Compartmentation of Glutathione: Implications for the Study of Toxicity and Disease (original) (raw)
Related papers
Cellular and clinical implications of glutathione
Indian journal of experimental biology, 2000
abundant intracellular thiol compound present in virtually all mammalian tissues -• Functions of GSH in reductive processes are essential for the synthesis and also degradation of proteins, formation of the deoxyribonucleotid precursors of deoxyribonucleic acid (DNA), regulation of enzymes, and protection of the cells against reactive oxygen species and free radicals produced even in normal metabolism•. By its multifunctional properties GSH attracts the interest of researchers in various subjects such as enzyme mechanisms, biosynthesis of macromolecules, intermediary metabolism, drug metabolism, radiation, cancer, oxygen toxicity, transport, immunology, endocrinology, environmental toxins, aging and exercise·-. Most of the new information about GSH biochemistry is produced with selective inhibitors of the enzymes involved in GSH turnover. Selective modulation of GSH metabolism also makes new therapeutic approaches possible• Glutathione metabolism still looks promising to scientists ...
The FASEB Journal, 2002
Fluorescence microscopy of A549 cells stained with a glutathione (L-γ-glutamyl-Lcysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis. However, confocal visualization of antibody-stained A549 cells in the z-plane revealed the majority of the perinuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z-plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z-section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near-complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z-plane. The antibody also provided initial visualization of the compartmentalization of protein-GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti-GSH antibody-stained Jurkat T lymhocytes, we also demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide. In addition, we showed cell-cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.
Selective glutathione depletion on function and structure of the isolated perfused rat kidney
Kidney International, 1983
Selective glutathione depletion on function and structure of the isolated perfused rat kidney. The role of glutathione (GSH) in the preservation of renal function and the pathogenesis of renal injury has been investigated using the isolated perfused rat kidney as a model. In kidneys perfused for 80 mm with 5 mi glucose as the only exogenous substrate, tissue GSH becomes depleted, renal function deteriorates, and a degenerative change appears, restricted to the medullary thick ascending limb. These abnormalities can be ameliorated by providing amino acid supplements or by adding GSH itself to the perfusion. To distinguish between the effects of amino acid supplementation and GSH depletion per se, selective depletion of GSH was accomplished in several different ways. Synthesis of GSH was inhibited by the addition of dl-buthionine-SRsulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase. GSH depletion was also produced by 2-cyclohexene-l-one and diethylmaleate, both known to diminish the concentration of GSH selectively without affecting protein thiols. Perfused kidneys selectively depleted of GSH showed significant impairment of concentrating ability, and less marked decreases in tubular reabsorption of sodium. The degenerative changes in the medullary thick ascending limb, on the other hand, were unaltered. While GSH depletion seriously impairs certain renal transport functions, it is probably not responsible for the anatomical disruption of the thick ascending limb that characterizes the isolated perfused rat kidney.
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]).
American Journal of Respiratory Cell and Molecular Biology, 1995
Cells in most culture media use cystine as the primary source of the cysteine precursor needed for glutathione (GSH) synthesis. As a result, GSH levels in many cultured cells may be limited by the rate of uptake of cystine into cells. We have shown that incubation with extracellular GSH can result in the reaction of GSH with cystine to generate cysteine, and that bovine pulmonary artery endothelial cells and lung type II epithelial cells transported cysteine more efficiently than cystine. Cystine transport was not affected by the presence of GSH. In cells incubated with GSH in RPMI-1640 there was a cystine-dependent increase in intracellular GSH levels. The increases in GSH were not prevented by the presence of acivicin, an inhibitor of they -glutamyl transpeptidase reaction. Incubation with oxidized glutathione (GSSG) did not result in significant increases in intracellular GSH levels. We conclude that a primary mechanism by which extracellular GSH may increase intracellular GSH levels in cultured cells is by reducing cystine to cysteine, which is then rapidly transported and used as a substrate for intracellular GSH synthesis. Multiple factors control intracellular glutathione (GSH) levels. Adenosine triphosphate-dependent-y-glutamylcysteine synthetase, the rate-limiting enzyme in the GSH synthesis pathway, is subject to feedback inhibition by GSH (1,2). Alternatively, aSH synthesis may be limited by the availability of substrate (1, 3-6), glutamate, cysteine, or glycine. Glutamate and glycine are rarely rate limiting (5, 7). However, cysteine may be limiting, particularly in cell cultures (3, 7). Cysteine is primarily taken up by a transport system shared with serine and alanine (system ASC) (5) and can also be obtained by reduction of cystine, which is transported into many cells by a sodium-independent transport system (x-c) shared with glutamate (3, 5-8). In addition, they -glutamyl transpeptidase reaction may be used to break down extracellular GSH and transport the resultant amino acids into the cell for use in the resynthesis ofGSH (9, 10). Extracellular GSH reacts with the membranebound enzyme, whereupon they -glutamyl moiety is transferred to an amino acid acceptor. In the presence of excess cysteine,-y-glutamylcysteine can be formed, which could be
Cellular and subcellular heterogeneity of glutathione metabolism and transport in rat kidney cells
Toxicology, 1998
Selective permeabilization of plasma membranes with digitonin produced separation of cytosolic and mitochondrial compartments of proximal tubular (PT) and distal tubular (DT) cells from a rat kidney. Subcellular distributions of several intracellular glutathione (GSH)-dependent enzymes were similar in the two cell types but specific activities were significantly higher in PT cells, indicating that DT cells, particularly in their mitochondrial fraction, have a diminished capacity to detoxify reactive oxygen species. To enable isolation of suspensions of mitochondria, renal cells were treated with digitonin followed by the bacterial protease nagarse and were filtered through polycarbonate membranes. Activity distributions of enzymatic markers for subcellular fractions were quantitated and uptake of GSH was studied in suspensions of PT and DT cell mitochondria. While PT cell mitochondria catalyzed rapid uptake of GSH that was inhibited by malate, indicating involvement of dicarboxylate carriers, DT cell mitochondria exhibited limited capacity for GSH uptake that was not inhibited by substrates for the two dicarboxylate carriers. This report provides the first description of methodology for the preparation of mitochondria from renal cells derived from specific nephron cell types and shows that mitochondria from DT cells have a significantly lower capacity to use GSH for detoxification and regulation of redox status. : S 0 3 0 0 -4 8 3 X ( 9 8 ) 0 0 0 9 3 -6 L. H. Lash et al. / Toxicology 130 (1998) 1-15
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.