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D J Reed

Publications and source records attributed to D J Reed.

At least 55 records · Page 3Linked to original sources
PubMed1991

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria.

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.

Animals↗
PubMed1991

Toxicity to isolated hepatocytes caused by the intracellular calcium indicator, Quin 2.

To determine whether incubation for several hours with intracellular Ca++ indicators caused toxicity to freshly isolated hepatocytes from rats, cells were incubated under 95% O2-5% CO2 in medium containing 2 mM Ca++ and the acetoxymethyl (AM) esters of Quin 2, Indo 1, Fluo 3, 5,5'-Dimethyl BAPTA, 4,4'-Difluoro BAPTA or Fura 2 for up to 5 hr. Quin 2-AM and Indo 1-AM (2.5 microM) induced lipid peroxidation in the cells after 1 or 3 hr of treatment, respectively. Additional experiments with Quin 2-AM (25 microM) revealed that it also caused lactate dehydrogenase leakage, cell blebbing and vitamin E loss in cells, but did not affect reduced glutathione or intracellular Ca++ content. The ability of Quin 2-AM to cause toxicity was dependent on the amount of Quin 2 which was present in the cell. Ca++ appeared to be involved in the mechanism of Quin 2-AM toxicity, for modulation of the extracellular Ca++ concentration partially inhibited lipid peroxidation, vitamin E loss, cell blebbing and lactate dehydrogenase leakage.

Aminoquinolines↗
PubMed1991

Involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes.

When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.

Aminoquinolines↗
PubMed1990

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E.

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.

Animals↗
PubMed1990

Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress.

t-Butyl hydroperoxide was utilized to alter the thiol homeostasis in rat brain mitochondria. Following exposure to t-butyl hydroperoxide (50-500 microM), intramitochondrial GSH content decreased rapidly and irreversibly with a major portion of the depleted GSH being accounted for as protein-SS-Glutathione mixed disulfide. Formation of GSSG was not observed nor was efflux of GSSG or GSH from the mitochondria detected in the incubation medium. The loss of intramitochondrial GSH was accompanied by loss of protein thiols. Unlike liver mitochondria, which can reverse t-butyl hydroperoxide induced formation of GSSG, addition of 50 microM t-butyl hydroperoxide resulted in irreversible loss; indicating greater susceptibility of brain mitochondria to oxidative stress than liver mitochondria.

Animals↗
PubMed1990

Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides.

The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

Cell Line↗
PubMed1990

Extracellular calcium effects on cell viability and thiol homeostasis.

Studies of chemically induced cell injury and death, which have used as model systems freshly isolated rat hepatocytes and hepatocytes in culture, are discussed. An important model uses the omission of Ca2+ from the medium during rat hepatocyte incubations. Ca2+ omission induces an intense oxidative stress within hepatocytes incubated in a 95% O2 + 5% CO2 atmosphere. The relationship of calcium homeostasis to the parameters of oxidative stress is important to understanding the progression from reversible to irreversible injury. In the Ca2+ omission model, the vitamin E (Vit. E) content of hepatocytes is important for the prevention of cell injury. Recent studies with rat hepatocytes show that ruthenium red (RR) and La3+, which block Ca2+ translocation through the mitochondrial uniport, can prevent malondialdehyde (MDA) formation, reduced glutathione (GSH), and protein-SH loss, Vit. E loss, and LDH leakage induced by Ca2+ omission from the incubation medium. Ca2+ omission promoted a marked loss of mitochondrial transmembrane potential (delta phi) that was prevented by RR, EGTA, Vit. E, and desferrioxamine. The absence of extracellular Ca2+ may cause mitochondrial Ca2+ cycling that contributes to the observed oxidative stress, resultant loss of cell viability, and protein thiol homeostasis. Chemical agents including a glutathione-depleting agent, ethacrynic acid, and a redox cycling agent, adriamycin, increase the loss of cell viability caused by a Ca2(+)-free medium, but they have some additional effects on cellular processes. The demise of cell viability by the agent is also preventable by Vit. E supplementation. Ca2+ has a role in cell injury that appears to uniquely involve mitochondrial homeostasis.

Animals↗
PubMed1989

Role of a partially purified glutathione S-transferase from rat liver nuclei in the inhibition of nuclear lipid peroxidation.

Glutathione protects isolated rat liver nuclei against lipid peroxidation by inducing a lag period prior to the onset of peroxidation. This GSH-dependent protection was abolished by exposing isolated nuclei to the glutathione S-transferase inhibitor S-octylglutathione. In incubations containing 0.2 mM S-octylglutathione, the GSH-induced lag period was reduced from 30 to 5 min. S-Octylglutathione (0.2 mM) also completely inhibited nuclear glutathione S-transferase activity and reduced glutathione peroxidase activity by 85%. About 70% of the glutathione S-transferase activity associated with isolated nuclei was solubilized with 0.3% Triton X-100. This solubilized glutathione S-transferase activity was partially purified by utilizing a S-hexylglutathione affinity column. The partially purified nuclear glutathione S-transferase exhibited glutathione peroxidase activity towards lipid hydroperoxides in solution. The data from the present study indicate that a glutathione S-transferase associated with the nucleus may contribute to glutathione-dependent protection of isolated nuclei against lipid peroxidation. Evidence was obtained which indicates that this enzyme is distinct from the microsomal glutathione S-transferase.

