1,721,021 research outputs found
Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon-gamma.
No full textAddition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival
Arachidonic acid inhibits neuronal nitric oxide synthase elicited by proinflammatory stimuli and promotes astrocyte survival with both exogenous and endogenous peroxynitrite via different mechanisms.
No full textCytosolic phospholipase A(2)-inhibited astrocytes respond to the cocktail lipopolysaccharide/interferon-gamma with an immediate formation of peroxynitrite (ONOO(-)) and a delayed lethal response. Low concentrations of arachidonic acid (ARA; i.e., 3 microM, promoted cytoprotection when applied to the cultures up to 50 min after the formation of endogenous ONOO(-) had been completed or up to 30 min after addition of exogenous ONOO(-). The mechanism(s) involved in these responses was, however, independent of tyrosine kinase activation and was in fact mediated by ARA metabolites of the lipoxygenase pathway. These results are consistent with a scenario in which astrocytes respond to low or high amounts of ARA with the triggering of different pathways involved in the inflammatory response. Early nNOS inhibition mediated by very low levels of ARA is indeed critical for nuclear factor-kappaB activation, which is otherwise effectively inhibited by constitutive nitric oxide, and for preventing early formation of ONOO(-). Greater ARA concentrations promote survival in astrocytes committed to death by ONOO(-), a species extensively released under inflammatory conditions, via a mechanism dependent on lipoxygenase metabolism and inhibition of downstream events leading to cell demise
tert-Butylhydroperoxide induces peroxynitrite-dependent mitochondrial permeability transition leading PC12 cells to necrosis
Full text linkA short-term exposure of PC12 cells to tertbutylhydroperoxide,
followed by recovery in fresh culture
medium, causes cell death and the extent of this response
progressively increases during the 120 min of posttreatment
incubation. Morphological and biochemical analyses
of these cells revealed that the mode of cell death was
necrosis. Cell killing induced by the hydroperoxide seems
to be in part mediated by peroxynitrite because the lethal
response was markedly and similarly reduced by the nitric
oxide synthase inhibitor Nv-nitro-L-arginine methylester
and by scavengers of nitric oxide or peroxynitrite. This
peroxynitrite-dependent mechanism of cytotoxicity was
blunted by antioxidants and inhibitors of mitochondrial permeability
transition and the onset of cell death was preceded
by mitochondrial depolarization and loss of cellular
ATP. We conclude that tert-butylhydroperoxide promotes
peroxynitrite-dependent PC12 cell necrosis causally linked
to peroxidation of membrane lipids and mitochondrial permeability
transition
Peroxynitrite-mediated release of arachidonic acid from PC12 cells
No full textA short term exposure of PC12 cells to a concentration of tert-butylhydroperoxide (tB-OOH) causing peroxynitrite-dependent DNA damage and cytotoxiticity promoted a release of arachidonic acid (AA) that was sensitive to phospholipase A(2) (PLA(2)) inhibitors and insensitive to phospholipase C or diacylglycerol lipase inhibitors. The extent of AA release was also mitigated by nitric oxide synthase (NOS) inhibitors and peroxpnitrite scavengers. Low levels (10 mu M) of authentic peroxynitrite restored the release of AA mediated by tB-OOH in NOS-inhibited cells whereas concentrations of peroxynitrite of 20 mu M, or higher, effectively stimulated a PLA(2) inhibitor-sensitive release of AA also in the absence of additional treatments. These results are consistent with the possibility that endogenous as well as exogenous peroxynitrite promotes activation of PLA(2)
The antioxidant butylated hydroxytoluene induces apoptosis in human U937 cells: the role of hydrogen peroxide and altered redox state.
No full textExposure of U937 cells to the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT), unlike exposure to other antioxidants such as N,N'-diphenyl-1,4-phenylenediamine, Trolox or alpha-tocopherol, promotes a time- and concentration-dependent induction of apoptosis. This response was prevented by the iron chelator o-phenanthroline and by the thiol reagent N-acetylcysteine but was increased remarkably in cells pre-exposed to the catalase inhibitor 3-amino-1,2,4-triazole or to L-buthionine-[S,R]-sulfoximine, a specific inhibitor of glutathione synthesis. Furthermore, the BHT-induced apoptotic response was markedly enhanced by cytochrome P450 inhibitors. Taken together, the experimental results presented in this study indicate that BHT efficiently induces apoptosis in U937 cells and that this response is not caused by products of cytochrome P450 metabolism. Instead, apoptosis appeared to be causally linked to an altered cellular redox state in which hydrogen peroxide plays a pivotal rol
Arachidonic acid: a key molecule for astrocyte survival to peroxynitrite.
