GCS Activity Research Articles

Effects of curcumin on oxidative stress status and thefunctions of astrocytes in the brain of aged rats

The change of oxidative balance and astrocyte in the cortex and hippocampus of aged SD rats were investigated by Western blotting,enzyme assay,immunofluorescence and HPLCMS.The data showed that curcumin reduced the level of MDA and 8-OHdG and enhanced the level of GSH and GCS activity,which indicated that curcumin inhibited the oxidative damage in the brian;the detection of the level of glial fibrillary acidic protein(GFAP)indicated curcumin inhibited the excessive activation of astrocytes and also the results of the activity of GS,the level of GDNF and D-serine showed that curcumin improved the function of astrocytes.

Ubiquitous transgene expression of the glucosylceramide-synthesizing enzyme accelerates glucosylceramide accumulation and storage cells in a Gaucher disease mouse model.

Gaucher disease is a lysosomal storage disease caused by defective activity of acid β-glucosidase (GCase), which leads to the accumulation of its major substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in many cells. To modulate cellular substrate concentration in viable mouse models of Gaucher disease (Gba1 mutants), a novel mouse model was created with enhanced glycosphingolipid biosynthesis. This was accomplished by cross-breeding Gba1 mutant mice with mice expressing a transgene (GCStg) containing the mouse glucosylceramide synthase (GCS, Ugcg) cDNA driven by the ROSA promoter, yielding GCStg/Gba1 mice. The GCStg rescued Ugcg null mice from embryonic lethality. GCStg/Gba1 mice showed 2–3 fold increases in tissue GCS activity as well as accelerated GlcCer accumulation and the appearance of lipid-laden CD68 positive macrophages in visceral organs. Although GlcCer/GlcSph concentrations were elevated in the brain, there was no neurodegenerative phenotype up to 1 yr of age conceivably due to the greater residual GCase hydrolytic activity in the brains than in the visceral tissues of 9V/null mice. These studies provide ‘proof of principle’ for threshold substrate flux that modifies phenotypic development in Gaucher disease and other lysosomal storage diseases.

Postprandial Insulin Resistance in Zucker Diabetic Fatty Rats is Associated with Parasympathetic‐Nitric Oxide Axis Deficiencies

The Zucker diabetic fatty (ZDF) rat is an obesity and type 2 diabetes model. Progression to diabetes is well characterised in ZDF rats, but only in the fasted state. We evaluated the mechanisms underlying postprandial insulin resistance in young ZDF rats. We tested the hypothesis that the overall postprandial action of insulin is affected in ZDF rats as a result of impairment of the hepatic parasympathetic-nitric oxide (PSN-NO) axis and/or glutathione (GSH), resulting in decreased indirect (PSN-NO axis) and direct actions of insulin. Nine-week-old male ZDF rats and lean Zucker rats (LZR, controls) were used. The action of insulin was assessed in the fed state before and after parasympathetic antagonism atropine. Basal hepatic NO and GSH were measured, as well as NO synthase (NOS) and γ-glutamyl-cysteine synthethase (GCS) activity and expression. ZDF rats presented postprandial hyperglycaemia (ZDF, 201.4 ± 12.9 mg/dl; LZR, 107.7 ± 4.3 mg/dl), but not insulinopaenia (ZDF, 5.9 ± 0.8 ng/ml; LZR, 1.5 ± 0.3 ng/ml). Total postprandial insulin resistance was observed (ZDF, 78.6 ± 7.5 mg glucose/kg; LZR, 289.2 ± 24.7 mg glucose/kg), with a decrease in both the direct action of insulin (ZDF, 54.8 ± 7.0 mg glucose/kg; LZR, 173.3 ± 20.5 mg glucose/kg) and the PSN-NO axis (ZDF, 24.5 ± 3.9 mg glucose/kg; LZR, 115.9 ± 19.4 mg glucose/kg). Hepatic NO (ZDF, 117.2 ± 11.4 μmol/g tissue; LZR, 164.6 ± 4.9 μmol/g tissue) and GSH (ZDF, 4.9 ± 0.3 μmol/g; LZR, 5.9 ± 0.2 μmol/g) were also compromised as a result of decreased NOS and GCS activity, respectively. These results suggest a compromise of the mechanism responsible for potentiating insulin action after a meal in ZDF rats. We show that defective PSN-NO axis and GSH synthesis, together with an impaired direct action of insulin, appears to contribute to postprandial insulin resistance in this model.

