Littérature scientifique sur le sujet « Cystine/glutamate transporter »

Cysteine is the limiting precursor for glutathione synthesis. Because of its low bioavailability, cysteine is generally produced from cystine, which may be taken up through two different transporters. The cystine/glutamate antiporter (x[Formula: see text] system) transports extracellular cystine in exchange for intracellular glutamate. The XAG transport system takes up extracellular cystine, glutamate, and aspartate. Both are sensitive to competition between cystine and glutamate, and excess extracellular glutamate thus inhibits glutathione synthesis, a nonexcitotoxic mechanism for glutamate toxicity. We demonstrated previously that human macrophages express the glutamate transporters excitatory amino acid transporter (EAAT)1 and EAAT2 (which do not transport cystine, X[Formula: see text] system) and overcome competition for the use of cystine transporters. We now show that macrophages take up cystine through the x[Formula: see text] and not the XAG system. We also found that glutamate, although competing with cystine uptake, dose-dependently increases glutathione synthesis. We used inhibitors to demonstrate that this increase is mediated by EAATs. EAAT expression in macrophages thus leads to glutamate-dependent enhancement of glutathione synthesis by providing intracellular glutamate for direct insertion in glutathione and also for fueling the intracellular pool of glutamate and trans-stimulating the cystine/glutamate antiporter.

Cysteine availability is rate limiting for the synthesis of glutathione, an important antioxidant in the lung. We used rat alveolar epithelial type II cells to study the mechanism of cysteine and cystine uptake. Consistent with carrier-mediated transport, each uptake process was saturable with Michaelis-Menten kinetics and was inhibited at 4°C and by micromolar levels of amino acids or analogs known to be substrates for a specific transporter. A unique system XAG was found that transports cysteine and cystine (as well as glutamate and aspartate, the only substrates previously described for system XAG). We also identified a second Na+-dependent cysteine transporter system, system ASC, and two Na+-independent transporter systems, system xc for cystine and system L for cysteine. In the presence of glutathione at levels measured in rat plasma and alveolar lining fluid, cystine was reduced to cysteine and was transported on systems ASC and XAG, doubling the transport rate. Cysteinylglycine, released from glutathione at the cell surface by γ-glutamyl transpeptidase, also stimulated uptake after reduction of cystine. These findings suggest that, under physiological conditions, cysteine and cystine transport is influenced by the extracellular redox state.

We have shown previously that extracellular cysteine is necessary for cellular responses to S-nitrosoalbumin. In this study we have investigated mechanisms involved in accumulation of extracellular cysteine outside vascular smooth muscle cells and characterized the role of cystine-cysteine release in transfer of nitric oxide (NO)-bioactivity. Incubation of cells with cystine led to cystine uptake, reduction, and cysteine release. The process was inhibitable by extracellular glutamate, suggesting a role for system xc− amino acid transporters. Smooth muscle cells express this transporter constitutively and induction of the light chain component (xCT) by either diethyl maleate or 3-morpholino-sydnonimine (SIN-1) led to glutamate-inhibitable cystine uptake and an increased rate of cysteine release from cells. Likewise, overexpression of xCT in smooth muscle cells or endothelial cells led to glutamate-inhibitable cysteine release. The resulting extracellular cysteine was found to be required for transfer of NO from extracellular S-nitrosothiols into cells via system L transporters leading to formation of cellular S-nitrosothiols. Cysteine release coupled to cystine uptake was also found to be required for cellular responses to S-nitrosoalbumin and facilitated S-nitrosoalbumin-mediated inhibition of epidermal growth factor signaling. These data show that xCT expression can constitute a cystine-cysteine shuttle whereby cystine uptake drives cysteine release. Furthermore, we show that extracellular cysteine provided by this shuttle mechanism is necessary for transfer of NO equivalents and cellular responses to S-nitrosoablumin.

Abstract Glutamine transporters transport different amino acids for cell growth and metabolism. In tumor cells, glutamine transporters are often highly expressed and play a crucial role in their growth. By inhibiting the amino acid transport of these transporters, the growth of cancer cells can be inhibited. In recent years, more and more attention has been paid to the study of glutamine transporter. In this article, the differences between the ASC system amino acid transporter 2 (ASCT2), L-type amino acid transporter 1 (LAT1), and the cystine–glutamate exchange (xCT) transporters research progress on the mechanism of action and corresponding small molecule inhibitors are summarized. This article introduces 62 related small molecule inhibitors of different transporters of ASCT2, LAT1, and xCT. These novel chemical structures provide ideas for the research and design of targeted inhibitors of glutamine transporters, as well as important references and clues for the design of new anti-tumor drugs.

