Inhibition Of Cystine Uptake Research Articles

Sulfasalazine inhibits the growth of primary brain tumors independent of nuclear factor-kappaB.

Nuclear factor-kappaB (NF-kappaB) is a pleiotropic transcription factor that generally enhances cellular resistance to apoptotic cell death. It has been shown to be constitutively active in some cancers and is being pursued as potential anticancer target. Sulfasalazine which is used clinically to treat Crohn's disease has emerged as a potential inhibitor of NF-kappaB and has shown promising results in two pre-clinical studies to target primary brain tumors, gliomas. Once digested, sulfasalazine is cleaved into sulfapyridine and 5-aminosalicylic acid (5-ASA; mesalamine) by colonic bacteria, and the latter, too, is reported to suppress NF-kappaB activity. We now show that glioma cells obtained from patient biopsies or glioma cell lines do not show significant constitutive NF-kappaB activation, unless exposed to inflammatory cytokines. This does not change when gliomas are implanted into the cerebrum of severe combined immun-deficient mice. Nevertheless, sulfasalazine but not its cleaved form 5-ASA caused a dose-dependent inhibition of glioma growth. This effect was entirely attributable to the inhibition of cystine uptake via the system x(c)(-) cystine-glutamate transporter. It could be mimicked by S-4-carboxy-phenylglycine (S-4-CPG) a more specific system x(c)(-) inhibitor, and lentiviral expression of a constitutively active form of IkappaB kinase b was unable to overcome the growth retarding effects of sulfasalazine or S-4-CPG. Both drugs inhibited cystine uptake causing a chronic depletion of intracellular GSH and consequently compromised cellular redox defense which stymied tumor growth. This data suggests that system x(c)(-) is a promising therapeutic target in gliomas and possibly other cancers and that it can be pharmacologically inhibited by Sulfasalazine, an FDA-approved drug.

Cell culture density affects the susceptibility of human glioma cells to β‐ODAP toxicity

β‐ODAP is a neurotoxin that is found in the seeds of the grass pea, Lathyrus satvius, and is reported to cause neurolathyrism. The mechanism by which β‐OD AP causes neurolathyrism is not well understood, but it has been shown to be a transportable inhibitor of the transporter, system xc−. System xc− is involved in the exchange of cystine and glutamate across the plasma membrane in order to supply the cell with the cysteine necessary to maintain cellular glutathione levels. Previous studies in our lab have shown that system xc− cell surface expression is regulated by the redox state of the cell and cell culture density. In this study, we used β‐ODAP to inhibit cystine uptake by system xc− in order to reduce cellular glutathione levels in cultured glioma cells, and we subsequently examined the susceptibility of these cells to oxidative cell death. We have shown that effect of β‐ODAP on cellular glutathione levels and susceptibility to oxidative cell death is also dependent on cell culture density, with confluent cultures demonstrating an increased capacity to maintain cellular gluathione levels and resist oxidative cell death compared to non‐confluent cells. We hypothesize that the observed differences in susceptibility of confluent and non‐confluent cultures to β‐ODAP are a result of reduced capacity of non‐confluent cells to increase cell surface expression of System xc− when the oxidative load of the cell is increased.

Water extract of propolis and its main constituents, caffeoylquinic acid derivatives, exert neuroprotective effects via antioxidant actions

