Cerebral cystine uptake: a tale of two transporters

McBean, Gethin J.

doi:10.1016/s0165-6147(02)02060-6

Cited by 146 publications

(107 citation statements)

References 28 publications

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“…This study did however describe low level GSH in other retinal neurons. It is recognized that system x c -has been detected more frequently in glial cells than other cell types [2], which has led to a model whereby system x c -is thought to be primarily a transporter active in glial cells. More recent studies detecting system x c -in neurons [10,12] requires that the model be revisited.…”

Section: Discussionmentioning

confidence: 99%

“…As reviewed by McBean [2], experimental evidence supports a model in which cystine is taken up by astrocytes for GSH formation, which is then released to provide precursors for neuronal GSH. The model is based on early observations that neurons did not accumulate cystine well, however new evidence has emerged from studies of both hippocampal [10] and cortical neurons [11], demonstrating uptake of cystine.…”

Section: Introductionmentioning

confidence: 87%

“…Reagents used in this study were obtained from the following sources: Dulbecco's modified Eagle's medium F12 (DMEM:F12) medium, Earl's Balanced Salt Solution, Dulbecco's Phosphate Buffered Saline, Neurobasal medium, TRIzol reagent, penicillin-streptomycin (Gibco-Life Technologies, Rockville, MD); [2, H]-glutamate (specific radioactivity 50.0 Ci/mmol) (Amersham Bioscience, Arlington Heights, IL), L-[4,5-3H(N)]-leucine (specific radioactivity 60 Ci/mmol) (New England Nuclear, Boston, MA), L-[2,3-3H]-alanine (specific radioactivity 40 Ci/mmol) (Moravek Biochemicals, Brea, CA); 4F2hc (CD98) goat polyclonal IgG, goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); Cy-3 conjugated anti-rabbbit IgG, peroxidaseconjugated AffiniPure rabbit anti-goat IgG, Alexa Fluor 488-conjugated anti-goat IgG (In Vitrogen, Carlsbad, CA); Hoeschst dye 33342 (Molecular Probes, Eugene, OR); ECL detection kit (Pierce Biotechnology, Inc., Rockford, IL); Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA); Power Block (BioGenex, San Ramon, CA); Glutathione (GSH) Detection Kit (Chemicon International, Temecula, CA); complete Mini Protease Inhibitor Cocktail tablets, GeneAmp RNA PCR Kit (Applied Biosystems/Roche Molecular System, Branchburg, NJ); TaKaRa Taq ™ (Takara Bio Inc, Otsu, Shiga, Japan); sense and antisense primers for xCT and 4F2hc (Integrated DNA Technologies, Inc, Coralville, IA); papain (Worthington, Lakewood, NJ), trypsin inhibitor (Roche Applied Science, Mannheim, Germany), rabbit anti-macrophage antiserum (Accurate Chemical Corp., Westbury, NY), AffiniPure donkey anti-rabbit IgG (H+L) (Jackson, ImmunoResearch, Westgrove, PA), purified rat anti-mouse Thy 1.2 antibody (Pharmingen, San Diego, CA), brain-derived neurotrophic factor, ciliary-derived neurotrophic factor (basic fibroblast growth factor (PeproTech Inc., Rocky Hill, NJ), 3-nitroso-N-acetylpenicillamine (SNAP), Snitrosoglutathione (SNOG), 3-Morpholinosydnonimine hydrochloride (SIN-1), menadione sodium bisulfite, hydrogen peroxide 30% (W/W) solution, xanthine sodium salt, xanthine oxidase, fetal bovine serum, monoclonal antibody to β-actin and all other chemicals (SigmaAldrich Chemical Co., St. Louis, MO); RGC-5 cells were the kind gift of Dr. Neeraj Agarwal, Department of Anatomy and Cell Biology, University of North Texas Health Science Center, Fort Worth, TX.…”

Section: Reagentsmentioning

confidence: 99%

“…The cystine-glutamate transporter, known as system x c -, mediates the Na + -independent, electroneutral exchange of cystine and glutamate [1,2]. System x c -is a member of the glycoprotein-associated amino acid transporter (gpaAT) family [3].…”

