title: Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide.
abstract: Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported. We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used. Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC. In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N(omega)-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha-induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 microM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.
authors: Laboratoire d'Anesthésie-Réanimation, Université Paris XI, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre; France.
authors: S
authors: S
authors: Corda
authors: C
authors: C
authors: Laplace
authors: E
authors: E
authors: Vicaut
authors: J
authors: J
authors: Duranteau
mesh_terms: D002518:Ceramides / Q000378:metabolism*; D004579:Electron Transport; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002518:Ceramides; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D013108:Sphingomyelin Phosphodiesterase