Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity

Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma. Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal-glial cell cultures. Cells (13-16 days in vitro) were exposed to 50 microM hydrogen peroxide (H2O2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure. The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury. This protection was not reversed by pre-treatment with receptor associated protein. The NMDA receptor antagonist, MK-801, also provided partial protection against H2O2 toxicity. The degree of protection was similar to that conferred by ApoE treatment. The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H2O2 exposure. ApoE treatment had no effect on H2O2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H2O2 treated cultures. Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release. Taken together, these findings suggest that ApoE protects mixed neuronal-glial cell cultures against irreversible oxidative injury from H2O2 by reducing secondary glutamate excitotoxicity.     

Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae

A number of studies indicate that reactive oxygen species (ROS) are involved in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The neuroprotective effects of salvianolic acid B (SalB) from Radix Salviae miltiorrhizae (RSM) against hydrogen peroxide (H2O2)-induced rat pheochromocytoma line PC12 injury were evaluated in the present study. Vitamin E, a potent antioxidant, was employed as a positive control agent. Following exposure of cells to H2O2 (150 microM), a marked decrease in cell survival and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. In parallel, H2O2 caused significant elevation in intracellular Ca2+ level and caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with SalB (0.1-10 microM) prior to H2O2 exposure blocked these H2O2-induced cellular events noticeably. Moreover, SalB exhibited significantly higher potency as compared to Vitamin E. The present findings indicated that SalB exerts neuroprotective effects against H2O2 toxicity, which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases.     

Inhibition of protein phosphatases impairs the ability of astrocytes to detoxify hydrogen peroxide

We have used protein phosphatase (PP) inhibitors and rat cerebellar glial cells in primary culture to investigate the role of PP activity in the ability of glial cells to detoxify exogenously applied hydrogen peroxide (H2O2). The marine toxin okadaic acid (OKA), a potent PP1 and PP2A inhibitor, caused a concentration-dependent degeneration of astrocytes and increased the formation of hydroperoxide radicals significantly. Subtoxic exposures to OKA significantly potentiated toxicity by exogenous H2O2. The concentration of H2O2 that reduced by 50% the survival of astrocytes after 3 h was estimated at 720+/-40 microM in the absence and 85+/-30 microM in the presence of the toxin. The PP inhibitors calyculin A and endothall also potentiated H2O2 toxicity in cerebellar astrocytes. OKA caused a time-dependent inhibition of both glial catalase and glutathione peroxidase, reducing by approximately 50% the activity of these enzymes after 3 h, whereas other enzymatic activities remained unaffected. Also, OKA reduced the cellular content of total glutathione and elevated oxidized glutathione to about 25% of total glutathione. OKA-treated astrocytes cleared H2O2 from the incubation medium approximately two times more slowly than control cultures. Our results suggest a prominent role for PP activity in the antioxidant mechanisms protecting astrocytes against damage by H2O2.     

Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108

A number of studies indicate that free radicals are involved in the neurodegeneration in Parkinson's and Alzheimer's diseases. EPS2, an exopolysaccharide with a mean molecular weight of 1.3 x 10(5) Da, was isolated by ion-exchange and sizing chromatography from the culture of Keissleriella sp. YS4108, a marine filamentous fungus. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The protective effects of EPS2 on peroxide hydrogen (H2O2)-induced cell lesion, level of lipid peroxidation, antioxidant enzyme activities were investigated in the rat pheochromocytoma line PC12 cells. Following a 1-h exposure of the cells to H2O2, a significant reduction in cell survival and activities of glutathione peroxidase (GSH-Px) and catalase (CAT), as well as increased levels in malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. However, preincubation of the cells with EPS2 prior to H2O2 exposure elevated the cell survival and GSH-Px and CAT activities, and decreased the level of MDA and LDH activity in a dose-dependent manner. In conclusion, EPS2 possesses pronounced protective effects against H2O2-induced cell toxicity. The finding is of a higher value in searching for new therapeutic agent for treating oxidative damage-derived neurodegenerative disorders.     

High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.     