No full textNontoxic concentrations of peroxynitrite nevertheless commit rat astrocytes to mitochondrial permeability transition-dependent toxicity, however prevented by a signaling response driven by arachidonic acid (ARA). The lipid
messenger was released upon peroxynitrite-dependent activation of cytosolic phospholipase A2 and its pharmacological inhibition, or knock-down, was invariably associated with a prompt apoptotic response sensitive to exogenous ARA, but insensitive to other polyunsaturated fatty acids, as eicosapentaenoic or linoleic acid. Interestingly, while microglia also used ARA to cope with peroxynitrite, cerebellar granule cells were killed by the same concentrations of peroxynitrite employed in astrocyte/microglia experiments via a mechanism sensitive to inhibition of ARA release. These results collectively support the notion that resistance of glial cells to peroxynitrite, a species extensively produced under neuroinflammatory conditions, is largely based on a critical survival signaling triggered by the inflammatory product ARA. In remarkable contrast with these results, the lipid messenger appears to mediate toxicity in neuronal cells
Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.
Full text linkA 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis
Susceptibility of rat astrocytes to DNA strand scission induced by activation of NADPH oxidase and collateral resistance to the effects of peroxynitrite.
No full textRat astrocytes accumulate extensive DNA single-strand breakage in response to agents promoting activation of NADPH oxidase. Proinflammatory stimuli, as bacterial lipopolysaccharide associated with interferon-γ, caused a rapid/robust burst of superoxide radicals, sensitive to NADPH oxidase inhibition, followed by dismutation to H2O2, the species resulting in DNA damage via a Fenton-type reaction. There was no contribution of superoxide radical/H2O2 of mitochondrial origin and there was no evidence for the formation/involvement of peroxynitrite. On the other hand, astrocytes were virtually invulnerable to the DNA-damaging effects of exogenous peroxynitrite, an agent causing DNA strand scission in other cell types, via the Ca2+-dependent mitochondrial formation of superoxide radical/H2O2. Resistance was not dependent on scavenging of peroxynitrite but, rather, on insufficient mitochondrial Ca2+ accumulation. Hence, different manipulations resulting in an increase of the mitochondrial Ca2+ pool were invariably associated with the formation of DNA-damaging levels of H2O2. In conclusion, it appears that the strategy adopted by astrocytes to avoid inflammation-dependent genotoxic events, in particular those mediated by peroxynitrite, is to prevent mitochondrial Ca2+ accumulation, critical for the formation of secondary species largely responsible for DNA damage induced by peroxynitrite
Peroxynitrite-induced mitochondrial translocation of PKCalpha causes U937 cell survival.
No full textOur previous work has shown that non-toxic concentrations of peroxynitrite nevertheless commit U937 cells to mitochondrial permeability-transition (MPT)-dependent necrosis that is however prevented by a parallel survival signaling pathway involving cytosolic phospholipase A(2) (cPLA2)-dependent arachidonic acid release and PKC alpha activation associated with the cytosolic translocation of Bad. The present study provides evidence of an early mitochondrial translocation of PKC alpha. Inhibition of the survival signaling at the level of either cPLA(2), or PKC, was invariably associated with prevention of the mitochondrial localization of PKC alpha, with the mitochondrial translocation of Bad and Bax and with a very rapid lethal response. Collectively, the results presented in this study demonstrate that peroxynitrite, while committing U937 cells to necrosis, triggers a parallel signaling response leading to the cytosolic localization of two important members of the Bcl-2 family implicated in the onset of MPT
The effect of hydrogen peroxide/L-histidine-induced DNA single- vs. double-strand breaks on poly(ADP-ribose)polymerase.
No full textL-Histidine markedly increases the ability of hydrogen peroxide to induce DNA cleavage and this effect is associated with a 3-aminobenzamide-inhibitable decline in NAD(+) levels, an event which very likely reflects an enhanced stimulation of the enzyme poly(ADP-ribose)polymerase. 3-Aminobenzamide slowed down the removal of alkaline elution-detected strand breaks induced by either H2O2 alone (producing only DNA single-strand breaks) or associated with L-histidine (resulting in the formation of both single-strand breaks and DNA double-strand breaks), and the extent of inhibition was similar under the two experimental conditions. 3-Aminobenzamide did not affect the rate of rejoining of DNA double-strand breaks generated by the cocktail H2O2/L-histidine. The above results suggest that these double-strand breaks have hardly any effect on the induction of poly(ADP-ribose)polymerase activity, a conclusion that is consistent with the observation that the activity of this enzyme appears to be basically identical under conditions that abolish the formation of DNA double-strand breaks, in the absence of measurable variations in the level of induction of DNA single-strand breaks (e.g. in the presence of an excess of L-glutamine, a competitive inhibitor of L-histidine uptake). Finally, 3-aminobenzamide did not affect the toxicity of the oxidant, both in the absence and presence of L-histidine
- …