Alterations observed in the endothelial HUVEC cell line exposed to pure Cylindrospermopsin

The cyanobacterial toxin Cylindrospermopsin (CYN) is receiving great interest due to its increasing presence in waterbodies, which has lead to recognize it as a potential threat to drinking water safety. CYN is a potent inhibitor of protein and glutathione synthesis. The present work studies for the first time the effects of CYN in endothelial cells. The basal cytotoxicity endpoints studied at 24 and 48h were total protein content (PC), neutral red (NR) uptake and the tretazolium salt, MTS, reduction. Moreover, the effect of subcytotoxic concentrations of CYN on the generation of intracellular reactive oxygen species (ROS), the activity of γ-glutamylcysteine synthetase (GCS) and glutathione (GSH) content have been investigated. In addition, morphological alterations of HUVEC cells subsequent to CYN exposure were recorded.The cytotoxicity endpoints revealed a decrease in the cellular viability in a time and concentration-dependent way. The most sensitive cytotoxicity endpoint was NR uptake assay, with reductions in cell viability of 95% at 48h of exposure to 40μgmL−1 CYN. Intracellular ROS production was increased only at the lowest concentration assayed, while GCS activity and GSH content underwent concentration-dependent enhancements. The most remarkable morphological alterations observed were: nucleolar segregation with altered nuclei, degenerated Golgi apparatus, increases in the presence of granules and apoptosis.

Biochemical and pathological toxic effects induced by the cyanotoxin Cylindrospermopsin on the human cell line Caco-2

Cylindrospermopsin (CYN), a cyanotoxin produced by several freshwater cyanobacteria, causes human intoxications and animal mortalities. The present study focuses on the cytotoxic effects of CYN on Caco-2 cells at 24 and 48 h. The basal cytotoxicity endpoints studied were total protein content (TP), neutral red uptake (NR) and reduction of the tetrazolium salt (MTS). The effect of non-cytotoxic concentrations of CYN on the generation of intracellular reactive oxygen species (ROS), γ-glutamylcysteine synthetase (GCS) activity and glutathione (GSH) content was also studied and the morphological alterations in the Caco-2 cells subsequent to CYN exposure were recorded.The most sensitive endpoint – the reduction of MTS – showed that the viability of Caco-2 cells after exposure to the highest concentration assayed (40 μg/mL CYN) was reduced by about 90%. Intracellular ROS production increased only when exposed to a concentration of 1.25 μg/mL CYN, while GSH content and GCS activity increased when exposed to 2.5 μg/mL CYN. The main insights provided by the present study are the ultrastructural alterations, which reveal lipid degeneration, mitochondrial damage and nucleolar segregation with altered nuclei. Therefore, it has been demonstrated that CYN can induce toxic effects in Caco-2 cells in a time-concentration dependent manner. Moreover, unlike the cytotoxic and biochemical alterations, which were only evident at higher concentrations, morphological damage at the ultrastructural level was noticeable even at the lowest concentration used.

Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.

Treatment from birth of nonketotic hyperglycinemia due to a novel GLDC mutation

To determine whether the devastating outcome of neonatal-onset glycine encephalopathy (NKH) could be improved by instituting treatment immediately at birth rather than after symptoms are already well established. A newborn with NKH diagnosed prenatally following the neonatal death of a previous affected sibling was treated from birth with oral sodium benzoate (250 mg/kg/day) and the NMDA receptor antagonist ketamine (15 mg/kg/day) immediately after sampling cord blood and cerebrospinal fluid (CSF) for glycine determination. Glycine cleavage system (CGS) activity was determined in placental tissue. Mutation analysis was performed by sequencing all GLDC, GCSH and AMT exons. CSF glycine (99 micromol/L, reference 3.8-8.0) was already markedly elevated at birth. GCS activity in placental tissue was severely reduced (2.6% of controls). A novel homozygous GLDC c.482A-->G(Y161C) missense mutation was identified. Neonatal hypotonia and apnea did not occur but the long-term outcome was poor, with intractable seizures and severe psychomotor retardation. This contrasts with the favorable outcome with early treatment in variant NKH with mild GCS deficiency (Ann Neuol 2004;56:139-143). Prospective treatment with this regimen can favorably modify the early neonatal course of severe NKH but does not prevent the poor long-term outcome, suggesting glycine-induced prenatal injury and/or ongoing postnatal damage.