Substance-use disorder is globally prevalent and responsible for numerous social and medical problems. Pregabalin (Lyrica), typically used to treat diabetic neuropathy, has recently emerged as a drug of abuse. Drug abuse is associated with several neuronal changes, including the downregulation of glutamate transporters such as glutamate transporter 1 and cystine/glutamate antiporter. We investigated the effects of N-acetylcysteine, a glutamate transporter 1 and xCT upregulator, on pregabalin addiction using a conditioned place preference paradigm. Pregabalin (60 mg/kg) was found to induce conditioned place preference when compared to a vehicle. A 100 mg/kg dose of N-acetylcysteine was found to block pregabalin-seeking behaviors. These results support previous findings showing that glutamate transporters play an important role in pregabalin-induced seeking behaviors. N-acetylcysteine may represent a beneficial agent in preventing the abuse potential of pregabalin.

l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using 14C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (Km=69.4 μM). Also, l(2)01810 uptakes glutamate in a Na+-independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na+-independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein's localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg146 in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.

AbstractMechanisms for maintenance of the extracellular level of glutamate in brain tissue and its regulation still remain almost unclear, and criticism of the current paradigm of glutamate transport and homeostasis has recently appeared. The main premise for this study is the existence of a definite and non-negligible concentration of ambient glutamate between the episodes of exocytotic release in our experiments with rat brain nerve terminals (synaptosomes), despite the existence of a very potent Na+-dependent glutamate uptake. Glutamate transporter reversal is considered as the main mechanisms of glutamate release under special conditions of energy deprivation, hypoxia, hypoglycemia, brain trauma, and stroke, underlying an increase in the ambient glutamate concentration and development of excitotoxicity. In the present study, a new vision on transporter-mediated release of glutamate as one of the main mechanisms involved in the maintenance of definite concentration of ambient glutamate under normal energetical status of nerve terminals is forwarded. It has been suggested that glutamate transporters act effectively in outward direction in a non-pathological manner, and this process is thermodynamically synchronized with uptake and provides effective outward glutamate current, thereby establishing and maintaining permanent and dynamic glutamatein/glutamateout gradient and turnover across the plasma membrane. In this context, non-transporter tonic glutamate release by diffusion, spontaneous exocytosis, cystine-glutamate exchanger, and leakage through anion channels can be considered as a permanently added ‘new’ exogenous substrate using two-substrate kinetic model calculations. Permanent glutamate turnover is of value for tonic activation of post/presynaptic glutamate receptors, long-term potentiation, memory formation, etc. Counterarguments against this mechanism are also considered.

In many cell lines, glutamate cytotoxicity is known to be mediated by an inhibition of cystine transport. Because glutamate and cystine share the same transporter, elevated levels of extracellular glutamate competitively inhibit cystine transport leading to depletion of intracellular glutathione. A glutathione-depleted state impairs cellular antioxidant defenses resulting in oxidative stress. It was therefore of interest to investigate whether proglutathione agents, e.g., N-acetylcysteine and lipoic acid, are able to protect against glutamate cytotoxicity. Both lipoic acid (100 μM-1 mM) and N-acetylcysteine (100 μM-1 mM) completely protected C6 cells from the glutamate-induced cell death. Both agents facilitate extracellular supply of cysteine, the reduced form of cystine, that is transported into the cell by a glutamate-insensitive transport mechanism. Protection by lipoic acid and N-acetylcysteine corresponded with a sparing effect on cellular glutathione, which is usually depleted after glutamate treatment. In the presence ofl-buthionine-( S, R)-sulfoximine, a γ-glutamylcysteine synthetase inhibitor, low doses (<100 μM) of lipoic acid and N-acetylcysteine did not protect cells against glutamate-induced cytotoxicity. At higher concentrations (>500 μM), however, both lipoic acid and N-acetylcysteine provided partial protection against glutamate cytotoxicity even in glutathione synthesis-arrested cells. These results indicate that at low concentrations the primary mechanism of protection by the thiol antioxidants was mediated by their proglutathione property rather than direct scavenging of reactive oxygen. At higher concentrations (>500 μM), a GSH-independent direct antioxidant effect of lipoic acid and N-acetylcysteine was observed. Dichlorofluorescin fluorescence, a measure of intracellular peroxides, increased sixfold after glutamate treatment of C6 cells. Lipoic acid and N-acetylcysteine treatment significantly lowered glutamate-induced dichlorofluorescin fluorescence compared with that of controls. Interestingly, α-tocopherol (50 μM) also suppressed glutamate-induced dichlorofluorescin fluorescence, indicating the peroxides detected by dichlorofluorescin were likely lipid hydroperoxides. Both thiol antioxidants, particularly lipoic acid, appear to have remarkable therapeutic potential in protecting against neurological injuries involving glutamate and oxidative stress.

