 123

DCF

2′-7′-dichlorofluorescein

DCFDA

2′-7′-dichlorofluorescin diacetate

MOPS

4-morpholinepropanesulfonic acid

NF-κB

nuclear factor κB

PC-PLC

phosphatidylcholine-dependent phospholipase C

Q

ubiquinone

ROS

reactive oxygen species

SMase

sphingomyelinase

TTFA

thenoyltrifluoroacetone.
↵² C. García-Ruiz, A. Morales, M. Marí, M. Miranda, A. Colell, and J. C. Fernández-Checa, manuscript in preparation.
- Received September 17, 1996.
- Revision received December 30, 1996.