Regulation of cysteine dioxygenase and γ-glutamylcysteine synthetase is associated with hepatic cysteine level

Two hepatic enzymes, cysteine dioxygenase (CDO) and γ-glutamylcysteine synthetase (GCS), play important regulatory roles in the response of cysteine metabolism to changes in dietary sulfur amino acid or protein levels. To examine the time-course of changes in CDO and GCS activities, CDO and GCS-catalytic or heavy subunit protein and mRNA levels, and cysteine and glutathione levels, we adapted rats to either a low protein (LP) or high protein (HP) diet, switched them to the opposite diet, and followed these parameters over 6 days. Hepatic CDO activity and amount, but not mRNA level, increased in response to higher protein intake; the t 1/2 of change for CDO activity or protein level was 22 h for rats switched from a LP to a HP diet and 8 h for rats switched from a HP to a LP diet, suggesting that the HP diet decreased turnover of CDO. Hepatic GCS activity, catalytic subunit amount and mRNA level decreased in response to a higher protein intake. GCS catalytic subunit level changed with a similar t 1/2 for both groups, but the change in GCS activity in rats switched from a LP diet to a HP diet was faster (∼16h) than for rats switched from a HP to a LP diet (∼74h). Hepatic cysteine and glutathione levels reached new steady states within 12 h (LP to HP) or 24 h (HP to LP). CDO activity appeared to be regulated at the level of protein, probably by diminished turnover of CDO in response to higher protein intake or cysteine level, whereas GCS activity appeared to be regulated both at the level of mRNA and activity state in response to the change in cysteine or protein availability. These findings support a role of cysteine concentration as a mediator of its own metabolism, favoring catabolism when cysteine is high and glutathione synthesis when cysteine is low.

Enhancement of Glutathione and γ-Glutamylcysteine Synthetase, the Rate Limiting Enzyme of Glutathione Synthesis, by Chemoprotective Plant-Derived Food and Beverage Components in the Human Hepatoma Cell Line HepG2

Glutathione (GSH) is an important antioxidant and cofactor of detoxifying metabolism. Therefore, elevation of GSH as achieved by inducing γ-glutamylcysteine synthetase (GCS), the limiting enzyme of GSH synthesis, may contribute to chemoprevention against cancer. In previous animal studies, increases in GCS were mainly found in liver and other organs that are not easily accessible in humans. Thus, employment and evaluation of alternative systems such as human-derived cell lines are encouraged. In the present experiment, we used the hepatoma cell line HepG2 to investigate the response of GCS and GSH to five plant-derived chemoprotectants contained in regularly consumed foodstuffs and beverages (kahweol/cafestol [K/C] [15.5-62.0 μM], α-angelicalactone [100-400 μM], benzyl isothiocyanate [1.7-5.0 μM], diallyl sulfide [175-700 μM], and quercetin [10-50 μM]). All treatments led to dose-dependent increases in both GCS activity and GSH concentration. Time course studies with K/C indicated that the enhancement of GCS preceded that of GSH, suggesting a causal relationship. K/C did not enhance γ-glutamyl transpeptidase, a further enzyme that assists GSH-related chemoprotection. Although GCS induction has been suggested to require an initial short-lived GSH depletion, we did not find any decrease in GSH after 3 h of incubation with K/C. In summary, HepG2 cells were shown to be a useful model to investigate the capacity of potential chemoprotectants to enhance GCS and GSH. To our knowledge, the present study is also the first to show increases in GCS by K/C and a-angelicalactone in vitro and by diallyl sulfide and quercetin in any system.

Cysteine regulates expression of cysteine dioxygenase and γ-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.

Age-associated decline in γ-glutamylcysteine synthetase gene expression in rats

Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.

Down-regulation of renal glutathione synthesis by systemic nitric oxide synthesis inhibition in spontaneously hypertensive rats

Nitric oxide stimulates in vitro the synthesis of glutathione, an abundant thiol with a number of functions such as detoxification of xenobiotics and reactive oxygen species. In order to study this relationship in an animal model of hypertension, we treated spontaneously hypertensive rats (SHR) either with a nitric oxide synthase inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME) or with a nitric oxide donor isosorbide-5-mononitrate (IS-5-MN). Inhibition of nitric oxide synthesis led to malignant hypertension and to a marked decrease in glutathione synthesis through down-regulation of the rate-limiting enzyme γ-glutamylcysteine synthetase (GCS). The reduction in GCS activity was further augmented in SHR on a high sodium diet. Renal GCS activity in untreated SHR was 234 ± 14 and 240 ± 18 nmol/min/mg protein (mean ±SD) on a low and high sodium diet, respectively. When l-NAME was included in the diet, the activities dropped to 173 ± 28 and 123 ± 28 for the low and high sodium diets, respectively. IS-5-MN attenuated the rise in blood pressure induced by sodium chloride, but did not affect the GCS activity. The mechanism of GCS stimulation by nitric oxide is not known, but our results combined with the literature suggest that a relatively high concentration of nitric oxide is needed.

