Gamma-glutamylcysteine Ligase Research Articles

The redox switch of γ‐glutamylcysteine ligase via a reversible monomer–dimer transition is a mechanism unique to plants

In plants, the first committed enzyme for glutathione biosynthesis, gamma-glutamylcysteine ligase (GCL), is under multiple controls. The recent elucidation of GCL structure from Brassica juncea (BjGCL) has revealed the presence of two intramolecular disulfide bridges (CC1, CC2), which both strongly impact on GCL activity in vitro. Here we demonstrate that cysteines of CC1 are confined to plant species from the Rosids clade, and are absent in other plant families. Conversely, cysteines of CC2 involved in the monomer-dimer transition in BjGCL are not only conserved in the plant kingdom, but are also conserved in the evolutionarily related alpha- (and some gamma-) proteobacterial GCLs. Focusing on the role of CC2 for GCL redox regulation, we have extended our analysis to all available plant (31; including moss and algal) and related proteobacterial GCL (46) protein sequences. Amino acids contributing to the homodimer interface in BjGCL are highly conserved among plant GCLs, but are not conserved in related proteobacterial GCLs. To probe the significance of this distinction, recombinant GCLs from Nicotiana tabacum (NtGCL), Agrobacterium tumefaciens (AtuGCL, alpha-proteobacteria) and Xanthomonas campestris (XcaGCL, gamma-proteobacteria) were analyzed for their redox response. As expected, NtGCL forms a homodimer under oxidizing conditions, and is activated more than threefold. Conversely, proteobacterial GCLs remain monomeric under oxidizing and reducing conditions, and their activities are not inhibited by DTT, despite the presence of CC2. We conclude that although plant GCLs are evolutionarily related to proteobacterial GCLs, redox regulation of their GCLs via CC2-dependent dimerization has been acquired later in evolution, possibly as a consequence of compartmentation in the redox-modulated plastid environment.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

A Possible Role of Nrf2 in Prevention of Renal Oxidative Damage by Ferric Nitrilotriacetate

To ascertain the possible roles of nuclear erythroid 2 p45-related factor 2 (Nrf2), a key transcription factor of phase 2 drug-metabolizing enzymes, in renal cellular defense against oxidative stress, wild-type and Nrf2-knockout -/- mice were treated with ferric nitrilotriacetate (Fe-NTA) at doses of 3 or 6 mg iron/kg body weight. After Fe-NTA treatment, Nrf2 -/- mice consistently showed lower levels of glutathione (GSH) in the kidney at the low dose and the liver at the high dose than the wild-type mice. Gamma-glutamylcysteine ligase (GCL) activity in the kidney and liver of Nrf2-/- mice was also consistently lower than in wild-type mice after the Fe-NTA treatment. Histopathological examination revealed that nephrotoxicity of Fe-NTA, reflected in necrosis of renal tubule epithelial cells following nuclear damage, was more severe in the Nrf2-/- mice than in their wild-type counterparts. Overall, the data suggest that Nrf2 -/- mice are unable to compensate for depletion of renal GSH because of oxidative stress, being more susceptible to Fe-NTA-induced nephrotoxicity. In conclusion, the present study showed that Nrf2 might play an important role in protecting cells from oxidative stress in the kidney through its regulation of antioxidant enzymes.

Potent Upregulation of Glutathione and NAD(P)H:Quinone Oxidoreductase 1 by Alpha-lipoic Acid in Human Neuroblastoma SH-SY5Y Cells: Protection Against Neurotoxicant-elicited Cytotoxicity

Alpha-lipoic acid (LA) has recently been reported to afford protective effects in neurodegenerative disorders. However, the mechanisms underlying LA-mediated neuroprotection remain to be investigated. This study was undertaken to determine whether LA treatment could increase endogenous antioxidants and phase 2 enzymes in cultured human neuroblastoma SH-SY5Y cells, and whether such increased cellular defenses could afford protection against cytotoxicity induced by neurotoxicants. Incubation of SH-SY5Y cells with micromolar concentrations of LA for 24 h resulted in a significant increase in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQQ1) in a concentration-dependent fashion. Treatment of the cells with LA also led to an increased mRNA expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. To determine the protective effects of the LA-induced cellular defenses on neurotoxicant-elicitedl cell injury, SH-SY5Y cells were pretreated with LA for 24 h and then exposed to acrolein, 4-hydroxy-2-nonenal (HNE), H(2)O(2) and the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1). We observed that LA pretreatment of SH-SY5Y cells led to a marked protection against acrolein, HNE, H(2)O(2 )and SIN-1-mediated cytotoxicity, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Taken together, this study demonstrates for the first time that LA can induce GSH and NQO1 in cultured human neuroblastoma cells and LA-upregulated cellular defenses are accompanied by a markedly increased resistance to cytotoxicity induced by various neurotoxicants. The results of this study may have important implications for the neuroprotective effects of LA.

