Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Control of extracellular cysteine/cystine redox state by HT-29 cells is independent of cellular glutathione

Human cell lines regulate the redox state (E(h)) of the cysteine/cystine (Cys/CySS) couple in culture medium to approximately -80 mV, a value similar to the average E(h) for Cys/CySS in human plasma. The mechanisms involved in regulation of extracellular E(h) of Cys/CySS are not known, but GSH is released from tissues at rates proportional to tissue GSH concentration, and this released GSH could react with CySS to contribute to maintenance of this balance. The present study was undertaken to determine whether depletion of cellular GSH alters regulation of extracellular Cys/CySS E(h). Decrease of GSH in HT-29 cells by inhibiting synthesis with l-buthionine-[S,R]-sulfoximine showed no effect on the rate of reduction of extracellular CySS to achieve a stable E(h) for Cys/CySS in the culture medium. Limiting Cys and CySS in the culture medium also substantially decreased cellular GSH but resulted in no significant effect on extracellular Cys/CySS E(h). Addition of CySS to these cells showed that extracellular Cys/CySS E(h) approached -80 mV at 4 h while cellular GSH and extracellular GSH/GSSG E(h) recovered more slowly. Together, these results show that HT-29 cells have the capacity to regulate the extracellular Cys/CySS E(h) by mechanisms that are independent of cellular GSH. The results suggest that transport systems for Cys and CySS and/or membranal oxidoreductases could be more important than cellular GSH in regulation of extracellular Cys/CySS E(h).     

Metabolic basis for differential glutamine requirements of human leukemia cell lines

We compared the ability of human leukemia cell lines of various origins to grow in glutamine-deficient media. The growth of B lymphoblastoid cell lines, including promyelocytic HL-60, is highly dependent on glutamine, whereas T-cell lines are able to proliferate in glutamine-free media. Such glutamine dependency has a good inverse correlation with the activity of glutamine synthetase. Moreover, glutamine synthetase can be induced in glutamine-deficient media, especially in glutamine-independent cells. In HL-60 cells, glutamine deprivation results in the decrease of both ATP and dATP levels. The addition of adenine to the culture medium abolishes these changes without restoring cell growth, indicating that the effects of glutamine deprivation on cell growth cannot be fully explained by the perturbation of adenine nucleotide pools.     

Regulatory T cells interfere with glutathione metabolism in dendritic cells and T cells

Naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) suppress proliferation of CD4(+)CD25(-) effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.     

The role of glutathione in the aerobic radioresponse. I. Sensitization and recovery in the absence of intracellular glutathione

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.     

The role of low molecular weight thiols in T lymphocyte proliferation and IL-2 secretion

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.     

Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion

Treatment of A549 human lung carcinoma cells with L-buthionine-[S,R]-sulfoximine (BSO) results concomitantly in cellular glutathione (GSH) depletion and growth inhibition. The nature of BSO effects on cell growth and the relationships between BSO inhibition of cell growth and BSO effects on cellular GSH levels were determined in this study. A dose dependent effect of BSO on cell growth was observed, but this effect was found not to correlate with BSO effects on cellular GSH levels. Treatment with BSO for 60 h at concentrations of 5 and 10 mM was found to deplete cellular GSH at similar rates and to an undetectable level (below 0.5 nmol/mg protein). However, cessation of growth occured in 10 mM BSO whereas growth continued at better than one half the control rate in 5 mM BSO. The results suggest there may be a distinct threshold level of intracellular G GSH (on the order of or less than 0.5 nmol/mg protein) required for cell growth and for cells to protect themselves from the antiproliferative effects of BSO. At a concentration of 10 mM, BSO inhibited both DNA and protein synthesis and arrested growth of A549 cells throughout rather than at a specific phase of the cell cycle. BSO inhibition of growth was not, as indicated by colony-forming efficiency (CFE) and electron microscopy studies, accompanied by indications of cytotoxic effects. A stimulatory effect of 0.1 mM BSO on the growth of A549 cells was found also.Abbreviations BSO L-buthionine-[S,R]-sulfoximine - GSH Glutathione (reduced form) - GSSG Glutathione disulfide - DTNB 5,5-dithiobis (2-nitrobenzoate) - PBS Phosphate buffered saline - BSA Bovine serum albumin - PI Propidium iodide - CFE Colony-forming efficiency - EM Electron microscopy     

Glutamine protects mitochondrial structure and function in oxygen toxicity

Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.     

Glutamine and α-ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α-ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α-ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)(-1) ·min(-1), respectively) compared with 0.52 ± 0.09 μmol·(L RBC)(-1) ·min(-1) for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)(-1) ·min(-1), while the initial rate for 0.8 mM α-ketoglutarate was 0.97 μmol·(L RBC)(-1) ·min(-1). However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α-ketoglutarate (0.31 and 0.25?μmol·(L RBC)(-1) ·min(-1), respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α-ketoglutarate and/or glutamine.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Effects of cysteine,asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.     

Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8+ T cell function by limiting GSH and Cys availability

NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8⁺ T cell activation by using an antigen-driven coculture model consisting of Nrf2^−/− and Nrf2^+/+ bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8⁺ T cells. OT-1 CD8⁺ T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257–264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257–264)-pulsed Nrf2^−/− BMDMΦ with transgenic OT-1 CD8⁺ T cells attenuated CD8⁺ T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8⁺ T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2^−/− cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2^−/− cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2^+/+ BMDMΦ mimicked the effect of Nrf2^−/− BMDMΦ on CD8⁺ T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2^−/− BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8⁺ T cell function.     

Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death

The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress.     

Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.     

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

Glutamine synthetase activity in WI-38 cells

In the presence of complete growth media (Eagle's MEM), human diploid WI-38 cells have a low level of glutamine synthetase activity. The activity could be increased by depriving the cells of exogenous glutamine; addition of hydrocortisone to either glutamine-deficient or complete medium had no effect on the activity of the enzyme. Cell growth ceased under conditions that enhanced glutamine synthetase activity, and hydrocortisone could not reverse this inhibition.     

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.