We investigated whether water extract of Brazilian green propolis (WEP) and its main constituents [caffeoylquinic acid derivatives (3,4-di- O-caffeoylquinic acid, 3,5-di- O-caffeoylquinic acid, chlorogenic acid) and cinnamic acid derivatives ( p-coumaric acid, artepillin C, drupanin, baccharin)] exert neuroprotective effects against the retinal damage induced by oxidative stress. Additionally, their neuroprotective effects were compared with their antioxidant effects. WEP, 3,4-di- O-caffeoylquinic acid, 3,5-di- O-caffeoylquinic acid, chlorogenic acid, and p-coumaric acid (but not artepillin C, baccharin, or drupanin) concentration-dependently inhibited oxidative stress-induced neurotoxicity [achieved using l-buthionine-( S, R)-sulfoximine (BSO) to deplete glutathione in combination with glutamate to inhibit cystine uptake] in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus). At their effective concentrations against oxidative stress-induced retinal damage, WEP, 3,4-di-caffeoylquinic acid, 3,5-di-caffeoylquinic acid, and chlorogenic acid (but not cinnamic acid derivatives) inhibited lipid peroxidation (LPO) in mouse forebrain homogenates. Thus, the neuroprotective effects of WEP and caffeoylquinic acid derivatives paralleled those against LPO. These findings indicate that WEP and caffeoylquinic acid derivatives have neuroprotective effects against retinal damage in vitro, and that these effects may be partly mediated via antioxidant effects.

Metallothionein, an Endogenous Antioxidant, Protects against Retinal Neuron Damage in Mice

To clarify the functional role of metallothionein (MT) in retinal damage in mice deficient in both MT-I and -II (MT-I/-II-deficient mice [C57BL/6J background]) and wild-type (C57BL/6J) mice and MT induction (zinc sulfate [ZnSO4] and 1alpha, 25-dihydroxyvitamin D3 [Vit. D3]). Retinal, cell damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 40 nmol/eye). Retinal MT-I, -II, and -III mRNA expression was monitored by real-time reverse-transcription-PCR of total retinal RNA from eyes injected or not injected with NMDA. In wild-type mice, MT-I and -II immunohistochemistry was performed (with antibody that recognizes both proteins) 12 and 24 hours after intravitreous NMDA injection. To examine the involvement of induced retinal MT, ZnSO4 (10 nmol/eye) or Vit. D3 (0.2 or 2 ng/eye) was intravitreously injected 24 hours before NMDA injection in wild-type or MT-I/-II-deficient mice, and ganglion cell layer (GCL) cell loss and inner plexiform layer (IPL) thinning were evaluated 7 days after the NMDA injection. The protective effect of Vit. D3 was assessed against the RGC-5 cell death induced by oxidative stress (using buthionine sulfoximine [BSO] to deplete glutathione in combination with glutamate to inhibit cystine uptake). In wild-type mice, MT-II mRNA expression was time-dependently elevated by NMDA (5.9 and 7.4 times versus the nontreated control at 4 and 12 hours, respectively, after injection), with the normal level being regained within 24 hours. In contrast, MT-I and -III showed persistent decreases (to <50% control) from 4 to 24 hours. In wild-type mice, MT-like immunoreactivity was increased in the inner retina (GCL and IPL) 12 and 24 hours after NMDA injection. At 7 days after NMDA injection in MT-I/-II-deficient mice (versus wild-type mice), GCL cell loss was increased, but IPL thickness was not different. Pretreatment with ZnSO4 or Vit. D3 increased inner retinal MT-like immunoreactivity 24 hours after NMDA injection and significantly attenuated NMDA-induced GCL cell loss in wild-type mice, but ZnSO4 pretreatment did not protect against such cell loss in MT-I/-II-deficient mice. In vitro, Vit. D3 pretreatment (100 nM) reduced BSO+glutamate-induced RGC-5 cell death. These findings suggest that MT, especially MT-II, protects against retinal neuron damage, by acting as an endogenous antioxidant.