Endothelial Dysfunction and Oxidant Status in Pediatric Patients with Hemoglobin E-β Thalassemia

Thalassemia major is characterized by anemia, iron overload, and oxidant damage to major organs, especially the cardiovascular system. Oxidative stress is ultimately involved in endothelial dysfunction, a condition which is evident in adults suffering from various cardiovascular diseases including thalassemia. We investigated endothelial function in pediatric patients with hemoglobin E-beta thalassemia (HbE-beta thalassemia), who have been exposed to excessive iron and oxidative stress for much shorter period than adults with thalassemia. We recruited 22 blood transfusion-dependent HbE-beta thalassemia patients aged 11.8 +/- 2.9 years and 20 healthy controls aged 12.1 +/- 1.7 years. Oxidant status was determined, and endothelial function was assessed by a forearm blood flow technique. Oxidative stress was increased in the thalassemic patients, as blood glutathione (GSH) and ratios of reduced GSH to GSH disulfide were markedly reduced, and superoxide anion released from blood cells was highly elevated. Oxidative stress response, assessed by gamma-glutamylcysteine ligase activity, was increased approximately twofold in thalassemia patients. Basal forearm blood flow was significantly increased in patients compared with controls (7.3 +/- 1.8 vs 6.0 +/- 1.8 ml/100 ml tissue/min, respectively), whereas forearm vasodilatory response to reactive hyperemia was depressed by 50% in patients compared with controls. Endothelial function is impaired in young thalassemia patients, and impaired endothelial function is associated with oxidant stress.

Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress

Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (γGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/MpJ-Fas lpr mice (MRL/ lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/ lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/ lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, γGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/ lpr mice.

Glutathione and glutathione-linked enzymes in normal human aortic smooth muscle cells: chemical inducibility and protection against reactive oxygen and nitrogen species-induced injury

Substantial evidence suggests a crucial role for glutathione (GSH) and GSH-linked enzymes in protecting against oxidative vascular disorders. However, studies on the chemical inducibility of these antioxidant defenses and their protective effects on oxidant injury in normal human vascular cells are currently lacking. Accordingly, this study was undertaken to investigate the inducibility of GSH, glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) by the chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) in cultured normal human aortic smooth muscle cells (HASMCs). HASMCs expressed measurable levels/activities of GSH, GR, GPx, and GST. Incubation of HASMCs with low micromolar concentrations of D3T resulted in a marked elevation in total cellular GSH content and GR activity. The protein and mRNA expression of gamma-glutamylcysteine ligase (GCL) and GR were also upregulated by D3T. In addition, D3T caused significant increases in mitochondrial GSH content and GR activity. In contrast, neither cellular GPx nor GST activity was altered after D3T treatment. Pretreatment of HASMCs with D3T afforded remarkable protection against reactive oxygen and nitrogen species (ROS/RNS)-mediated cell injury. Depletion of cellular GSH by pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis led to marked potentiation of the ROS/RNS-induced cell injury. Moreover, co-treatment of HASMCs with BSO was found to completely abolish the D3T-mediated GSH elevation, and remarkably reverse D3T cytoprotection against the ROS/RNS-elicited injury. Taken together, this study demonstrates that both GSH/GCL and GR in normal HASMCs are inducible by D3T, and that upregulation of GSH biosynthesis appears to be the predominant mechanism underlying D3T-mediated cytoprotection against ROS/RNS-elicited injury to human vascular smooth muscle cells.

Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Faslpr mice

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.

Lipoic acid supplementation prevents cyclosporine-induced hypertension and nephrotoxicity in spontaneously hypertensive rats

