Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes.

Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.     

Redox regulation of copper-metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios 相似文献   

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.     

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.     

Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.