Subcellular compartmentation of glutathione and glutathione precursors (original) (raw)
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The glutathione content of retinal Müller (glial) cells: effect of pathological conditions
Neurochemistry International, 2000
Maintenance of isolated retinal MuÈ ller (glial) cells in glutamate-free solutions over 7 h causes a signi®cant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of MuÈ ller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe eects are caused by high extracellular K + (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal MuÈ ller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig MuÈ ller cells using the glutathione-sensitive¯uorescent dye monochlorobimane. The purpose was to ®nd out how the glial glutathione content is aected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate de®ciency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate± glutamine pathway, at the expense of glutathione. This mechanism may contribute to the ®nding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K + , decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended.
The physiological consequences of glutathione variations
Life Sciences, 1992
~,mrnRry The major low molecular weight thiol inside cells, the tripeptide glutathione (GSH), is of importance for protection of the cell against oxidative challenge, for thiol homeostasis required to guarantee basic functions, and for defence mechanisms against xenobiotics. Since the pathophysiological significance of a perturbed GSH status in human disease is less clear, this review evaluates the consequences of/n viuo variations of GSH. Owing to intracellular GSH concentrations above 2 mM depletion of GSH as such has little metabolic consequences unless an additional stress is superimposed. The kinetic properties of GSH-dependent enzymes imply that loss of up to 90% of intraceUular GSH may still be compatible with cellular integrity. Mitochondrial GSH, which accounts for about 10% of total cellular GSH, may define the threshold beyond that toxicity commences. Thus, in cases of severe GSH-depletion a substitution of GSH as a therapeutic measure seems justified. Such a severe depletion of GSH has been described for some diseases such as liver dysfunction, AIDS or pulmonary fibrosis. I. Scope The intracellular redox balance of mammalian cells is maintained by a homeostatic mechanism which links small pools of coenzymes and cofactors to a large redox buffer with common chemical properties, i.e. the thiol system. The overwhelming part of intra-as well as extracellular soluble thiols is represented by the tripeptide glutathione (GSH) which occurs in any eukaryotic cell in high concentrations, i.e. 2-10 retool/1. The intactness of this glutathione system is essential for maintainment of physiological functions. The continuing research interest in glutathione is documented by a publication rate of two scientific papers per day with an increasing frequency of monographies published [Cited in ref. [1] and [2]).
Glutathione: interorgan translocation, turnover, and metabolism
Proceedings of the National Academy of Sciences, 1979
Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one...
Antisera to Glutathione: Characterization and Immunocytochemical Application to the Rat Cerebellum
European Journal of Neuroscience, 1994
Rabbits were immunized with reduced glutathione (7-glutamyl-cysteinyl-glycine) coupled to bovine serum albumin by glutaraldehyde or a mixture of glutaraldehyde and formaldehyde. The antisera that were formed were tested qualitatively, by screening them against more than 50 amino acids and peptide conjugates that had been immobilized on cellulose discs (spot test), and quantitatively, by immunogold analysis of test conjugates that had been embedded in an epoxy resin. It was shown that the antisera selectively recognized the reduced and oxidized forms of glutathione and that they did not exhibit any significant crossreactivity with glutamate, cysteine, glycine, y-glutamyl-cysteine or cysteinyl-glycine. lmmunocytochemistry of Vibratome sections of rat cerebellum suggested that glutathione occurs in glial cells as well as in neurons. This was confirmed by electron microscopic, immunogold cytochemistry of tissue from rat cerebellum that had been freeze-substituted and embedded in Lowicryl under low temperature. Gold particles were concentrated over Golgi epithelial cells and perivascular glial processes, but also occurred over several types of neuronal profile including Purkinje and granule cell bodies, and mossy fibre terminals. At the subcellular level, glutathione-like immunoreactivity was found in the cytoplasmic matrix, mitochondria and nuclei. The immunolabelling intensity was strongly reduced in animals that had been pretreated with buthionine sulphoximine, which is known to depress the level of glutathione by inhibiting 7-glutamyl-cysteine synthetase. The availability of antisera to glutathione is likely to further our understanding of the physiological and pathophysiological roles of this tripeptide.