Cyclosporine (CsA) has significantly improved long-term survival after organ transplantations. Hypertension and nephrotoxicity are common side effects during CsA treatment and are aggravated by high salt intake. To examine whether lipoic acid (LA), a natural antioxidant that scavenges reactive oxygen species and regenerates/recycles endogenous antioxidants, could prevent CsA-induced hypertension and nephrotoxicity. Six-week-old spontaneously hypertensive rats (SHR) on a high-sodium diet (NaCl 6%) received CsA [5 mg/kg subcutaneously (s.c.)] alone or in combination with LA (0.5% w/w) for 6 weeks. Blood pressure, arterial functions, and tissue morphology were determined. Immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography were used for kidney and heart samples. CsA induced severe hypertension, cardiac hypertrophy, endothelial dysfunction, and pronounced albuminuria. Histologically, the kidneys showed severe thickening of the media of the afferent arteries with fibrinoid necrosis, perivascular monocyte/macrophage infiltration and nitrotyrosine overexpression. CsA induced the expression of fibrogenic connective tissue growth factor both in the heart and kidneys. The detrimental effects of CsA were associated with upregulation of myocardial atrial natriuretic peptide (ANP) mRNA expression, paradoxical activation of the renin-angiotensin system (RAS), induction of renal reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and overexpression of oxidative stress-induced transcription factor NRF2. LA lowered blood pressure, ameliorated cardiac hypertrophy and endothelial dysfunction, and totally normalized albuminuria. In LA-treated rats, renal and cardiac morphologies were indistinguishable from those of SHR controls. CsA-induced myocardial ANP and connective tissue growth factor (CTGF) mRNA overexpression, RAS activation, NADPH oxidase induction, and NRF2 overexpression were prevented by LA. LA induced the mRNA expression of gamma-glutamylcysteine ligase, the rate-limiting enzyme in glutathione synthesis, and markedly increased hepatic cysteine and glutathione concentrations. Our findings suggest a salutary role for lipoic acid supplementation in the prevention of CsA-induced hypertension and nephrotoxicity, and underscore the importance of increased oxidative stress in the pathogenesis of CsA toxicity.

Astrocyte control of fetal cortical neuron glutathione homeostasis: up‐regulation by ethanol

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron gamma-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of gamma-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrocyte up to 517% (p < 0.05). Ethanol increased both gamma-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the gamma-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.

(R)-α-Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage

To determine whether (R)-alpha-lipoic acid (LA) protects cultured human fetal retinal pigment epithelial (hfRPE) cells against oxidative injury and identify the pathways that may mediate protection. Cultured hfRPE cells were pretreated with various concentrations of LA for 14 to 16 hours followed by treatment with a chemical oxidant, tert-butylhydroperoxide (t-BuOOH; 0.8 mM, 3 hours). Reactive oxygen species (ROS) production and cell viability were measured using H(2)DCF and MTT assays, respectively. RPE cells were evaluated with fluorescent dyes (SYTOX Orange and SYTO Green; Molecular Probes, Eugene, OR), which differentiate between live and dead cells. Apoptosis was visualized by using the TUNEL assay. Changes in mitochondrial membrane potential were detected by JC-1 dye. Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by HPLC. Regulation of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme of GSH production, was assayed by RT-PCR. Pretreatment of hfRPE cells with LA, 0.2 mM and 0.5 mM, significantly reduced the levels of t-BuOOH-induced intracellular ROS, by 23% and 49%, respectively. LA (0.5 mM) prevented oxidant-induced cell death and apoptosis and also increased the viability of oxidant-treated hfRPE cells from 38% to 90% of control. LA upregulated the mRNA expression of GCL, and was protective against t-BuOOH-induced decreases in both mitochondrial membrane potential and intracellular levels of GSH and GSH/GSSG. The present study suggests that the protective effect of LA involves multiple pathways and that LA could be effective against age-associated increase in oxidative stress and mitochondrial dysfunction in RPE cells.

Prevention of Oxidative Stress by Adenoviral Overexpression of Glutathione‐Related Enzymes in Pancreatic Islets

Chronic exposure to supraphysiologic glucose concentrations causes functional damage to cells and tissues, a process known as glucose toxicity. Recent research indicates that one important mechanism for glucose toxicity is oxidative stress. Glucose has been shown to form reactive oxygen species through several metabolic pathways. The pancreatic islet is distinguished by its relatively low antioxidant enzyme content and activity, which render it especially susceptible to oxidative stress. Adenoviral overexpression of glutathione peroxidase as well as gamma-glutamylcysteine ligase have been shown to protect the islet against oxidative stress. Antioxidants have been shown to brake the worsening of diabetes by improving beta cell function in animal models. These observations suggest that enhancing antioxidant defense mechanisms in pancreatic islets may be a valuable pharmacologic approach to managing diabetes.

Kinetic measurement by LC/MS of γ-glutamylcysteine ligase activity

gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.