Gypenosides protect primary cultures of rat cortical cells against oxidative neurotoxicity

Gypenosides (GPs) were tested for their ability to protect primary cultures of immature cortical cells against oxidative glutamate toxicity. In immature neural cells, glutamate cytotoxicity is known to be mediated by the inhibition of cystine uptake, leading to depletion of intracellular glutathione (GSH). The depletion of GSH impairs cellular antioxidant defenses resulting in oxidative stress and cell death. We found that pretreatment with GPs (100–400 μg/ml) significantly protected cells from glutamate-induced cell death. It was therefore of interest to investigate whether GPs protect cortical cells against glutamate-induced oxidative injury through preventing GSH depletion. Results show that GPs significantly up-regulated mRNAs encoding γ-glutamylcysteine synthetase (γ-GCS) and glutathione reductase (GR) and enhanced their activities for GSH synthesis as well as recycle. Furthermore, GPs lowered the consumption of GSH through decreased accumulation of intracellular peroxides, leading to an increase in the intracellular GSH content. GPs were also found to prevent lipid peroxidation and reduce the influx of Ca 2+ which routinely follows glutamate oxidative challenge. GPs treatment significantly blocked glutamate-induced decrease in levels of Bcl-2 and increase in Bax, leading to a decrease in glutamate-induced apoptosis. Thus, we conclude that GPs protect cortical cells by multiple antioxidative actions via enhancing intracellular GSH, suppressing glutamate-induced cytosolic Ca 2+ elevation and blocking glutamate-induced apoptosis. The novel role of GPs implies their remarkable preventative and therapeutic potential in treatment of neurological diseases involving glutamate and oxidative stress.

Oxytosis: A novel form of programmed cell death.

Extensive nerve cell death occurs during the development of the central nervous system as well as in episodes of trauma and in neurodegenerative disease. The mechanistic details of how these cells die are poorly understood. Here we describe a unique oxidative stress-induced programmed cell death pathway called oxytosis, and outline pharmacological approaches which interfere with its execution. Oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake through the cystine/glutamate antiporter leading to a depletion of glutathione, is used as an example of oxytosis. It is shown that there is a sequential requirement for de novo macromolecular synthesis, lipoxygenase activation, reactive oxygen species production, and the opening of cGMP-gated channels which allow the influx of extracellular calcium. The translation initiation factor elF2alpha plays a central role in this pathway by regulating the levels of glutathione. Finally, examples are given in which the reduction in glutathione, the production of reactive oxygen species, and calcium influx can be experimentally manipulated to prevent cell death. Data are reviewed which suggest that oxytosis may be involved in nerve cell death associated with nervous system trauma and disease.

The Consequences of Methylmercury Exposure on Interactive Functions between Astrocytes and Neurons

Methylmercury (MeHg) is a highly neurotoxic, environmentally ubiquitous chemical that exerts its toxic effects by largely unknown mechanisms. Maintenance of optimal intracellular concentrations of glutathione (GSH) is vital for cellular defenses against damage from free radicals. Since astrocytes play an essential role in providing GSH precursors to neurons, studies were directed at the effect of MeHg on cystine transport in both cell types. Astrocytes accumulated cystine via three independent transporters, referred to as system X AG−, system X C−, and γ-glutamyltranspeptidase (GGT). In contrast, neurons accumulated cystine exclusively via system X C− and GGT. MeHg potently inhibited cystine uptake in astrocytes (but not in neurons), and this effect could be fully accounted for by inhibition of the system X AG− transporter. The transport of glutamate in astrocytes is also inhibited by reactive oxygen species (ROS). Accordingly, additional studies examined the ability of thiol reducing or oxidizing agents to inhibit the astrocytic transport of H 3 - d-aspartate, a glutamate analog. The antioxidant catalase significantly attenuated MeHg-induced inhibition of astrocytic H 3 -aspartate uptake. Combinedly, these studies suggest that inhibition of cystine uptake and decreased astrocytic GSH levels and efflux reduce the availability of precursors for GSH synthesis in neurons. In addition, MeHg-induced generation of H 2O 2 plays a role in the inhibition of astrocytic glutamate transport. These effects likely increase neuronal vulnerability to MeHg-induced oxidative stress, and excess N-methyl d-aspartate (NMDA) receptor activation leading to neuronal demise.