Clioquinol inhibits peroxide-mediated toxicity through up-regulation of phosphoinositol-3-kinase and inhibition of p53 activity

A growing body of evidence supports a central role for biometals in neurodegenerative disorders. Biometals induce oxidative stress through the generation of reactive oxygen species and contribute to neuronal cell dysfunction in Alzheimer's disease (AD), prion disorders and Parkinson's disease (PD). Therapies based on modulation of biometal metabolism are currently being developed and the metal ligand, 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol or CQ) has been investigated for the treatment of AD. CQ has also shown therapeutic benefits in an animal model of PD. However, little is known about the neuroprotective processes of CQ in vivo. In this study, we examined the effect of CQ in BE(2)-M17 human neuroblastoma cells exposed to increased oxidative stress (hydrogen peroxide (H2O2) treatment). Although CQ alone induced a moderate toxic effect on cells, when added to H2O2-treated M17 cells, CQ induced a significant inhibition of H2O2 toxicity. This correlated with up-regulation of phosphoinositol-3-kinase (PI3K) activity in CQ-treated cells. The protective action of CQ was not observed in murine N2a neuroblastoma cells treated with H2O2 and this cell-line did not reveal CQ-mediated increases in PI3K activation. The protective effect was specific for CQ and was not induced by a number of different metal ligands. Inhibition of PI3K activity with LY294002 prevented CQ protection against H2O2 toxicity, demonstrating a crucial role for CQ activation of PI3K in protection against oxidative stress. Furthermore, CQ inhibited H2O2-mediated up-regulation of p53 activity in the M17 cells and this was dependent on PI3K activation. Our studies demonstrate that in human M17 cells, CQ can protect against oxidative stress by activating the PI3K-dependent survival pathway and blocking p53-mediated cell death. These findings have important implications for the development of protective metal ligand-based therapies for treatment of disorders involving oxidative stress.     

Evaluation of the protective effect of oestradiol against toxicity induced by 6-hydroxydopamine and 1-methyl-4-phenylpyridinium ion (Mpp+) towards dopaminergic mesencephalic neurones in primary culture

Recent findings suggest that gonadal steroid hormones are neuroprotective and may provide clinical benefits in delaying the development of Parkinson's disease. In this report we investigated the ability of oestradiol to protect mesencephalic dopaminergic neurones cultured in serum-free or serum-supplemented medium from toxicity induced by 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion (MPP+). The efficiency of both toxins and oestradiol was evaluated by tyrosine hydroxylase (TH) immunocytochemistry, [3H]dopamine ([3H]DA) uptake, length of dopaminergic processes and lactate dehydrogenase (LDH) release measurement. In cultures grown in serum-supplemented medium, a 2-h pre-treatment with high concentrations (10-100 microM) of 17beta-oestradiol or 17alpha-oestradiol, the stereoisomer with weak oestrogenic activity, protected both dopaminergic and non-dopaminergic neurones from toxicity induced by 6-hydroxydopamine (6-OHDA; 40 or 100 microM) and by the high MPP+ concentrations (50 microM) necessary to obtain significant neuronal death under those culture conditions. At these concentrations, MPP+ was no longer selective for dopaminergic neurones but affected all cells present in the culture. In contrast, the hormonal treatments did not protect against selective degeneration of dopaminergic neurones induced by lower MPP+ concentrations (below 10 microM), related to inhibition of complex I of respiratory chain. In cultures grown in serum-free medium, oestradiol concentrations higher than 1 microM induced neuronal degeneration and no protection against 6-OHDA or MPP+ toxicity was observed at lower concentrations of the steroid. The neuroprotective effects of 17alpha- or 17beta-oestradiol evidenced in this model might be due to the antioxidant properties of these compounds. However, other non-genomic effects of the steroids cannot be excluded.     

Glutathione and catalase provide overlapping defenses for protection against respiration-generated hydrogen peroxide in Haemophilus influenzae

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H2O2) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H2O2 scavenging activity and, accordingly, substantial amounts of H2O2 accumulate in the growth medium. H. influenzae generates H2O2 at rates similar to those reported for Escherichia coli, but the toxicity of this harmful metabolite is averted by glutathione-based H2O2 removal, which appears to be the primary system for protection against H2O2 endogenously generated during aerobic respiration. When H2O2 concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.     