Bcl-2 Attenuation of Oxidative Cell Death Is Associated with Up-regulation of γ-Glutamylcysteine Ligase via Constitutive NF-κB Activation

Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis, which is regulated by many functional genes and their protein products. The evolutionarily conserved protein Bcl-2 blocks apoptosis induced by a wide array of death signals. Despite extensive research, the molecular milieu that characterizes the anti-apoptotic function of Bcl-2 has not been fully clarified. In this work, we have investigated the role of bcl-2 in protecting against oxidative death induced by H(2)O(2) in cultured rat pheochromocytoma PC12 cells. Transfection with the bcl-2 gene rescued PC12 cells from apoptotic death caused by H(2)O(2). Addition of NF-kappaB inhibitors such as pyrrolidine dithiocarbamate and N-tosyl-l-phenylalanine chloromethyl ketone to the medium aggravated oxidative cell death. PC12 cells overexpressing bcl-2 exhibited relatively high constitutive DNA binding and transcriptional activities of NF-kappaB compared with vector-transfected control cells. Western blot analysis and immunocytochemistry revealed that bcl-2-transfected PC12 cells retained a higher level of p65 (the functionally active subunit of NF-kappaB) in the nucleus compared with vector-transfected controls. In addition, sustained activation of ERK1/2 (upstream of NF-kappaB) was observed in bcl-2-overexpressing cells. In contrast, the cytoplasmic inhibitor IkappaBalpha was present in lower amounts in cells overexpressing bcl-2. The ectopic expression of bcl-2 increased the cellular glutathione level and gamma-glutamylcysteine ligase expression, which were attenuated by NF-kappaB inhibitors. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against H(2)O(2)-induced apoptosis in PC12 cells through augmentation of antioxidant capacity.

Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation.

BackgroundThe major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD).MethodsHippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO2) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O2.-), hydroxyl radicals (. OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression.ResultsAll the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia.ConclusionsRC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage.

Insulin signaling regulates gamma-glutamylcysteine ligase catalytic subunit expression in primary cultured rat hepatocytes.

Decreased glutathione (GSH) levels and gamma-glutamylcysteine ligase (GCL) activity have been observed in diabetic patients, and insulin reportedly increases GSH synthesis via increased GCL catalytic subunit (GCLC) gene expression. The signaling pathways responsible for mediating insulin effects on GCLC expression and GSH levels, however, are unknown. The signaling pathways involved in the regulation of GSH synthesis in response to insulin were examined in primary cultured rat hepatocytes. GSH levels, GCL activity, GCLC protein, and mRNA levels were increased to 140, 160, 600, and 340% of that monitored in untreated cells, respectively, in hepatocytes cultured with 100 nM insulin. The phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002 [2-(4-morpholinyl)-9-phenyl-4H-1-benzopyran-4-one], dominant-negative Akt, or rapamycin, an inhibitor of mTOR (mammalian target of rapamycin) and ribosomal p70 S6 kinase (p70S6K) phosphorylation, inhibited the insulin-mediated increase in GCLC protein and GSH levels. Although the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase, p38 MAPK, and JNK (c-Jun N-terminal kinase) were activated in response to insulin, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of JNK, and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], an inhibitor of p38 MAPK, failed to inhibit the insulin-mediated increase in GCLC protein levels. In conclusion, these data show that insulin signaling pathways involving PI3K/Akt/p70S6K, but not MAPKs, are active in the insulin-mediated regulation of GSH synthesis via increased GCLC expression.

Protecting Against Peroxynitrite-Mediated Cytotoxicity in Vascular Smooth Muscle Cells Via Upregulating Endogenous Glutathione Biosynthesis by 3H-1,2-dithiole-3-thione

Peroxynitrite (ONOO(-)) is critically involved in the pathogenesis of cardiovascular diseases. Reaction with glutathione (GSH) was proposed to be a major detoxification pathway of ONOO(-) in the biological system. This study was undertaken to determine if chemically elevated intracellular GSH affords protection against ONOO(-)-mediated toxicity in vascular cells. Incubation of aortic smooth muscle A10 cells with 3H-1,2-dithiole-3-thione (D3T) led to a concentration- and time-dependent elevation of cellular GSH. Treatment of the cells with D3T also augmented protein and gene expression of gamma-glutamylcysteine ligase. To examine the effects of D3T-induced GSH on ONOO(-)-mediated toxicity, we pretreated A10 cells with D3T and then exposed them to either authentic ONOO(-) or the ONOO(-) generator, 3-morpholinosydnonimine. We observed that D3T pretreatment of A10 cells resulted in a significant protection against ONOO(-) cytotoxicity. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of ONOO(-) cytotoxicity. To further demonstrate the causal involvement of GSH induction in D3T cytoprotection, we cotreated A10 cells with BSO to abolish D3T-induced GSH elevation. BSO cotreatment was found to greatly reverse the protective effects of D3T on ONOO(-)-elicited cytotoxicity. Taken together, our results demonstrate that upregulating GSH biosynthesis by D3T results in a marked protection against ONOO(-)-induced toxicity in vascular cells.