DIRECT EFFECT OF CERAMIDE ON THE MITOCHONDRIAL ELECTRON-TRANSPORT CHAIN LEADS TO GENERATION OF REACTIVE OXYGEN SPECIES - ROLE OF MITOCHONDRIAL GLUTATHIONE

Ceramide is a sphingolipid that is generated in the signaling of infla mmatory cytokines such as tumor necrosis factor (TNF), which exerts ma ny functional roles depending on the cell type where it is produced. S ince TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in g eneration of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C-2- ceramide) to mitochondria led to an increase of fluorescence of dihydr orhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 m u M and maximal at 1-5 mu M C-2, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosi ne at the same concentration range, whereas the immediate precursor of C-2, C-2-dihydroceramide increased hydrogen peroxide at 1-5 mu M. Sph ingosine generated hydrogen peroxide at concentrations greater than or equal to 10 mu M, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C-2 was not tri ggered by mitochondrial permeability transition as C-2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C-2; ho wever, interruption of electron flow at complex III by antimycin A pot entiated the inductive effect of C-2. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of h ydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted m itochondria. Mitochondria isolated from TNF-treated cells showed an in crease (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a t arget of ceramide produced in the signaling of TNF whose effect on mit ochondrial electron transport chain leads to overproduction of hydroge n peroxide and consequently this phenomena may account for the generat ion of reactive oxygen species during TNF cytotoxicity.