Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by glutamate. Neither PC12 nor PC12/bcl-2 cells showed a significant incidence of apoptosis in response to glutamate. Conventional phospholipid analysis by high-performance TLC and phosphorous determination showed no significant changes in the phospholipid composition of either cell line incubated with 5 mM glutamate. The peroxyl radical initiator 2,2'-azobis(2,4-dimethylvaleronitrile) caused a pronounced loss of all major phospholipid classes in PC12 cells, but no loss of cell viability. No phospholipid peroxidation was detected in PC12/bcl-2 cells incubated with 相似文献   

Red blood cell glutathione and plasma sulfhydryls in chronic lung disease of the newborn

We compared the postnatal changes (days 1-28) in red blood cell glutathione and plasma sulfhydryl content in preterm babies developing chronic lung disease (CLD, n = 13) to those in babies with respiratory distress syndrome (RDS, n = 13) and control babies (n = 21). There were no initial differences in these measurements between the three groups. However, on day 28 in CLD and RDS the red blood cell glutathione was decreased compared to the control babies (p < 0.05). In CLD, there was a significant correlation between reduced/oxidized glutathione and (i) maximal FiO2 (r = -0.69, p < 0.05) and (ii) minimal a/A ratio (r = +0.73, p < 0.005). On day 28, although the plasma sulfhydryl level did not differ between the groups, the sulfhydryl/total protein ratio was decreased in CLD (p < 0.05). The late decrease in both glutathione and sulfhydryl/total protein ratio in babies with CLD suggests that oxidative stress is still ongoing at 28 days after birth and that the antioxidant capacity of their blood is still diminished at this time.     

Destabilization and denaturation of cellular protein by glutathione depletion

There is an increasing realization that every effort should be made to minimize the exposure to ionizing radiation of patients and members of the dental team. There are, however, few published guidelines to assist the general practitioner in deciding whether or not radiographs are needed. There is even less information concerning the taking of dental radiographs of children. Using the Delphi Technique this study set out to seek a consensus amongst Consultant Paediatric Dentists (n = 36) working in the UK as to when dental radiographs should and should not be taken of children cared for within general dental services. A two-stage questionnaire was used and a response of 94% and 76% was obtained in the first and second questionnaire respectively. A consensus view was reached regarding whether or not radiographs should be taken in a series of 17 specific clinical situations.     

Irreversible inactivation of protein kinase C by glutathione

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca2+- and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca2+- and PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca2+- and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-alpha remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-alpha was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-alpha and the tripeptide, but it was associated with an increase in the susceptibility of PKC-alpha to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.     

Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.     

Crystal structure of the S-nitroso form of liganded human hemoglobin

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.     

Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37 degrees C under 100% O2. Lipid peroxidation was induced by adding 400 microM adenosine 5'-triphosphate, 2.5 to 20 microM FeCl3, and 450 microM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992) Arch. Biochem. Biophys. 293, 103-109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases

Thymine hydroperoxide (5-hydroperoxymethyluracil), a model compound representing products of oxidative damage to DNA, is a substrate for glutathione peroxidase and some isoforms of glutathione transferase. In this paper, we show that selenium-dependent human phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx) exhibits about four orders of magnitude higher activity on thymine hydroperoxide than that of other human enzymes such as selenium-dependent glutathione peroxidase and various representatives of glutathione transferases. The results indicate that Se-PHGPx may be an important enzyme in repairing oxidatively damaged DNA.     

Ca2+ pool emptying stimulates Ca2+ entry activated by S-nitrosylation

The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how "store-operated" entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate that S-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism.     

Suppression of nitric oxide-induced apoptosis by N-acetyl-L-cysteine through modulation of glutathione, bcl-2, and bax protein levels

It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N-acetyl-L-cysteine (LNAC) and free-radical spin traps (5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6,-tetramethyl-1-piperidinyloxy) against NO-induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose-dependent manner, promoted long-term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl-2 oncoprotein level in the LNAC-pretreated cells was significantly elevated in a time-dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells.     

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Intracameral ascorbic acid, glutathione and protein levels in albino and pigmented rabbits

We performed a comparative investigation into differences between albino and pigmented rabbits in respect of intracameral concentrations of ascorbic acid, glutathione and total protein with high-performance liquid chromatography. The intracameral concentrations of reduced ascorbic acid, total ascorbic acid, reduced glutathione, total glutathione and protein in albino rabbits were similar (p > 0.05) to those in pigmented rabbits.     