The Activation of Dopamine D4 Receptors Inhibits Oxidative Stress-Induced Nerve Cell Death

Oxidative stress is thought to be the cause of nerve cell death in many CNS pathologies, including ischemia, trauma, and neurodegenerative disease. Glutamate kills nerve cells that lack ionotropic glutamate receptors via the inhibition of the cystine-glutamate antiporter x(c)(-), resulting in the inhibition of cystine uptake, the loss of glutathione, and the initiation of an oxidative stress cell death pathway. A number of catecholamines were found to block this pathway. Specifically, dopamine and related ligands inhibit glutamate-induced cell death in both clonal nerve cell lines and rat cortical neurons. The protective effects of dopamine, apomorphine, and apocodeine, but not epinephrine and norepinephrine, are antagonized by dopamine D4 antagonists. A dopamine D4 agonist also protects, and this protective effect is inhibited by U101958, a dopamine D4 antagonist. Although the protective effects of some of the catecholamines are correlated with their antioxidant activities, there is no correlation between the protective and antioxidant activities of several other ligands. Normally, glutamate causes an increase in reactive oxygen species (ROS) and intracellular Ca(2+). Apomorphine partially inhibits glutamate-induced ROS production and blocks the opening of cGMP-operated Ca(2+) channels that lead to Ca(2+) elevation in the late part of the cell death pathway. These data suggest that the protective effects of apomorphine on oxidative stress-induced cell death are, at least in part, mediated by dopamine D4 receptors via the regulation of cGMP-operated Ca(2+) channels.

Methylmercury inhibits the in vitro uptake of the glutathione precursor, cystine, in astrocytes, but not in neurons

Maintenance of adequate intracellular glutathione (GSH) levels is vital for intracellular defense against oxidative damage. The toxic effects of methylmercury (MeHg) are attributable, at least in part, to elevated levels of reactive oxygen species, and thus decreases in GSH synthesis may increase methylmercury toxicity. Astrocytes have recently been proposed to play an essential role in providing GSH precursors to neurons. Therefore, cystine transport, a prerequisite to GSH production, was characterized in cultured astrocytes and neurons, and the effects of methylmercury on this transport were assessed. Astrocytes and neurons both possessed temperature dependent transport systems for cystine. Astrocytes accumulated cystine by Na +-independent (X C−) and -dependent (X AG−) systems while neurons used exclusively Na +-independent systems. Inhibition of the X AG− transport system decreased cystine transport in astrocytes to levels equivalent to those in sodium-depleted conditions, suggesting that cystine is carried by a glutamate/aspartate transporter in astrocytes. Inhibition of the multifunction ectoenzyme/amino acid transporter γ-glutamyltranspeptidase (GGT) decreased cystine transport in both neurons and astrocytes. Inhibition of System X C− with quisqualate also decreased cystine uptake in both astrocytes and neurons. These data demonstrate that cultured astrocytes accumulate cystine via three independent mechanisms, System X AG−, System X C−, and GGT, while cultured hippocampal neurons use System X C− and GGT exclusively. Inhibition of cystine uptake in astrocytes by methylmercury appears to be due to actions on the System X AG− transporter.

Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer’s and Parkinson’s. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca 2+ influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca 2+ despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.

The role of Bax in glutamate-induced nerve cell death.

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate–cystine antiporter (system X c – ), whereas, others suggest that it is taken up by the glutamate transporter (system X AG ). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b o,+ and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-β-hydroxamate (AβH) and L- trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-α-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 μM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.