Effect of hydrogen peroxide on reoxygenation-induced Ca2+ accumulation in rat cardiomyocytes

Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca(2+) homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H(2)O(2)) on Ca(2+) accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca(2+) ([Ca(2+)](i)) and Mg(2+)([Mg(2+)](i)), and mitochondrial membrane potential (Deltapsim). Cell Ca(2+) was determined by (45)Ca(2+) uptake. Free [Mg(2+)](i) and [Ca(2+)](i) and Deltapsim were measured by flow cytometry. Reoxygenation-induced Ca(2+) accumulation was attenuated by 23 and 34% by 10 and 25 microM H(2)O(2), respectively, added at reoxygenation. H(2)O(2) at 100 and 250 microM increased cell Ca(2+) by 50 and 83%, respectively, whereas 500 microM H(2)O(2) decreased cell Ca(2+) by 20%. H(2)O(2) at (25 microM) reduced LDH release and [Mg(2+)](i) and increased Deltapsim, indicating cell protection, whereas 250 microM H(2)O(2) increased LDH release and [Mg(2+)](i) and decreased Deltapsim, indicating cell damage. Clonazepam (100 microM) attenuated the increase in Ca(2+) accumulation, the elevation of [Ca(2+)](i), and the decrease in Deltapsim induced by 100 and 250 microM H(2)O(2) during reoxygenation. We report for the first time that 25 microM H(2)O(2) attenuates Ca(2+) accumulation, LDH release, and dissipation of Deltapsim during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.     

H2O2 mediates O2 toxicity in cultured fetal rat distal lung epithelial cells.

It is unknown which of the reactive oxygen species is primarily responsible for the cytotoxicity of 95% O2 for rat distal fetal lung epithelial cells in vitro. Incubation of cells with 25 U/ml polyethylene glycol (PEG)-conjugated SOD and 50 U/ml PEG-catalase, but not PEG-SOD or SOD mimics alone, significantly reduced 95% O2-mediated cytotoxicity. Liposome-entrapped catalase, without SOD, also significantly reduced 95% O2-mediated cytotoxicity. Increased formation of lipid hydroperoxides, as assessed by the formation of 8-isoprostane and aldehydes, was attenuated by both 100 microM Trolox, a vitamin E analogue, and by 5 microM U74389G, an amino steroid. Trolox, but not U74389G, prevented an increase in cell-derived H2O2, hydroxyl radical and 95% O2-mediated cytotoxicity. An increase in hydroxyl radical formation, but not cell death, observed in 95% O2, was prevented by 0.1 microM phenanthrolene, a cell permeant iron chelator. DNA extracts of rat distal fetal lung epithelial cells maintained under serum-free conditions had an electrophoretic pattern consistent with some degree of apoptosis. However, no increase in laddering was seen with exposure to 95% O2. These data are consistent with hydrogen peroxide, but not lipid hydroperoxides or hydroxyl radical, being a critical effector of O2-mediated necrotic cell death in distal lung epithelial cells.     

An endogenous metabolite of dopamine, 3,4-dihydroxyphenylethanol, acts as a unique cytoprotective agent against oxidative stress-induced injury

A natural compound contained in olive oil, 3,4-dihydroxyphenylethanol (DOPE), is also known as an endogenous metabolite of dopamine. The role of DOPE in oxidative stress-induced cell damage was investigated using differentiated PC12 cells. Superoxide (O(2)(-)) and H(2)O(2) induced a dose-dependent leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by MTT assay. While O(2)(-) -induced cell damage was not affected by DOPE, pretreatment of the cells with DOPE dose-dependently prevented the leakage of LDH induced by H(2)O(2). In these cells, augmented activity of catalase was demonstrated, while the levels of glutathione and glutathione peroxidase activity remained unchanged. The effect of DOPE was abolished when an inhibitor of catalase 3-amino-l, 2,4-triazole, was included in the medium. DOPE also protected against cell damage induced by H(2)O(2), and Fe(2+). In the hydroxyl radical ((.-)OH) assay using p-nitroso-N, N-dimethylaniline (PNDA), oxidation of PNDA by (.-)OH generated by the Fenton reaction was significantly attenuated in the presence of DOPE. By an electron spin resonance spin trapping study that represents the direct activity of DOPE to scavenge (.-)OH, however, limited scavenging activity was demonstrated for DOPE. Taken together, DOPE may act as a unique cytoprotective compound in nerve tissue subjected to oxidative stress.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process

Nitric oxide (*NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an *NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after *NO exposure. Altered thiol metabolism was observed immediately after *NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, gamma-glutamylcysteine, and cysteine), which then reaccumulated during the *NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to *NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in *NO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that *NO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.     

