1,721,175 research outputs found
Dexamethasone Promotes Toxicity in U937 Cells Exposed to Otherwise Non-toxic Concentrations of Peroxynitrite: Pivotal Role for Lipocortin 1 Mediated Inhibition of Cytosolic Phospholipase A2
No full textPretreatment with dexamethasone (Dex) was not toxic for U937 cells but caused a rapid lethal response upon subsequent exposure to otherwise nontoxic concentrations of peroxynitrite. This effect was not associated with enhanced formation of hydrogen peroxide taking place after peroxynitrite and was shown previously to play a pivotal role in the ensuing lethal response. Further analyses revealed that although Dex did not affect cytosolic phospholipase A(2) (cPLA(2)) expression, it markedly reduced the extent of arachidonic acid (AA) release mediated by peroxynitrite-dependent stimulation of cPLA(2). This event, as well as the enhanced toxicity, was abolished by mifepristone, a glucocorticoid receptor antagonist. The outcome of various approaches, using phospholipase A(2) inhibitors, cPLA(2) antisense oligonucleotide-transfected cells, and supplementation with exogenous AA, led to the demonstration that inhibition of cPLA(2) activity is causally linked to the increased susceptibility to peroxynitrite caused by Dex. Finally, the effects of Dex were shown to be mediated by enhanced expression of lipocortin 1 (LC1), a cPLA(2) inhibitory protein. These results indicate that Dex promotes toxicity in U937 cells exposed to otherwise nontoxic concentrations of peroxynitrite and that this event is causally linked to enhanced expression of LC1 leading to inhibition of cPLA(2). Thus, the increased lethal response arises because of LC1-dependent impairment of the AA-induced cytoprotective mechanism triggered by peroxynitrite
Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2.
No full textExposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A(2) [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539-1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain, In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A(2). This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A, inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A, inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A(2). In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA, isoform, probably localized in the mitochondria
Mitochondrial H2O2 limits U937 cell survival to peroxynitrite by promonting ERK1/2 dephosphorylation.
No full textSequential activation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), critically regulated by extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent phosphorylation, mediates U937 cell survival to peroxynitrite. In contrast, a limiting factor is represented by the parallel mitochondrial formation of H2O2 leading to suppression of the survival signaling. We now report that the inhibitory effects of H2O2 are at the level of ERK1/2 phosphorylation and involve activation of orthovanadate-sensitive phosphotyrosine protein phosphatase(s). Under these conditions, the otherwise stimulatory effects of peroxynitrite on ERK1/2 phosphorylation are concealed by phosphatase-dependent dephosphorylation and the activities of cPLA2 and 5-LO are significantly reduced or suppressed, respectively. The ensuing inhibition of downstream events preventing mitochondrial permeability transition rapidly leads these cells to death. Thus, endogenous H2O2 limits U937 cell survival to peroxynitrite via activation of phosphotyrosine protein phosphatase(s) promoting upstream inhibition of the survival signaling critically regulated by the extent of ERK1/2 phosphorylation
Peroxynitrite damages U937 cell DNA via the intermediate formation of mitochondrial oxidants.
No full textEight years ago we published in this journal the first evidence that peroxynitrite does not directly produce DNA single-strand breakage in intact U937 cells (Guidarelli et al., IUBMB Life 50. 195-201). This event was rather attributed to the secondary reactive species produced at the mitochondrial level via a Ca2+-dependent reaction in which ubisemiquinone serves as an electron donor. Under these conditions, electrons are directly transferred to molecular oxygen and superoxide/H2O2, and the ensuing DNA damage can therefore be produced in a time dependent manner for at least 30 min. Formation of H2O2 and DNA single-strand breaks was therefore dependent on interference with electron transport at the complex Ill level as well as on mitochondrial Ca2+ accumulation. Further studies led to the demonstrations that peroxynitrite mobilizes Ca2+ from the ryanodine receptor. Finally. in U937 cells, a pro-monocytic cell line sharing with monocytes/macrophages the same signaling events to survive to peroxynitrite. mitochondrial H2O2 promotes inhibition of survival via tyrosine phosphatase activation, leading to ERK1/2 dephosphorylation and thus to upstream inhibition of the survival signaling
Correlations of DNA strand breaks and their repair with cell survival following acute exposure to mercury(II) and X-rays.
No full textAnalysis of the induction of alkali sensitive sites in the DNA by chromate and other agents that induce single strand breaks.
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Delayed formation of hydrogen peroxide mediates the lethal response evoked by peroxynitrite in U937 cells
Full text linkThe toxicity paradigm used in the present study involves exposure of U937 cells to a concentration of authentic peroxynitrite, leading to a rapid necrotic response mediated by mitochondrial permeability transition. We found that addition of catalase after treatment with peroxynitrite specifically prevents the loss of mitochondrial membrane potential and the ensuing lethal response. The protective effects of catalase were mimicked by the cocktail glutathione peroxidase/reduced glutathione. A defensive role of intracellular catalase was implied by experiments showing that catalase-depleted cells are hypersensitive to peroxynitrite and that cells with an increased catalase content, selected for their resistance to H2O2, are cross-resistant to peroxynitrite. Further experiments demonstrated that H2O2 formation takes place after peroxynitrite exposure. Various approaches using inhibitors of the mitochondrial respiratory chain as well as respiration-deficient cells revealed that the oxidant is produced upon dismutation of superoxides generated at the level of complex III. Interestingly, respiration-deficient cells were found to be resistant to peroxynitrite toxicity, and all those treatments increasing formation of H2O2produced a parallel increase in toxicity. In conclusion, the results presented in this study indicate that peroxynitrite-induced impairment of electron transport from cytochrome b to cytochromec1 leads to delayed formation of hydrogen peroxide, which plays a pivotal role in the ensuing necrotic response
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