Modulation of glutathione synthetic enzymes by acidic fibroblast growth factor

Increasing evidence suggests that glutathione (GSH) synthesis is a regulated process. Documented increases in gamma-glutamylcysteine synthetase (GCS) occur in response to oxidants, in tumors, on plating cells at a low cell density, and with nerve growth factor stimulation, suggesting that GSH synthesis may be related to the cell growth and transformation. Previously, extracellular acidic fibroblast growth factor (FGF-1) has been demonstrated to cause transformation and aggressive cell growth in murine embryonic fibroblasts. In the present investigation, we sought to determine whether FGF-1, with its growth inducing properties, resulted in the modulation of GSH biosynthetic enzymes, GCS and GSH synthetase. Murine fibroblasts transduced with (hst/KS)FGF-1, a chimeric human FGF-1 gene containing a signal peptide sequence for secretion, displayed elevated gene expression of both heavy and light subunits of GCS. Activity of GSH synthetase was also elevated in these cells compared with control cells. Nonetheless, GSH was decreased in the FGF-1-transduced cells along with high energy phosphates, adenine nucleotides, NADH, and the redox poise. However, GSSG was not elevated in these cells. Fibroblasts stably expressing human immunodeficiency virus type 1 Tat, which induces intrinsic FGF-1 secretion, resulted in similar changes in GCS, GS, and GSH. The results suggest that although increases in the enzymes of GSH synthesis are a common response to growth factors, an increase in GSH content per se is not required for altered cell growth.     

Stimulation of glutathione synthesis in iron-loaded mice

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89-94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and gamma-glutamylcysteine synthetase (L-glutamate: L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 +/- 12 nmol/g wet wt.) than in the controls (164 +/- 8 nmol/g wet wt.), and gamma-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 +/- 3.2 nmol/mg protein per min) compared with the controls (28.6 +/- 3.8 nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high gamma-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.     

Induction of gamma-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of gamma-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of gamma-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced gamma-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.     

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.     

Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.

Glutathione (GSH), a major cellular antioxidant, is elevated 2- to 3-fold in kidneys of rats during prolonged treatment with mercury as methyl mercury hydroxide (MMH). Increased renal GSH is accompanied by a dose- and time-related elevation in the relative abundance of mRNA hybridizable to a cDNA probe which encodes renal gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Renal GCS mRNA is maximally elevated 4.4-fold at 3 weeks following initiation of MMH treatment. Enhancement of GSH and GCS mRNA content corresponds to a relative sparing of renal cells from oxidative tissue damage during MMH exposure. These observations suggest that increased synthesis of GSH at the genetic level occurs as an initial adaptive response to mercury-induced oxidative stress in kidney cells.     

Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis

1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.     

The function of gamma-glutamylcysteine and bis-gamma-glutamylcystine reductase in Halobacterium halobium

gamma-Glutamylcysteine and bis-gamma-glutamylcystine reductase appear to function in the halobacteria in a fashion analogous to GSH and glutathione reductase in other cells. Bis-gamma-glutamylcystine reductase (GCR), a NADPH-dependent dimer of Mr 122,000 recently purified to homogeneity from Halobacterium halobium (Sundquist, A.R., and Fahey, R.C. (1988) J. Bacteriol., 170, 3459-3467), was found to be highly specific for bis-gamma-glutamylcystine and to be present in cell extract at a level sufficient to maintain gamma-glutamylcysteine predominantly in its thiol form [( thiol]/[disulfide] approximately 50). Bis-gamma-glutamylcystine reductase is similar to glutathione reductase in many respects; GCR demonstrated a FAD:subunit stoichiometry of 1, inhibition by heavy metal ions, and a pH optimum near neutrality. However, GCR exhibited no activity with GSSG and was most active at salt levels exceeding 2 M. A turnover number of 1,700 mumol min-1 mumol-1 FAD and apparent Km values of 0.8 mM for bis-gamma-glutamylcystine and 0.29 mM for NADPH were determined for GCR. The effect of salt on the autoxidation rates of gamma-glutamylcysteine, GSH, and Cys was also studied. In the absence of added salt, Cys oxidized more rapidly than gamma-glutamylcysteine, which in turn oxidized more rapidly than GSH. The presence of 4.3 M chloride (K+ and Na+) significantly slowed the autoxidation of all three thiols. The rate of autoxidation of gamma-glutamylcysteine in 4.3 M chloride proved slower than that of GSH in the absence of added chloride. Thus, gamma-glutamylcysteine is at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize gamma-glutamylcysteine rather than GSH.     

Glutathione-degrading enzymes of microvillus membranes

Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro

The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2).     

External and internal forms of yeast aminopeptidase II.

1. Intact cells of Saccharomyces cerevisiae catalyze the hydrolysis of various aminopeptidase substrates. This activity is not due to permeation of substrates and products but exerted by an external enzyme. 2. From its substrate specificity and the effects of pH and inhibitors the enzyme was identified as aminopeptidase II. 3. About 40% of total aminopeptidase II activity is detectable with untreated exponentially growing cells. Up to two thirds of the external enzyme is released into the medium during enzymic digestion of the cell wall, while little enzyme is liberated by osmotic shock. Membrane preparations contained only small amounts of aminopeptidase II; thus, the localization of the external enzyme appears to be similar to that of the so-called 'periplasmic' yeast hydrolases. 4. By cytochemical methods the presence of aminopeptidase II in the cell envelope was visualized. 5. In contrast to aminopeptidase II, yeast dipeptidase is an entirely intracellular enzyme.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

Metabolism of extracellular glutathione in rat small-intestinal mucosa

Metabolism of exogenous glutathione was investigated in suspensions of freshly isolated rat small-intestinal mucosal cells. The cells catalyzed the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG). Neither serine . borate nor methionine significantly influenced this reaction. Formed GSSG was further metabolized as indicated by its disappearance from the medium. Degradation of GSSG was stimulated by methionine and inhibited by serine . borate. Separation and identification of GSSG metabolites were achieved by high performance liquid chromatography. The results indicate that the preferred route for GSSG metabolism to the constituent amino acids in small intestine, is by hydrolytic removal of the two gamma-glutamyl groups of GSSG to yield cystinyl-bisglycine which is subsequently hydrolyzed to cystine. gamma-Glutamyltransferase activity was compared in isolated intestinal, kidney and liver cells using gamma-glutamyl-p-nitrocarboxyanilide as substrate. Kidney cells were approximately 5-fold and 150-fold more active than intestinal and liver cells, respectively. Serine . borate markedly inhibited, and glycyl-glycine stimulated, hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide in all cell types confirming the involvement of gamma-glutamyltransferase in the reaction. The hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide was inhibited to approximately the same extent by either GSH or GSSG suggesting that both compounds interact at the donor site of gamma-glutamyltransferase. Comparison of the rates of glutathione metabolism by isolated intestinal and kidney cells suggests that the intestinal contribution to the degradation of extracellular glutathione may be physiologically more important than has previously been assumed.     

Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.