Constitutive hsp70 attenuates hydrogen peroxide-induced membrane lipid peroxidation

Thermal pretreatment improves cardiac recovery from subsequent ischemia/reperfusion. Induction of heat shock proteins (hsps) may contribute to this protection. We have demonstrated that augmentation of the constitutive hsp70 (hsc70) in H9c2 heart myoblasts promotes oxidative resistance. We employed a model oxidant to explore potential target(s) of protection by hsc70. Upon exposure to 54 microM of hydrogen peroxide (H(2)O(2)), hsc70-overexpressing cells exhibited a lower lipid peroxidation than the sham-transfected control. Constitutive hsc70 overexpression, however, did not protect against H(2)O(2)-induced depletion of ATP and glutathione (GSH). Lipid protection also occurred in cells preconditioned at 39 degrees C (selectively induces hsc70) during H(2)O(2) exposure. Interestingly, the protection conferred by hsc70 was comparable in magnitude to that provided by alpha-tocopherol, and was followed with a reduced release of lactate dehydrogenase and a unaltered calcium uptake during H(2)O(2) challenge. Collectively, our observations suggest that hsc70 may preserve membrane function via attenuation of lipid peroxidation during oxidative insult.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart

Adult (6 months) and senescent (greater than 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of 1/sec, cardiac relaxation (90% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P less than 0.05). Additional experiments indicated that H2O2 (500 microM), a powerful oxidant, increased resting force and decreased developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 81 +/- 6% and senescent = 42 +/- 27% of pre-H2O2 values; P less than 0.05). Age-related differences in effects of H2O2 did not result simply from a decreased ability of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H2O2-mediated decreases in GSH were similar in adult and senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway.     

热休克反应对培养的大鼠脑星形胶质细胞的保护作用及对其IL-6分泌的影响

采用ＭＴＴ还原法和乳酸脱氢酶释放法研究热休克２反应对新生大鼠脑星形胶质细胞２的保护作用。结果表明,热休克反应能增强星形胶持细胞对Ｈ２Ｏ２耐受力。实验还测定了热休克反应对新生大鼠脑星形胶质细胞白细胞介素－６释放的影响。结果显示,热休克反应后６ｈ,星形胶质的细胞ＩＬ－６释放明显增多。     

Proteasome inhibition induces glutathione synthesis and protects cells from oxidative stress: relevance to Parkinson disease

The cause of selective dopaminergic neuronal degeneration in Parkinson disease has still not been resolved, but it has been hypothesized that oxidative stress and the ubiquitin-proteasome system are important in the pathogenesis. In this report, we investigated the effect of proteasome inhibition on oxidative stress-induced cytotoxicity in PC12 cells, an in vitro model of Parkinson disease. Treatment with proteasome inhibitors provided significant protection against toxicity by 6-hydroxydopamine and H(2)O(2) in a concentration-dependent manner. The measurement of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate demonstrated that lactacystin, a proteasome inhibitor, significantly reduced 6-hydroxydopamineand H(2)O(2)-induced reactive oxygen species production. Proteasome inhibitors elevated the amount of glutathione and phosphorylated p38 mitogen-activated protein kinase (MAPK) prior to glutathione elevation. The treatment with lactacystin induced the nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased the level of mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme in glutathione synthesis. Furthermore, SB203580, an inhibitor of p38 MAPK, abolished glutathione elevation and cytoprotection by lactacystin. These data suggest that proteasome inhibition afforded cytoprotection against oxidative stress by the elevation of glutathione content, and its elevation was mediated by p38 MAPK phosphorylation.