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Query: UNIPROT:P06889 (Mol)
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This study was performed to determine whether depletion of myocardial glutathione would impair recovery of left ventricular function of blood-perfused, isolated hearts after reversible ischaemic injury. Cats were treated with either vehicle or buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of glutathione. The feline isolated hearts were perfused with the blood of normal donor cats before and after 40 min of global myocardial ischaemia. The myocardial concentration of glutathione of the BSO group, 178 +/- 38 ng/mg tissue, was significantly less than that of the control group, 292 +/- 38 ng/mg tissue (P < 0.05). The peak left ventricular developed pressure (LVDP) 1 h after reperfusion, expressed as a fraction of the peak LVDP before ischaemia, was 0.87 +/- 0.10 for the control group and 0.64 +/- 0.08 for the BSO group (P = 0.05 vs. control). The peak left ventricular dP/dt after reperfusion, expressed as a fraction of the peak dP/dt before ischaemia, was 1.08 +/- 0.14 for the control group and 0.78 +/- 0.09 for the BSO group (P = 0.05 vs. control). The myocardial creatine kinase activity of the BSO group, 1046 +/- 46 U/g tissue, was not significantly different from that of the control group, 1038 +/- 17 U/g tissue (P = 0.87). Thus, depletion of myocardial glutathione resulted in impaired post-ischaemic contractile function that cannot be attributed to a greater extent of irreversible cell injury.
J Mol Cell Cardiol 1992 Nov
PMID:Myocardial glutathione depletion impairs recovery of isolated blood-perfused hearts after global ischaemia. 136 26

Endogenous hydrogen peroxide (H2O2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H2O2 or menadione. Extracellular H2O2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H2O2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H2O2. Extracellular H2O2 release was 0.079 +/- 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or gamma-glutamylcysteine synthetase did not change the rate of extracellular release of H2O2. Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2O2 release or intracellular H2O2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H2O2 (30 microM), extracellular H2O2 was scavenged primarily by the glutathione redox pathway. Exogenously added H2O2 (100 microM) changed intracellular H2O2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 microM), which undergoes intracellular redox cycling, increased extracellular H2O2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H2O2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H2O2 release during menadione treatment, indicating that H2O2 can diffuse across the plasma membrane during oxidant stress.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Feb
PMID:Regulation of hydrogen peroxide generation in cultured endothelial cells. 154 Mar 80

Several thiols, including homocysteine and cysteamine, have been shown to increase glutathione levels in C3H/10T1/2 Cl 8 cells [Biochem. Pharmacol. 39:421-429 (1990)]. The present paper shows that cysteamine also increases homocysteine export from these cells. Cellular glutathione content and export of glutathione and homocysteine increased with increasing doses of cysteamine. Twenty-four hours after addition, 300 microM cysteamine increased both glutathione content and homocysteine export 3-4-fold. No change in the ratio between reduced and oxidized glutathione could be detected, suggesting that the cysteamine effect was not due to reduction of pools of oxidized glutathione. The elevation of glutathione occurred rapidly but declined between 24 and 48 hr after addition of cysteamine, whereas the homocysteine export increased momentarily after cysteamine exposure and then proceeded at a rate similar to that from untreated control cells. The cysteamine-induced increase in glutathione was completely prevented by the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine but was not affected by inhibition of homocysteine formation by 3-deazaaristeromycin. Buthionine sulfoximine did not prevent the increase in homocysteine export by cysteamine, and only a small increase in homocysteine export was observed when the cells were exposed to 3-deazaaristeromycin before treatment with cysteamine. Two major conclusions were drawn. 1) Increase of glutathione content and homocysteine export by cysteamine were independent events, indicating that glutathione status and homocysteine formation are regulated by independent mechanisms in C3H/10T1/2 Cl 8 cells. 2) S-Adenosylhomocysteine catabolism was the main source of the homocysteine export induced by cysteamine.
Mol Pharmacol 1990 Sep
PMID:Cysteamine increases homocysteine export and glutathione content by independent mechanisms in C3H/10T1/2 cells. 240 25

We investigated the effects of glutathione-S-transferase (GST) inhibitor treatment on human colon HT29 cell mRNA levels of dihydrodiol dehydrogenase (DDH), glyoxalase I, and gamma-glutamylcysteine synthetase. Time- and concentration-dependent increases in both DDH and gamma-glutamylcysteine synthetase mRNAs resulted from treatment with ethacrynic acid, ethacrynic acid/glutathione conjugate, and T.199 (gamma-glutamyl-S-(benzyl)-cysteinyl-R(-)-phenyl glycine diethyl ester), a selective GST pi inhibitor. In contrast, glutathione analogue GST alpha- and GST mu-selective inhibitors did not induce expression of these genes. Treatment with ethacrynic acid or T.199 had no effect on the mRNA levels of the glutathione-dependent glyoxalase I gene. Pretreatment of cells with buthionine-DL-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor and glutathione depleter, coupled with ethacrynic acid, ethacrynic acid/glutathione conjugate, or T.199 resulted in greater levels of gamma-glutamylcysteine synthetase and DDH induction compared with single treatments. Treatment with buthionine-DL-sulfoximine alone resulted in modest increases in both gamma-glutamylcysteine synthetase and DDH expression. Analyses of DDH induction by both differential Northern hybridization with specific oligonucleotides as probes and reverse transcriptase-polymerase chain reaction amplification of products, followed by diagnostic restriction digestion with endonucleases, showed that ethacrynic acid induced multiple DDH transcripts in HT29 cells and human HepG2 and SKHep1 hepatoma cells. Possible induction mechanisms include the alteration of sulfhydryl status by the electrophilic properties of EA or by elevations of endogenously generated oxidative stress via transient removal of GST pi from the cytosolic GST pool.
Mol Pharmacol 1995 Oct
PMID:Modulation of detoxification gene expression in human colon HT29 cells by glutathione-S-transferase inhibitors. 747 89