Brain γ‐glutamyl cysteine synthetase (GCS) mRNA expression patterns correlate with regional‐specific enzyme activities and glutathione levels

The first and rate-limiting reaction in the formation of glutathione is catalyzed by γ-glutamylcysteine synthetase (GCS), a dimer composed of a catalytic heavy and a regulatory light subunit. We previously found that heavy subunit GCS mRNA appears to be expressed at high levels in the hippocampus, cerebellum, and cortex of murine brain and at lower levels in the neostriatum (Kang et al. [1997] NeuroReport 8:2053). Here we report that variations in expression of light subunit GCS mRNA in murine brain resembles that of the heavy subunit mRNA with a few minor exceptions. Moreover, levels of GCS activity and glutathione levels in various brain regions appear to correspond to levels of expression of both GCS mRNA subunits. Based on these data, differences in the distribution of expression of the GCS subunits in the brain may therefore have major implications for the susceptibility of various brain regions to oxidative stress and/or mitochondrial damage. J. Neurosci. Res. 58:436–441, 1999. © 1999 Wiley-Liss, Inc.

Effect of Thioacetamide on the Hepatic Expression of γ-Glutamylcysteine Synthetase Subunits in the Rat

Glutathione (GSH) is the main nonprotein thiol important in antioxidant defense and maintenance of the intracellular redox state. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GCS). A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally. cis-Acting elements important for the oxidative stress-induced transcriptional up-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1) site. The nuclear factor-κB (NF-κB) binding site may also regulate the heavy subunit. Increased GSH and γ-glutamyltranspeptidase are often observed in preneoplastic hepatocyte nodules and may be important in hepatocarcinogenesis. The current work examined the effect of a commonly used hepatocarcinogen, thioacetamide (TAA), on the expression of GCS subunits. After 3 weeks of TAA treatment, liver GSH level remained unchanged despite significant oxidative stress as measured by the thiobarbituric acid reactive substance assay. The mRNA levels of GCS-HS and GCS-LS increased six- and fourfold, respectively, and the protein level of GCS-HS and GCS activity all increased. Electrophorectic mobility shift assay showed binding to ARE, AP-1, and NF-κB probes all increased. These results suggest TAA treatment increased hepatic GCS subunit expression and GCS activity by inducing oxidative stress and increasing the binding to redox-sensitive cis-acting elements important for transcriptional up-regulation of GCS. This is the first in vivo study that examined the effect of a hepatocarcinogen on GCS expression.

Regulation of γ-glutamylcysteine synthetase regulatory subunit ( GLCLR) gene expression: identification of the major transcriptional start site in HT29 cells

γ-Glutamylcysteine synthetase (GCS) is of major importance in glutathione homeostasis. The GCS heterodimer is composed of catalytic (heavy subunit, GCS h) and regulatory (light subunit, GCS l) subunits. Regulation of the human GCS l subunit gene ( GLCLR) expression was studied as GCS l has a critical role in glutathione synthesis. The minimal basal expression of GLCLR was found to be mediated by a region between nt −205 and −318. The major transcriptional start site in HT29 cells was located within this region at nt −283. A region between nt −411 and −447 was identified as having a potential involvement in the negative regulation of GLCLR expression. In order to study the transcriptional regulation of GCS l by oxidant stress, HepG2 cells were treated with sodium nitroprusside (SNP). SNP (1.5 mM) was found to increase glutathione levels by 2-fold, as well as GCS activity by 6-fold. This is accompanied by a co-ordinate increase in the levels of the both the GCS l and GCS h subunits, each by approximately 2-fold. The transcriptional activity of the GLCLR gene was increased by approximately 2.5-fold in SNP-treated cells.

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.

Elevation of glutathione and related enzyme activities in high-grade and metastatic extremity soft tissue sarcoma