The uptake of L-cystine into cultured human umbilical vein endothelial cells has been shown to occur by a Na+-independent system which is inhibited by L-glutamate and L-homocysteine, but not by other amino acids. It is likely that the system transporting L-cystine is shared by L-glutamate. Thiol groups associated with membrane bound components appear to be essential for L-cystine uptake but it is not yet evident whether these constitute an integral part of the transporter per se.

2007/2008
Summary This doctoral thesis covers three years period (2006-2008) during which I have investigated the bilirubin neurotoxicity in the neuroblastoma SH-SY5Y cell line, a neuronal cell model widely used in the study of the pathogenesis and in the development of new therapeutic compounds for neurodegenerative diseases. In the first chapter is summarized the current knowledge about bilirubin chemistry and metabolism including disorders of bilirubin metabolism and the neuronal disturbances associated. In addition, the main discoveries in bilirubin toxicity mechanisms are described. Chapter two describes how we have chosen the cellular model to study the unconjugated bilirubin (UCB) damage. We first compared the bilirubin accumulation and cell viability in two neuronal cell lines (2a1 mouse neuronal progenitor cell line and SH-SY5Y cell line) and one non neuronal cell line (HeLa cells). In addition, we performed studies on cellular localization of Mrp1 (involved in UCB extrusion) and mRNA expression. We observed that SH-SY5Y cells show higher accumulation of bilirubin and lower survival than 2a1 and HeLa cells. SH-SY5Y cells shows a clear localization of Mrp1 at membrane level. Based on these observations we selected the SH-SY5Y cell line as our experimental model, and we characterized this cell line for molecular events linked with bilirubin neurotoxicity. Chapter three revises original data published by mainly our group, about “the free bilirubin hypothesis”. It has been suggested that cell injury correlates better with free unconjugated bilirubin (Bf) than total unconjugated bilirubin (BT). To directly test this hypothesis we evaluated cell viability in four cell lines (SH-SY5Y, MEF, HeLa and 2a1 cell lines) after incubation with different Bf/BT ratios, obtained by mixing varied UCB concentrations and albumins with different binding affinities (bovine, fetal calf and human); Bf was measured in each solution by the peroxidase method. Our data show that the loss of viability is dependent on the Bf but not on BT although bilirubin sensitivity varied with the different cell line tested. This in vitro study reinforces the proposal that Bf or Bf combined with total serum bilirubin should improve risk assessment for neurotoxicity in both term and premature infants. Chapter four describes our studies about the biochemical and molecular changes in SH-SY5Y cells exposed to a rather high Bf (140 nM) for 24 hours. Biochemical changes (cell viability, proliferation, cellular redox environment -ROS and GSH content) and gene expression profile were evaluated in the cells which survived after the treatment. Results suggest that the surviving cells become more resistant to a second oxidative exposition (Bf or H2O2) and this was associated with an increases expression of various genes involved both in ER stress response and in the transport system Xc- (cystine-glutamate exchanger). This transport system is of great relevance in maintaining the redox homeostasis within the cell, and together with the ER stress genes may contribute to the activation of an adaptative response to bilirubin damage. Further studies will be necessary to elucidate the molecular mechanisms that confer resistance to bilirubin toxicity; these mechanisms could help understanding the different sensitivity of the cells to bilirubin damage, and why some neuronal cells die (as the Purkinje cells) while others don’t. Furthermore, these studies may achieve to the identification of target proteins useful to develop new drugs: this may be the case of the system Xc-.