The YAP1 and YAP2 genes encode yeast transcription factors of the c-jun family. We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae. They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary-phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes. However, resistance towards H2O2 under these conditions is significantly reduced. We show that expression of the yeast GSH1 gene (encoding gamma-glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants. Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion. However, we show that Yap1 is not required for the superoxide anion-mediated induction of GSH1 gene expression. Furthermore, while the H2O2-mediated induction of SSA1 expression is shown to by YAP1 dependent, the heat-shock-mediated induction of the SSA1 gene does not require YAP1. We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes.
Mol Microbiol 1995 May
PMID:The role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae. 756 3

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, is under both hormonal and cell density regulation in cultured rat hepatocytes. Specifically, the addition of insulin or hydrocortisone to culture media or the lowering of the initial plating cell density increased cell GSH by increasing the activity of GCS. In the present study, we examined the molecular mechanism of these effects. To determine whether the increase in GCS activity is associated with an increase in GCS heavy subunit (GCS-HS) mRNA expression, the steady state mRNA levels of GCS-HS were examined with the use of Northern blots. After 24-hr treatment of high density (0.6 x 10(5) cells/cm2) cultured rat hepatocytes with insulin (1 micrograms/ml) or hydrocortisone (50 nM), the steady state GCS-HS mRNA level increased by approximately 1-2 fold. When the plating density was decreased to 0.1 x 10(5) cells/cm2, the steady state GCS-HS mRNA level also increased by 1-2 fold 24 hr later. An increase in the steady state GCS-HS mRNA level was found within 4 hr of either hormonal treatment or cell density manipulation. The increase in steady state GCS-HS mRNA level resulted from increased gene transcription, as the transcriptional rates of GCS-HS after hormonal or cell density manipulation were increased by 2-3-fold, whereas the rates of GCS-HS mRNA degradation remained unchanged. Western blotting confirmed the increase in GCS-HS protein level after hormone treatment or lowering of plating cell density. When examined in vivo, the steady state GCS-HS mRNA level decreased by 50% in a rat in which diabetes had been induced with streptozotocin for 1 week; this was prevented with insulin replacement. In summary, GCS-HS gene expression is under both hormonal and cell density regulation.
Mol Pharmacol 1995 Aug
PMID:Hormonal and cell density regulation of hepatic gamma-glutamylcysteine synthetase gene expression. 765 54

Rat liver and kidney gamma-glutamylcysteine synthetase (gamma GCS) had similar catalytic properties and consisted of heavy and light subunits, but the molecular structure of the two enzymes was not the same as evidenced by the results of SDS-PAGE and disc gel electrophoresis. Unlike kidney enzyme, most of liver gamma GCS was in a reduced enzyme form which did not have disulfide linkage between heavy and light subunits. Although the oxidized form of the two enzymes which subunits were linked with disulfide bond(s) could be dissociated to a similar extent by GSH, liver gamma GCS was inhibited by GSH to a much greater extent. These results suggest that the relative sensitivity of the gamma GCS enzymes to inhibition by GSH might be related to the inherent dissociability of heavy and light subunit of gamma GCS.
Biochem Mol Biol Int 1994 Mar
PMID:Biochemical regulation of the activity of gamma-glutamylcysteine synthetase from rat liver and kidney by glutathione. 791 45

Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-1 are known. Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein. GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro. Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage. A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal. Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance.
Mol Cell Biol 1994 Sep
PMID:GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation. 791 5

Tumor cell resistance to many chemotherapeutic agents, including alkylating agents, cisplatin, and doxorubicin, is frequently associated with increased intracellular levels of the nonprotein sulfhydryl glutathione (GSH). Recent evidence has demonstrated that increased GSH levels can be accompanied by an increase in the activity of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in de novo synthesis of GSH, and by an increase in the steady state level of mRNA for the catalytic subunit of GCS. Using melphalan-resistant DU 145/M4.5 human prostate carcinoma cells, which express elevated GSH levels, GCS enzyme activity, and GCS mRNA levels, we sought to determine the mechanism(s) responsible for the increased GCS mRNA expression. As determined by Northern analyses and RNase protection assays, the steady state level of GCS message in the resistant cells was increased 10-20-fold, in comparison with the drug-sensitive parent DU 145 cells. No significant difference in gene copy number or evidence of rearrangement was detected in the resistant cell line by Southern analyses. The GCS-specific mRNA isolated from the resistant cells was less stable than that isolated from the drug-sensitive cells (half-lives of 6 hr and 9 hr, respectively), indicating that this difference does not contribute to the increased steady state levels in the resistant cells. Nuclear run-on experiments revealed that the GCS transcription rate in the DU 145/M4.5 cells was increased approximately 12-fold, in comparison with that detected in the DU 145 cells. This difference in transcription rate was comparable in magnitude to the difference in steady state mRNA levels detectable in the two cell populations. Similar correlations between steady state GCS mRNA levels and transcription rates were also observed in other DU 145 lines expressing intermediate degrees of resistance to melphalan and correspondingly intermediate GCS mRNA elevations. These data suggest that GCS expression is transcriptionally regulated in these melphalan-resistant tumor cells.
Mol Pharmacol 1994 Nov
PMID:Transcriptional up-regulation of gamma-glutamylcysteine synthetase gene expression in melphalan-resistant human prostate carcinoma cells. 796 79

Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
Mol Cell Biol 1994 Sep
PMID:Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. 806 32


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