Mixed disulfides of homocysteine and cysteine inhibit cystine uptake, decreasing intracellular glutathione: [formula omitted], Bonita L. Moses, and Yunxia Q. O'Malley Department of Veterans Affairs Medical Center and the Department of Medicine, University of Iowa, Iowa City, Iowa

Mixed disulfides of homocysteine and cysteine inhibit cystine uptake, decreasing intracellular glutathione: [formula omitted], Bonita L. Moses, and Yunxia Q. O'Malley Department of Veterans Affairs Medical Center and the Department of Medicine, University of Iowa, Iowa City, Iowa

Glutamate Toxicity on a PC12 Cell Line Involves Glutathione (GSH) Depletion and Oxidative Stress

The effect of antioxidants and reducing agents on glutamate-induced cytotoxicity was examined using PC12 cells. The antioxidants vitamin E, idebenone, and selegiline protected cells against the cytotoxicity observed 24 h after exposure to 0.5 or 10 mM glutamate, as determined by lactate dehydrogenase leakage, even when added 3 h after glutamate. The reducing agents, glutathione (GSH) and dithiothreitol (DTT), also provided protection against the cytotoxicity of glutamate. Preincubation of PC12 cells with the antioxidants mentioned above, or the incubation with those antioxidants after exposure to glutamate for 3 h, prevented the reduction of viability caused by glutamate. Cystine uptake was inhibited by exposure of cells to glutamate, as determined by L-[35S]-cystine uptake. Incubation of cells with 0.5 or 10 mM glutamate caused a marked decrease in cellular GSH levels, not prevented by antioxidants. The activity of GSSG reductase was decreased by glutamate and this inhibition was reverted in the presence of the reducing agents GSH and DTT. These results indicate that glutamate toxicity on PC12 cells results from the inhibition of cystine uptake with consequent GSH depletion and oxidative stress, suggesting that antioxidants may reduce the cellular damage in pathologic conditions associated with excessive glutamate release.

The Role of Monoamine Metabolism in Oxidative Glutamate Toxicity

Glutamate kills neuronal cells by either a receptor-mediated pathway or the inhibition of cystine uptake, the "oxidative pathway." Antioxidants can block cell death initiated by either pathway, suggesting that toxicity is dependent on the production of free radicals. We provide evidence that in a neuronal cell line, glutamate toxicity via the oxidative pathway requires monoamine metabolism as a source of free radicals. Glutamate toxicity is inhibited by monoamine oxidase (MAO) type-A-specific inhibitors, but only at concentrations much higher than those required to inhibit classical type-A MAO. Toxicity is not inhibited by MAO type-B-specific inhibitors at any concentration. Furthermore, treatment of cells with agents that block monoamine uptake inhibits glutamate toxicity. These results suggest that an enzyme distinct from MAO is involved in monoamine metabolism and demonstrate a relationship between glutamate toxicity and monoamine metabolism. These data also have implications for the understanding and treatment of neurodegenerative disorders in which glutamate toxicity is thought to be involved.

A role for γ-glutamyl transpeptidase in the transport of cystine into human endothelial cells: relationship to intracellular glutathione