Sulfane-activated reduction of cytochrome c by glutathione

Apolipoprotein E (Apo E) is a component of VLDL and HDL and plays a significant role in the regulation of cholesterol concentration. An improvement in isoelectric focusing for Apo E phenotyping is presented: the plasma Apo E was dissociated from lipoproteins by the use of Tween 20; the optimal concentration of type V neuraminidase was determined (1 U/ml); up to 48 samples were analyzed per plate and revealed by immunoblotting. Using this method, we have determined Apo E phenotypes and estimated their association with total cholesterol and Apo B levels in 498 healthy blood donors in Paris (France). The relative frequencies of Apo E alleles epsilon 2, epsilon 3 and epsilon 4 in this population were 0.079, 0.801 and 0.120, respectively. The association between Apo E phenotypes and concentration of Apo B-containing lipoproteins was confirmed (Apo B (g/l): E4/E3 subjects, 1.10 +/- 0.29; E3/E2 subjects, 0.93 +/- 0.22; both significantly different from E3/E3 subjects, 0.99 +/- 0.28). Total cholesterol (mmol/l): E4/E3 subjects, 5.43 +/- 1.15; E3/E2 subjects, 4.79 +/- 0.83; both significantly different from E3/E3 subjects, 5.03 +/- 1.11.     

Cooperation between glutathione depletion and protein synthesis inhibition against naturally occurring neuronal death

It is generally agreed that naturally-occurring neuronal death in developing animals is dependent on the synthesis of proteins. Oxidative stress, as when intracellular concentrations of free radicals are raised or when cell constituents such as membrane lipids or protein thiols are oxidized, is also involved in various types of neuronal death. In the present report, we show that the number of naturally dying retinal cells in the chick embryo can be reduced by intraocular injections of cycloheximide, an inhibitor of protein synthesis. L-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, can either enhance or diminish the cell death, depending on the conditions of treatment. Moreover, when the two inhibitors are combined, L-buthionine-[S,R]-sulfoximine potentiates the neuroprotective effects of cycloheximide. Measurements of retinal glutathione concentration and protein synthesis show the specificity of the treatments: buthionine-sulfoximine diminishes glutathione concentrations but not protein synthesis whereas cycloheximide inhibits protein synthesis without decreasing glutathione concentrations. Naturally-occurring neuronal death thus seems to involve the synthesis of proteins, and is also influenced by oxidative phenomena. Our results extend previous data in tectal-lesioned embryos, and suggest that a moderate, non-lethal oxidative stress can enhance the resistance of ganglion cells that might otherwise have died (spontaneously or following axotomy) owing to insufficient retrograde trophic support.     

Oxidative stress: role of mitochondria and protection by glutathione

We have monitored estrous cycle and measured serum estradiol, GH, IGF-1, T4 and T3 levels in adult hypothyroid female rats which were divided into four groups: H group, hypothyroid rats without treatment; H-T4 group, hypothyroid rats injected daily with T4; HT4-PTU group, hypothyroid rats injected daily with T4 plus PTU (propylthiouracil), and H-T4-IOP group, hypothyroid rats injected daily with T4 plus IOP (iopanoic acid); Euthyroid rats (E group) were used as control. Our results indicate that the lack of sexual cycle in H animals was associated with lower values of estradiol, GH and IGF-1 in comparison to E group; the restoration of sexual cycle in H-T4 group was associated with values of estradiol, GH and IGF-1 higher than those of H group, whereas in H-T4-PTU and H-T-IOP groups the restoration was associated with higher values of GH and IGF-1 and values of estradiol similar to those of H group. These data could suggest a potential role of GH/IGF-1 axis, at least in part, in the lack of sexual cycle in H group and in the ovulation induction in H-T4, H-T4-PTU and H-T4-IOP groups.     

Prevention of acetaminophen- and naphthalene-induced cataract and glutathione loss by CySSME

PURPOSES: To assess the efficacy of 2-mercaptoethanol/L-cysteine mixed disulfide (CySSME) as an L-cysteine prodrug suitable for glutathione biosynthesis in rat lenses in vitro, as an agent for the prevention of acetaminophen- and naphthalene-induced murine cataract in genetically-susceptible mice, and as an agent for maintenance of near-normal glutathione levels in lenses and livers of mice subjected to acetaminophen and naphthalene at cataractogenic doses. METHODS: Synthetic CySSME was added as a prodrug to rat lens culture medium devoid of L-cystine and L-methionine but containing [14C(U)]-glycine. After a 48-hour period of incubation, extracts of rat lenses were prepared for separation of [14C]-glutathione by high-performance liquid chromatography (HPLC) with a radioisotope detector to determine the extent of its biosynthesis. Cytochrome P-450 isozymes were induced in C57 bl/6 mice by either beta-naphthoflavone or phenobarbital. Cataracts were induced by administration of either acetaminophen or naphthalene to the pretreated mice. CySSME was coadministered with either acetaminophen or naphthalene to other groups of mice. Both oxidized and reduced glutathione were determined in extracts of livers and lenses using the HPLC method above. RESULTS: CySSME served as an effective L-cysteine precursor for glutathione biosynthesis in cultured rat lenses. This L-Cysteine prodrug was also highly effective in preventing acetaminophen- and naphthalene-induced cataract in mice and in maintaining near-normal glutathione levels in lenses and livers of such treated animals. CONCLUSIONS: This investigation demonstrates that maintenance of adequate physiological levels of glutathione in the presence of specific known cataractogenic agents by pharmacologic intervention with CySSME, an L-cysteine prodrug, is sufficient to prevent cataract formation.     