Riassunto Questo lavoro di tesi è il frutto delle ricerche svolte nei tre anni del mio dottorato (2006-2008), durante i quali mi sono occupato dello studio della neurotossicità da bilirubina nella linea cellulare di neuroblastoma umano SH-SY5Y; si tratta di un modello cellulare neuronale ampiamente utilizzato nello studio della patogenesi di malattie neurodegenerative, nonché nello sviluppo di composti neuroprotettivi. Nel primo capitolo si trovano riassunte le conoscenze attuali riguardanti la chimica della bilirubina, il suo metabolismo ed eventuali disordini ed i disturbi neuronali associati ad essa; inoltre, sono descritte le principali scoperte sui suoi meccanismi di tossicità. Nel secondo capitolo viene descritta come è stata effettuata la scelta di un modello cellulare adeguato allo studio del danno da bilirubina non coniugata (UCB). A questo scopo sono stati confrontati l’accumulo in bilirubina triziata e la vitalità cellulare dopo un trattamento con bilirubina libera, in due linee cellulari neuronali (progenitori neuronali di striato di topo -cellule 2a1- , neuroblastoma umano -cellule SH-SY5Y-) ed in una linea cellulare non neuronale (cellule HeLa). Oltre a ciò, sono stati eseguiti alcuni studi sulla localizzazione del trasportatore Mrp1 (coinvolto nell’estrusione di UCB), e sull’espressione dei geni Mrp1 ed Mdr1 (il cui prodotto proteico è un possibile trasportatore di bilirubina). Abbiamo osservato che le cellule SH-SY5Y presentano un accumulo di bilirubina più elevato ed una più bassa sopravvivenza rispetto alle cellule 2a1 ed HeLa, sebbene nelle cellule SH-SY5Y la localizzazione di Mrp1 risulti essere a livello di membrana plasmatica. Basandoci su queste osservazioni abbiamo scelto di lavorare con il modello cellulare già noto SH-SY5Y, e ci siamo occupati di caratterizzarlo per la neurotossicità da bilirubina. Nel terzo capitolo vengono presentati dati sperimentali pubblicati dal nostro gruppo a supporto dell’ “ipotesi della bilirubina libera”, la quale postula che il danno cellulare da bilirubina correli in modo migliore con la concentrazione di bilirubina libera (Bf) piuttosto che con quella di bilirubina totale (BT). Al fine di testare quest’ipotesi abbiamo valutato la vitalità in quattro diverse linee cellulari (SH-SY5Y, MEF, HeLa e 2a1) dopo aver incubato le cellule in soluzioni con un diverso rapporto Bf/BT. Tali soluzioni sono state ottenute sciogliendo diverse quantità di UCB in terreno con diversi tipi di albumina (bovina, umana e di siero fetale bovino); questi binders possiedono differenti affinità per la bilirubina. La Bf è stata determinata in ciascuna soluzione utilizzando il metodo della perossidasi. I dati ottenuti suggeriscono che, sebbene la sensibilità alla bilirubina vari nelle diverse linee cellulari, la riduzione in vitalità dipenda dalla Bf e non dalla BT. Quindi, questi studi in vitro costituiscono un’evidenza in più a favore della teoria della bilirubina libera, e sostengono la necessità di valutare il rischio di Kernittero mediante la misura della Bf serica e non solo della bilirubina totale. Nel quarto capitolo si descrivono le modificazioni a livello biochimico e molecolare nella linea cellulare SH-SY5Y dovute ad un trattamento di 24 ore in presenza di un’elevata concentrazione di bilirubina libera. Nelle cellule sopravvissute al trattamento abbiamo valutato diversi parametri biochimici tra cui vitalità e proliferazione cellulare ed ambiente redox cellulare (contenuto di ROS e GSH), nonché il pattern di espressione genica indotto dalla bilirubina. I risultati ottenuti suggeriscono che le cellule SH-SY5Y sopravvissute siano più resistenti all’esposizione ad un secondo stress ossidativo (Bf o H2O2), inoltre queste cellule mostrano un’aumentata espressione di diversi geni coinvolti nella risposta allo stress di reticolo endoplasmatico e dei geni i cui prodotti proteici fanno parte del sistema di trasporto Xc- (antiporto cistina-glutammato). Questo sistema di trasporto è estremamente importante nel mantenimento dell’omeostasi redox cellulare, ed insieme ai geni dello stress di ER potrebbe contribuire all’attivazione di una risposta adattativa al danno da bilirubina. Ulteriori studi che ci consentano di comprendere i meccanismi molecolari che conferiscono resistenza alla neurotossicità da bilirubina potrebbero aiutarci a capire la differenza di sensibilità dei diversi tipi di cellule alla bilirubina stessa, ed il motivo per cui alcune cellule neuronali muoiano (come ad esempio le cellule di Purkinje) mentre altre no. Inoltre questi studi possono portarci all’identificazione di target proteici utili allo sviluppo di nuovi farmaci, quale può essere ad esempio il caso del trasportatore Xc-.
XXI Ciclo
1978

A computational model of glutamate dynamics in the PFC-NAc syapse is developed. The mechanisms considered are release of glutamate into the synapse, diffusion of synaptic glutamate into the extracellular space, Glu added by cystine-glutamate exchanger, Glu removal via transporters, and binding to mGluR's. The model will be used to determine the relative impact of the different mechanisms on Glu homeostasis, by using information about Glu levels and ranges for the known parameters and kinetic constants. The model will then be integrated with a PFC cell firing model to investigate the effects of cocaine-induced cellular adaptations in the PFC-NAc glutamatergic pathway.