Oxidative stress can activate the epidermal platelet-activating factor receptor.
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Platelet-activating factor (1-alkyl-2-acetyl-glycero-phosphocholine) is a lipid mediator that has been implicated in keratinocyte function and cutaneous inflammation. Keratinocytes both synthesize platelet-activating factor and express functional platelet-activating factor receptors linked to calcium mobilization. Oxidative stress to various cells including keratinocytes can also result in the mobilization of intracellular Ca2+, a known stimulus for platelet-activating factor biosynthesis. The ability of the epidermal platelet-activating factor receptors to modulate oxidant-induced signaling was investigated using a unique model system created by retroviral-mediated transduction of the platelet-activating factor receptor-negative epithelial cell line KB with the platelet-activating factor receptor. Treatment of KB cells with the lipid pro-oxidant tert-butyl hydroperoxide induced transient increases in intracellular Ca2+ in a concentration-dependent fashion. Expression of the platelet-activating factor receptor in KB cells lowered the threshold for tert-butyl hydroperoxide-induced Ca2+ flux by an order of magnitude (10 microM in control KB versus 1 microM in KB cells expressing the platelet-activating factor receptors) and increased the peak change in intracellular Ca2+ concentration in response to this lipid hydroperoxide. This augmentation of tert-butyl hydroperoxide-induced Ca2+ mobilization was inhibited by pretreatment with the two competitive platelet-activating factor receptor antagonists CV-6209 and WEB 2086, as well as by the antioxidants vitamin E and 1,1,3,3-tetramethyl-2-thiourea. KB cells synthesized platelet-activating factor and the platelet-activating factor receptor agonist 1-palmitoyl-2-acetyl-glycero-phosphocholine in response to tert-butyl hydroperoxide treatment, suggesting the augmentation of oxidative stress-induced signaling seen in platelet-activating factor receptor-expressing cells was due in part to endogenous platelet-activating factor biosynthesis. These studies suggest involvement of the epidermal platelet-activating factor receptors in oxidant-mediated signaling. (+info)
Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells.
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PURPOSE: To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS: Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t-butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtdelta psi) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS: t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL-positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtdelta psi was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS: Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins. (+info)
Roles of oxygen radicals and elastase in citric acid-induced airway constriction of guinea-pigs.
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Antioxidants attenuate noncholinergic airway constriction. To further investigate the relationship between tachykinin-mediated airway constriction and oxygen radicals, we explored citric acid-induced bronchial constriction in 48 young Hartley strain guinea-pigs, divided into six groups: control; citric acid; hexa(sulphobutyl)fullerenes + citric acid; hexa(sulphobutyl)fullerenes + phosphoramidon + citric acid; dimethylthiourea (DMTU) + citric acid; and DMTU + phosphoramidon + citric acid. Hexa(sulphobutyl)fullerenes and DMTU are scavengers of oxygen radicals while phosphoramidon is an inhibitor of the major degradation enzyme for tachykinins. Animals were anaesthetized, paralyzed, and artificially ventilated. Each animal was given 50 breaths of 4 ml saline or citric acid aerosol. We measured dynamic respiratory compliance (Crs), forced expiratory volume in 0.1 (FEV0.1), and maximal expiratory flow at 30% total lung capacity (Vmax30) to evaluate the degree of airway constriction. Citric acid, but not saline, aerosol inhalation caused marked decreases in Crs, FEV0.1 and Vmax30, indicating marked airway constriction. This constriction was significantly attenuated by either hexa(sulphobutyl)fullerenes or by DMTU. In addition, phosphoramidon significantly reversed the attenuating action of hexa(sulphobutyl)fullerenes, but not that of DMTU. Citric acid aerosol inhalation caused increases in both lucigenin- and t-butyl hydroperoxide-initiated chemiluminescence counts, indicating citric acid-induced increase in oxygen radicals and decrease in antioxidants in bronchoalveolar lavage fluid. These alterations were significantly suppressed by either hexa(sulphobutyl)fullerenes or DMTU. An elastase inhibitor eglin-c also significantly attenuated citric acid-induced airway constriction, indicating the contributing role of elastase in this type of constriction. We conclude that both oxygen radicals and elastase play an important role in tachykinin-mediated, citric acid-induced airway constriction. (+info)
Thioredoxin peroxidase in the Cyanobacterium Synechocystis sp. PCC 6803.