Cysteine and glutathione secretion in response to protein disulfide bond formation in the ER

Protein folding in the endoplasmic reticulum (ER) often involves the formation of disulfide bonds. The oxidizing conditions required within this organelle were shown to be maintained through the release of small thiols, mainly cysteine and glutathione. Thiol secretion was stimulated when proteins rich in disulfide bonds were translocated into the ER, and secretion was prevented by the inhibition of protein synthesis. Endogenously generated cysteine and glutathione counteracted thiol-mediated retention in the ER and altered the extracellular redox. The secretion of thiols might link disulfide bond formation in the ER to intra- and intercellular redox signaling.     

Nephrotoxicity of cadmium-metallothionein: protection by zinc and role of glutathione

BACKGROUND: In vitro studies have shown an antiproliferative effect of tumor necrosis factor (TNF) against various nonsmall cell lung carcinoma (NSCLC) cell lines. However, clinical trials of combined interleukin-2 and TNF-alpha in patients with advanced NSCLC have demonstrated both conflicting and disappointing results. METHODS: Immunohistochemical (IHC) staining was performed on formalin fixed, paraffin embedded tissues from 39 bronchogenic adenocarcinomas and 32 squamous cell carcinomas using polyclonal antibodies against TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 proteins. IHC positivity was correlated with tumor stage, grade, and patient survival. RESULTS: Significant coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 was observed in NSCLC (significance range, P < 0.001-0.02). Although immunoreactivity for TNFs remained high in all tumor stages, a loss of TNF-R expression was found in advanced NSCLC (P < 0.006 for TNF-R1 and P < 0.003 for TNF-R2), suggesting down-regulation of TNF-Rs in the process of tumor progression. When all stages were considered together, immunoreactivity for TNF-beta(P < 0.001), TNF-R1, and TNF-R2 (both P < 0.001) significantly correlated with favorable outcome in univariate analysis. However, when stages were studied separately, an association between immunopositivity for TNF-Rs and favorable prognosis was found only in NSCLC without distant metastasis (P < 0.04 and P < 0.005 for TNF-R1 and TNF-R2 in Stage I [according to the American Joint Committee on Cancer staging system] disease, and P < 0.03 and P < 0.02 for TNF-R1 and TNF-R2 in Stage III disease). On multivariate analysis, increased expression of TNF-R1 (P < 0.003) and TNF-R2 (P < 0.001) as well as tumor stage (P < 0.001) independently predicted favorable outcome in patients with NSCLC. CONCLUSIONS: Although NSCLC exhibits strong coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2, there is a loss/down-regulation of TNF receptors in high stage tumors. TNF-R1 and TNF-R2 positivity independently predicts favorable outcome in NSCLC, particularly in tumors with no clinically distant metastasis. The current study supports a role for TNFs and their receptors in the evolution and progression of NSCLC.     

Synthesis and glutathione S-transferase structure-affinity relationships of nonpeptide and peptidase-stable glutathione analogues

A series of nonpeptidic glutathione analogues where the peptide bonds were replaced by simple carbon-carbon bonds or isosteric E double bonds were prepared. The optimal length for the two alkyl chains on either side of the mercaptomethyl group was evaluated using structure-affinity relationships. Affinities of the analogues 14a-f, 23, and 25 were evaluated for a recombinant GST enzyme using a new affinity chromatography method previously developed in our laboratory. Analysis of these analogues gives an additional understanding for GST affinity requirements: (a) the carbon skeleton must conserve that of glutathione since analogue 14a showed the best affinity (IC50 = 5.2 microM); (b) the GST G site is not able to accommodate a chain length elongation of one methylene group (no affinity for analogues 14c,f); (c) a one-methylene group chain length reduction is tolerated, much more for the "Glu side" (14d, IC50 = 10.1 microM) than for the "Gly side" (14b, IC50 = 1800 microM); (d) the mercaptomethyl group must remain at position 5 as shown from the null affinity of the 6-mercaptomethyl analogue 14e; (e) the additional peptide isosteric E double bond (25) or hydroxyl derivative (23) in 14e did not help to retrieve affinity. This work reveals useful information for the design of new selective nonpeptidic and peptidase-stable glutathione analogues.