The mechanism of uptake of cystine into human umbilical vein endothelial (HUVE) cells was further investigated. Previous experiments have suggested that the uptake of cystine into HUVE cells is mediated by the X − c anionic amino acid transporter. However, in our experiments, the accumulation of cystine into confluent HUVE cells was not fully inhibited by anionic amino acids, such as glutamate, aminoadipate and homocysteate. On the other hand, inhibitors of γ-glutamyl transpeptidase (γ-GT), such as anthglutin and AT-125, were also effective inhibitors of cystine accumulation in these human cells. Indeed, incubation of HUVE cells with anthglutin effectively depleted cellular glutathione (GSH) in association with inhibition of cystine uptake. A dose-dependent inhibitory effect of anthglutin on the initial rate of accumulation of cystine into control HUVE cells was also confirmed, which was also dependent on the carrier concentration of the disulfide. Additionally, in an effort to relate the uptake of cystine and the levels of GSH in these human cells, the incubation of HUVE cells with diethylmaleate (DEM, 25 μM) was shown to stimulate the uptake of cystine by up to 200% over the first hour of incubation, irrespective of whether DEM was coincubated or preincubated with the cells for 60 min before the addition of cystine. Under these conditions the stimulation was not associated with extensive depletion of cellular GSH. Depletion of HUVE cell GSH by an 18 h incubation in M199 medium lacking sulfur amino acids (M199-medium) also stimulated the uptake of cystine by up to 300%, which was dependent on de novo protein biosynthesis. On the other hand, the depletion of HUVE cell GSH by an 18 h preincubation with buthionine sulfoximine did not stimulate cystine uptake above controls. Again, both the anionic amino acids and the inhibitors of γ-GT were effective in inhibiting the accumulation of cystine into M199-medium-pretreated HUVE cells, in association with impaired resynthesis of GSH. Anthglutin also dose-dependently inhibited the initial cystine uptake rate into these M199-medium-pretreated cells by a proportionally similar extent to its effects in control cells. This also indicates that γ-GT-dependent uptake of cystine accounts for a portion of the induced uptake of this disulfide into these M199-medium-pretreated cells. These results indicate that both the X − c carrier and γ-GT are involved in the uptake of cystine into these human endothelial cells, illustrating another important physiological role for γ-GT in the uptake of cystine from the circulation into the human endothelium. This may be of importance in providing cysteine equivalents for protein and GSH synthesis, both within the endothelial cells themselves, and in other cells associated with endothelial cells which are unable to utilise cystine. The data also show that HUVE cells can up-regulate the uptake of cystine in response to different stress stimuli which are associated with the depletion of cellular GSH, but that the depletion of the GSH per se is not sufficient for this response. Finally, inhibitors of γ-GT may be useful in studies of the functions of GSH in HUVE cells.

Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes

HepG2 cells are widely used as a model of human hepatocytes for studies of drug metabolism and toxicity. However, GSH metabolism in HepG2 cells is poorly characterized. This report describes the utilization of sulfur amino acids for GSH synthesis in HepG2 cells. In contrast to primary cultures of rat hepatocytes, which rely mostly on methionine for GSH synthesis, HepG2 cells use cystine. Their inability to utilize methionine for GSH synthesis was not due to lack of methionine uptake or low cellular ATP levels, but rather to the lack of S-adenosyl-methionine synthetase activity. When HepG2 cells were cultured overnight in medium containing cystine as the only sulfur amino acid, addition of glutamate or acivicin had minimal to no effect on cell GSH; however, addition of threonine significantly depleted cell GSH. When cystine (0.18 mM) uptake was measured, glutamate (2.5 mM), which inhibited cystine uptake in cultured rat hepatocytes, had a minimal effect in HepG2 cells. Instead, threonine (20 mM) strongly inhibited the apparent uptake of cystine by HepG2 cells. Strong inhibition by threonine of apparent cystine uptake was actually due to inhibition of cysteine uptake, which resulted from GSH-cystine mixed disulfide exchange. Radio-HPLC confirmed this. After incubating cells with [ 35S]cystine (0.18 mM) for 10 min, the total counts inside the cell matched the counts in the uptake medium in the form of GSH-cysteine mixed disulfide. Finally, HepG2 cells took up cysteine by both Na +-dependent and -independent mechanisms. The former exhibited high affinity and low capacity, whereas the latter exhibited the opposite. At a physiologic concentration of cysteine (10 μM), 68% of cysteine uptake occured via the Na +-dependent system and 32% via system L 1.

Cystine/glutamate antiporter expression in retinal mu¨ller glial cells: Implications for dl-alpha-aminoadipate toxicity