Animals↗
PubMed1989

Cell calcium, vitamin E, and the thiol redox system in cytotoxicity.

The controversial role of extracellular Ca2+ in toxicity to in vitro hepatocyte systems is reviewed. Recent reports demonstrate that extracellular Ca2+-related cytotoxicity is dependent on Ca2+-influenced vitamin E (alpha-tocopherol) content of isolated hepatocytes. Based on a Ca2+-omission model of in vitro oxidative stress, the role of vitamin E in cytotoxicity is further explored. This model demonstrates the interdependence of the GSH redox system and vitamin E as protective agents during oxidative stress. Following chemical oxidant-induced depletion of intracellular GSH, cell morphology and viability are maintained by the continuous presence of cellular alpha-tocopherol above a threshold level of 0.6-1.0 nmol/10(6) cells. alpha-Tocopherol threshold-dependent cell viability is directly correlated with the prevention of the loss of cellular protein thiols in the absence of intracellular GSH. Potential mechanisms for this phenomenon are explored and include a direct reductive action of alpha-tocopherol on protein thiyl radicals, and the prevention of oxidation of protein thiols by scavenging of lipid peroxyl radicals by alpha-tocopherol. It is suggested that in light of the threshold phenomenon of vitamin E prevention of potentially severe oxidative stress-induced cytotoxicity, its use as a protective agent against an oxidative challenge in vivo should be reassessed.

Calcium↗
PubMed1989

The Indiana Speech Training Aid (ISTRA). I: Comparisons between human and computer-based evaluation of speech quality.

Experimental comparisons are reported between computer-based and human judgments of speech quality for the same sets of utterances. Speech stimuli were recorded from two normal talkers, who intentionally varied the quality of their speech, and from a hearing-impaired child who was receiving speech therapy on the Indiana Speech Training Aid (ISTRA). The tape recordings were submitted for evaluation to a naive jury, an expert jury, and the ISTRA System, a microcomputer equipped with a speaker-dependent speech recognition board that generated scores representing how well utterance matched a stored template. Correlational analyses of these data indicated that humans were slightly better at judging speech quality than was the computer, but that the computer was much more reliable. These results demonstrate that computer-based speech evaluation may be a reasonable substitute for human judgments for certain types of speech drill.

Computer Systems↗
PubMed1989

Effects of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation.

Endogenous alpha-tocopherol levels in isolated rat liver nuclei were determined to be 0.045 mol% (mol alpha-tocopherol per mol phospholipid x 100). This value corresponds to 970 polyunsaturated fatty acid (PUFA) moieties to one molecule of alpha-tocopherol in the nuclear membrane. Isolated nuclei, when incubated with various concentrations of exogenous alpha-tocopherol, took up only a small percent of initial levels of alpha-tocopherol present in the incubation media. Exogenous alpha-tocopherol, when incorporated in isolated nuclei above a threshold value of 0.085 mol%, effectively inhibited NADPH-induced lipid peroxidation. The addition of 1 mM glutathione lowered the threshold levels of alpha-tocopherol needed to inhibit lipid peroxidation to about 0.040 mol%. We suggest the data indicate a glutathione-dependent enhancement of the ability of alpha-tocopherol to inhibit nuclear lipid peroxidation.

Animals↗
PubMed1988

The glutathione status of rat kidney nuclei following administration of buthionine sulfoximine.

Rat kidney nuclei were isolated by techniques designed to limit the loss of small water-soluble metabolites. Lyophilized rat kidney powder was disrupted with a Tekmar Tissumizer, and nuclei were purified by discontinuous gradient centrifugation through a glycerol-metrizamide solution at 4 degrees C. Purified nuclei exhibited similar DNA to protein ratios as reported for other rat nuclei isolated by nonaqueous isolation techniques. Results suggest that in control animals the concentration of glutathione in the kidney nucleus is similar to that in the cytoplasm. However, following treatment with buthionine sulfoximine, rat kidney nuclear glutathione levels were less than the corresponding cytoplasmic glutathione levels.

Animals↗
PubMed1988

Mitochondrial glutathione status during Ca2+ ionophore-induced injury to isolated hepatocytes.

In this study the Ca2+ ionophore, A23187, was used to determine the effects of disrupted Ca2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca2+ and A23187 to induce accumulation or loss of cellular Ca2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca2+, the antioxidants alpha-tocopherol and diphenyl-p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca2+. In the presence of extracellular Ca2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca2+. Leupeptin, an inhibitor of Ca2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of cellular Ca2+ homeostasis.

Animals↗