Subcellular glutathione contents in isolated hepatocytes treated with L-buthionine sulfoximine
Biochemical and Biophysical Research Communications, 1984
The glutathione contents of the mitochondrial and cytosolic fractions and extracellular space of isolated hepatocytes decrease when glutathione synthesis is inhibited with L-buthionine sulfoximine. Mitochondrial glutathione is depleted to 50 % of its initial value whereas the cytosolic pool is cc~pletely emptied after 2 h incubation in the presence of inhibiter. The mitochondrial glutathione content was only fully depleted when L-buthionine sulfc~imine was added together with phorone (2,6-dimethyl-2,5-heptadiene-4-one), a substrate of the glutathione S-transferases (E.C. 2.5.1.18). © ,984 Academic Press. Inc. The existence of more than one pool of intracellular glutathione in liver was firstly suggested in 1952 (I) and has been recently reviewed (2,3). The 'stable pool' of glutathione reported by Higashi et el. (4) was suggested to be located in mitochondria. However, mitochondrial glutathione was found to be 0.20-0.45 mmol x g-1 (5-7) in isolated hepatocytes or liver, one order of magnitude lower than the size of the 'stable pool' (3 ~mol x g-l). At variance with the 2 h half-life of liver glutathione in (8), a half-life of 30 h was reported for the mitochondrial glutathione pool in isolated hepatocytes, based on studies with radiolabelled precursor of glutathione, I 3SSlmethi°nine (9). We have recently described a lower rate of replenishment of the mitochondrial glutathione as when compared to the cytosolic (7). The conditions used in this previous report allowed synthesis of GSH after iOn leave frcm Dept.
Chemico-Biological Interactions, 1989
One of the most widely used mechanisms by which the role of glutathione (GSH) in cellular functions has been withdrawn, is to deplete GSH intracellularly. The importance of the procedure and xenobiotic chosen to get it is discussed. Mitochondrial GSH plays certainly an important role in maintaining cellular homeostasis. This contribution varies depending on the tissue and the conclusions obtained about the functions of this GSH pool in one organ may not be applied to others. Original data on the subcellular distribution of GSH in myocardial tissue of the rat are presented, and the effect of phorone on both cardiac GSH pools is compared with the effect in liver. The mechanical failure of myocardium after ischemic or reperfusion damage might involve mitochondrial GSH, in view of the literature data referring to the role of thiol groups in energy transfer from mitochondria to cytosol.
Experimental Eye Research, 1992
A method for specific determination of glutathione (GSH) is described. This method utilizes the enzymatic conjugation of GSH to 1-chloro-2,4&nitrobenzene through reaction catalyzed by glutathione Stransferase. The recovery of GSH as determined by this method is comparable to that in currently used methods. The method is specific for GSH determination. Other sulihydryl (-SH) compounds including the protein-SH or p-mercaptoethanol, which are often included in tissue homogenates, do not interfere with GSH determination. Acid extraction of the tissue is not required in this method and comparatively smaller amounts of tissue samples (as little as 20 ,~l of a 10 y0 w/v tissue homogenate) are needed for the analyses. The method when applied for GSH determination in ocular tissues yielded results in agreement with the reported values in literature. Evidence for the sensitivity, accuracy, and convenience of the method is provided by analysing the sample containing GSH in the range of l-200 nmol by this method.
S-D-Lactoylglutathione can be an alternative supply of mitochondrial glutathione
Free Radical Biology and Medicine, 2014
The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, in defending mitochondrial DNA integrity and protecting mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. To demonstrate SLG transport from cytosol to mitochondria we used radiolabeled compounds and the results showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. Also, the incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. As well mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of Dlactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II in inter-membrane space and mitochondrial matrix and we investigated the role of SLG in whole cells. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.
Biochemistry, 1993
We show that y-glutamyl transpeptidase (GGT) is a glutathionase that enables cells to use extracellular glutathione as a source of cysteine. We transfected NIH/3T3 mouse fibroblasts with a plasmid containing cDNA for human GGT, and obtained stably transformed cell lines that expressed GGT in its proper orientation on the outer surface of the cell. NIH/3T3 fibroblasts require cysteine for growth and are unable to use extracellular glutathione as a source of cysteine. We demonstrate GGT-positive fibroblasts are able to grow in cysteine-free medium supplemented with glutathione. Cysteine derived from the cleavage of extracellular glutathione can be used to maintain intracellular levels of glutathione. GGT-positive NIH/3T3 cells were able to replenish intracellular glutathione when incubated in cysteine-free medium