Glutathione is a free radical scavenger implicated in the chemoresistance of certain tumors. As treatment with chemotherapy has added little to improved survival in adult soft tissue sarcoma and little is known concerning the mechanisms of chemoresistance in sarcoma, we studied concentrations of glutathione (nmol/mg protein) and activities of gamma-glutamylcysteine synthetase (GCS; nmol/mg protein/h) and gamma-glutamyl transpeptidase (GGTP; U/mg protein) in extremity soft tissue sarcoma. Tumor specimens (n = 65) were frozen in liquid nitrogen at the time of resection. Fourteen low-grade tumors, 40 high-grade tumors, and 11 pulmonary metastases were analyzed. Glutathione concentrations and GGTP activity were significantly lower in low-grade (3.97 +/- 0.7 nmol/mg protein and 1.07 +/- 0.2 U/mg protein) than in high-grade (8.98 +/- 1.2 nmol/mg protein, p < 0.001; 2.10 +/- 0.4 U/mg protein, p < 0.002) tumors and pulmonary metastases (10.05 +/- 1.8 nmol/mg protein, p < 0.008; 3.14 +/- 2.8 U/mg protein, p < 0.04). While GCS activity was lower in low-grade (0.81 +/- 0.3 nmol/mg protein/h) than high-grade (1.49 +/- 0.5 nmol/mg protein/h) tumors and pulmonary metastases (1.03 +/- 0.2 nmol/mg protein/h), these differences were not significant. In those patients with a high-grade tumor presenting with a local recurrence, glutathione levels were higher in those patients who had received preoperative doxorubicin-based chemotherapy (9.25 +/- 1.7 nmol/mg protein; n = 7) than in those who had no preoperative chemotherapy (4.71 +/- 3.1 nmol/mg protein; n = 4, p = 0.08). In extremity soft tissue sarcoma, glutathione concentration and GGTP activity are significantly elevated in patients with high-grade and metastatic sarcomas. In addition, there is a trend for increased glutathione levels in tumors previously exposed to doxorubicin-based chemotherapy. Glutathione may play a role in soft tissue sarcoma chemoresistance.

S-(1,2-Dicarboxyethyl)Glutathione, Intrinsic Tripeptide In Liver, Heart And Lens, Elevates γ-Glutamylcysteine Synthetase .Activity

Abstract: The effect of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) on glutathione (GSH) synthesis, especially γ -glutamylcysteine synthetase (γ-GCS), was studied in rat liver homogenate . It was found that the feedback inhibition of the γ-GCS by GSH was released by the addition of DCE-GS in the dose dependent manner. Namely, the activity of y-GCS decreased to 17% of the normal level when GSH at the concentration of 10 mM was added to the reaction mixture. However, the addition of DCE-GS offset the inhibition at 20 –μM and increased the γ -GCS activity to 1.7 and 3 times from the normal level at 50 and 100 -μM, respectively .These results suggest that DCE-GS plays a role in stimulating the GSH synthesis. Abstract: The effect of S-(1,2-dicarboxyethyl)glutathione(DCE-GS) on glutathione (GSH) synthesis, especially γ-glutamylcysteine synthetase (γ-GCS), was studied in rat liver homogenate . It was found that the feedback inhibition of the γ-GCS by GSH was released by the addition of DCE-GS in the dose dependent manner. Namely, the activity of γ-GCS decreased to 17% of the normal level when GSH at the concentration of 10 mM was added to the reaction mixture. However, the addition of DCE-GS offset the inhibition at 20 μM and increased the γ-GCS activity to 1.7 and 3 times from the normal level at 50 and 100 μM, respectively .These results suggest that DCE-GS plays a role in stimulating the GSH synthesis.

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Cholestatic Liver Injury Down-Regulates Hepatic Glutathione Synthesis

Hepatocellular injury caused by cholestasis may be caused in part by oxidant stress. The purpose of this study was to establish how acute cholestasis might alter hepatic glutathione homeostasis and to determine whether injured hepatocytes are capable of reverting to normal glutathione homeostatic mechanisms. Acute cholestasis was achieved by surgical ligation of the common bile duct in rats. Bile duct ligation induced a 3.7-fold increase in hepatic glutathione content over 4 days. This increase was not due to increased hepatic activity of γ-glutamylcysteine synthetase (GCS); on the contrary, whole-liver GCS activity was substantially diminished in the bile duct-ligated liver to 34 and 11% of normal after 4 and 7 days, respectively. To determine if hepatocytes removed from the cholestatic environment maintained these changes in glutathione homeostasis, hepatocytes were isolated from bile duct-ligated livers and established in primary culture. In cells isolated after 4 days of bile duct ligation, the elevated hepatocyte glutathione content decreased and the low GCS activity increased over 2 days in culture. More importantly, the ability of postcholestatic hepatocytes to substantially increase their glutathione synthetic capacity by increasing GCS activity in response to stress was preserved. This compensatory increase was due primarily to new protein synthesis. Together, these observations suggest that acute cholestasis impairs the ability of the liver to synthesize glutathione by down-regulating the key regulatory enzyme for its synthesis in response to acutely elevated glutathione levels and that the impaired glutathione synthetic capacity is corrected after cells are removed from the cholestatic environment.