A cytotoxicity of glutamate or related amino acids (10 mM) mediated by a cystine/glutamate antiporter (system Xc¯) has recently been demonstrated in N18 neuroblastoma-rat retina hybrid (N18RE105) cells and C6 glioma cells. The antiporter usually transports glutamate outside and cystine inside, thereby maintaining cellular concentrations of glutathione. High concentrations of glutamate inhibit cystine uptake and lead to depletion of cellular levels of glutathione. Among related amino acids, dl-alpha-aminoadipic acid ( dl-α-AAA), which is well known as a selective gliotoxin in the retina, is also toxic to these cells. However, this does not explain why dl-α-AAA acts gliospecifically on the retina. To answer this question we first examined the effects of dl-α-AAA on the [ 35S]cystine uptake with parental N18 neuroblastoma cells and rat retina of the hybrid cells. dl-α-AAA showed a competitive inhibition of [ 35S]cystine uptake in the rat retina but not in the N18 cells. Such a competitive inhibition of cystine uptake by dl-α-AAA could also be seen in the carp retina. The cystine uptake with carp retina was mainly Na +-independent and Cl −-dependent as already described as a characteristic ion dependency of the Xc¯antiporter. We next examined the effects of exogenous cystine on the glutamate release from the retina. Cystine (1 mM) actually induced a glutamate release approximately twice that of the control. Furthermore, the glutamate release induced by cystine was also Na +-independent and Cl −-dependent, and was blocked by dl-α-AAA. An autoradiogram of [ 35S]cystine uptake in the carp retina showed typical radial glial Mu¨ller cells. A large incorporation of [ 35S]cystine into retinal glutathione fraction was detected by a high pressure liquid chromatography method during a 1–4-h incubation. A significant or large decrease of retinal levels of glutathione was observed one day after an intravitreal injection of 8 μmol dl-α-AAA or l-α-AAA, respectively. Buthionine sulfoximine (2.5 μmol), a specific inhibitor of glutathione synthesis, induced a large decrease of retinal levels of glutathione and a loss of electroretinographic b-wave 20–30 h after treatment. Taken together, our present data with rat and carp retinas strongly indicate that the expression of cystine/glutamate antiporter is enriched in the retina, particularly in the glial Mu¨ller cells which have a rapid turnover pool for glutathione. The gliotoxin dl-α-AAA inhibits cystine uptake through this antiporter on the glial cells and elicits reduction of cellular levels of glutathione. The site-specific action of gliotoxin dl-α-AAA in the retina will be discussed on the basis of the hypothesis that the expression of cystine/glutamate antiporter is enriched in the glial Mu¨ller cells.

A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion

We have demonstrated that addition of l-glutamate in millimolar amounts to a culture of C6 glioma cells induced cell death within 24 h. The glutamate-induced toxicity in the C6 glioma cells was completely suppressed by adding l-cystine (0.4–1.0 mM), while the C6 cells degenerated in l-cystine-deprived culture medium. Kinetic studies of [ 35S]cystine and [ 3H]glutamate uptake showed that cystine competitively inhibited glutamate uptake, and conversely glutamate inhibited cystine uptake competitively, suggesting that C6 cells have a cystine/glutamate antiporter (system CG or Xc¯) similar to that already described in the periphery. Exogenous cystine (1 mM) stimulated a release of endogenous glutamate from C6 cells in a Na +-independent Cl −-dependent fashion. Thus, the antiporter normally transports glutamate out of and cystine into the cells. With the glutamate analogues tested, there was a good correlation between cytotoxicity and inhibition of cystine uptake. The de novo synthesis of glutathione was largely dependent upon the uptake of extracellular cystine. Intracellular levels of glutathione were dramatically decreased within 8–10 h by culture in glutamate-added or cystine-free medium. Vitamin E (100 μM), an antioxidant, rescued the death of C6 cells induced by glutamate exposure or by culture in cystine-deprived medium, but did not restore the apparent decrease of intracellular glutathione. Taken together, the present data strongly indicate that glutamate-induced cell death is initially due to inhibition of cystine uptake through the antiporter Xc¯system; such inhibition leads to glutathione depletion exposing the cells to oxidative stress. Excess of extracellular glutamate introduced from endogenous or exogenous roots might disorder this mechanism, resulting in cell death.

Differences between OK and LLC-PK1 cells: cystine handling

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.