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The amino acid sequence deduced from the open reading frame designated sll0755 in Synechocystis sp. PCC 6803 is similar to the amino acid sequences of thioredoxin peroxidases from other organisms. In the present study, we found that a recombinant SLL0755 protein that was expressed in Escherichia coli was able to reduce H2O2 and tertiary butyl hydroperoxide with thioredoxin from E. coli as the electron donor. Targeted disruption of open reading frame sll0755 in Synechocystis sp. PCC 6803 cells completely eliminated the H2O2-dependent and tertiary butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II. These results indicate that the product of open reading frame sll0755 is a thioredoxin peroxidase whose activities are coupled to the photosynthetic electron transport system in Synechocystis sp. PCC 6803. (+info)
Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium.
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Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase. (+info)
Propofol prevents peroxide-induced inhibition of glutamate transport in cultured astrocytes.
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BACKGROUND: Glutamate transporters located in the plasma membrane of cerebral astrocytes take up excitatory neurotransmitters from the synaptic cleft. In diseases characterized by oxidative stress, the extracellular glutamate concentration increases and contributes to neuronal death. The authors wanted to determine whether propofol defends brain cells against oxidant-induced changes in their transport of glutamate. METHODS: Primary cultures of rat cerebral astrocytes were exposed to tert-butyl hydroperoxide (1 mM) to serve as an in vitro model of oxidative stress. Astrocytes were incubated with propofol for 2 h and tert-butyl hydroperoxide was added for the final hour. Alternatively, astrocytes were incubated with tert-butyl hydroperoxide for 30 min and then with propofol for another 30 min. Control cells received drug vehicle rather than propofol. The rate of uptake of glutamate, the efflux of the nonmetabolizable analog D-aspartate, and the intracellular concentration of the endogenous antioxidant glutathione were measured. RESULTS: Tert-butyl hydroperoxide decreased the glutathione concentration and inhibited glutamate uptake but had a negligible effect on D-aspartate efflux. At clinically relevant concentrations, propofol did not affect the glutathione concentration but did prevent the effect of tert-butyl hydroperoxide on glutamate transport. Furthermore, the addition of propofol after tert-butyl hydroperoxide reversed the inhibition of glutamate uptake. CONCLUSIONS: Propofol prevents and reverses the inhibition of excitatory amino acid uptake in astrocytes exposed to tert-butyl hydroperoxide. The ability of propofol to defend against peroxide-induced inhibition of glutamate clearance may prevent the pathologic increase in extracellular glutamate at synapses, and thus delay or prevent the onset of excitotoxic neuronal death. (+info)
Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.
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The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis. (+info)
Effects of oleic acid, docosahexaenoic acid and eicosapentaenoic acid on background and genotoxin-induced frequencies of SCEs in Indian muntjac fibroblasts.
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Muntjac cells were cultured at 5 X 10(5) cells/10 cm Petri dish for 24 h prior to addition of fatty acids (50 microM) which were delivered to the cells complexed with 2% bovine serum albumin (fatty acid-free) and incubated for a further 24 h. Parallel dishes were processed for lipid extraction and GC analysis. This analysis showed highly significant (P < 0.01) uptake by the cells of each fatty acid. Genotoxins (75 microM hydrogen peroxide, 20 microM t-butylhydroperoxide and 2.4 microM mitomycin C) were added to the cells for 1 h prior to the end of the 24 h fatty acid incubation period. Control (no genotoxin or fatty acid) treatments were included. No difference was observed in background frequencies of SCEs between controls and fatty acid treatments, thus indicating that these fatty acids per se do not cause DNA damage. The cells incubated with the genotoxins showed increased (P < 0.05) frequencies of SCEs when compared with control frequencies. Cells incubated with genotoxins in the presence of fatty acids also showed significantly higher (P < 0.05) levels of SCEs when compared with control frequencies. When cells supplemented with genotoxins in the presence of fatty acids were compared with cells treated with genotoxins alone, higher levels of SCEs were observed in the former, suggesting that the fatty acids exacerbate DNA damage caused by these genotoxins. (+info)