Section: Introductionmentioning

confidence: 99%

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Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

et al. 2006

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Purpose-The cystine-glutamate transporter, system x c -, mediates the Na + -independent exchange of cystine into cells coupled to the efflux of intracellular glutamate. Within cells, cystine is reduced to cysteine, a component of glutathione, thus system x c -plays a critical role in glutathione homeostasis. Studies in brain had initially suggested that system x c -was present primarily in astrocytes and not neurons, but more recent work suggests that certain brain neurons have an active system x c -. In the retina, system x c -has been demonstrated in retinal Müller and RPE cells. Recent immunohistochemical work from our lab suggested that the two protein components of system x c -, xCT and 4F2hc, are present in ganglion cells of the intact retina. The purpose of this investigation was to determine whether system x c -was present in primary ganglion cells isolated from neonatal mouse retinas and, if so, to study its regulation by oxidative stress in a retinal ganglion cell line, RGC-5.Methods-The presence of xCT and 4F2hc in RGC-5 cells was established by RT-PCR, immunoblotting and immunohistochemistry and in primary mouse retinal ganglion cells by immunoblotting and immunohistochemistry; the function of the transporter in RGC-5 cells was established by measuring radiolabeled glutamate uptake in the absence of Na + . To assess regulation of system x c -by oxidative stress in ganglion cells, RGC-5 cells were incubated in the presence or absence of nitric oxide (NO) donors (3-nitroso-N-acetylpenicillamine (SNAP), Snitrosoglutathione (SNOG), or 3-morpholinosydnonimine hydrochloride (SIN-1) and reactive oxygen species (ROS) donors menadione sodium bisulfite, hydrogen peroxide and xanthine sodium salt/xanthine oxidase and system x c -was analyzed using functional assays, RT-PCR and immunoblotting.Results-RGC-5 cells and primary ganglion cells isolated from mouse retina express xCT and 4F2hc as demonstrated by RT-PCR, immunoblotting and immunohistochemistry. RGC-5 cells take up glutamate in the absence of Na + and this uptake was blocked by known inhibitors of system x c -, glutamate, cysteine, and cystine as well as quisqualic acid. Treatment of RGC-5 cells with NO donors and donors of ROS led to an increase in the functional activity of system x c -. Kinetic analysis of SNAP-treated RGC-5 cells compared to control cells showed that the increase was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. Molecular analyses showed that the upregulation is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. Conclusions-RGC-5 cells and ganglion cells isolated from neonatal mice express the cystine/ glutamate transporter x c -(the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. Oxidative stress upregulates this transport system in RGC-5 cells and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 87%

Section: Reagentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

et al. 2006

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“…Cystine is transported by sodium dependent and sodium independent systems in tissues. 1,2) Sodium dependent cystine transport is mediated by the glutamate transporters whereas in sodium independent transport systems glutamate and cystine are exchanged. Once the cystine is inside the cells it is reduced to two molecules of cysteine.…”

Section: Introductionmentioning

confidence: 99%

Hyperosmolarity Induced Cystine Transport and Cystine-cysteine Cycle between Erythrocytes and the Plasma

Yıldız

Cakir

2008

JOURNAL OF HEALTH SCIENCE

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The objective of the present study was to determine if a cystine-cysteine cycle operates between the erythrocytes and the plasma. In the present study we incubated the erythrocytes in krebs ringer phosphate buffer with different osmolarity containing different amounts of cystine. Our results show that erythrocytes do not uptake cystine from the environment when the osmolarity of the buffer is 310 mOsmol/l. Erythrocytes also do not operate a cystinecysteine cycle in this isoosmolar buffer. However, when exposed to hyperosmolar buffer in the ranges that occur in the kidney medulla which is in between 1200-1400 mOsmol/l erythrocytes start to uptake cystine from the environment and induce a cystine-cysteine cycle. The cystine uptake and cystine-cysteine cycle were characterized by measurement of changes in the free -SH concentrations in erythrocytes and in the buffer. Following incubation of erythrocytes in 1 mM cystine containing 1250 and 1300 mOsmol/l buffer, the free -SH concentrations in the buffer reached to 0.102 ± 0.002 and 0.241 ± 0.013 µmol/ml erythrocyte respectively. Our results demonstrate that erythrocytes display a cystine-cysteine cycle in hyperosmolar environment which is prevailed mainly in the kidney medulla. Our results also display that this process is biologically active and energy dependent. The observed cystine-cysteine cycle is inhibited when the erythrocytes are incubated at lower temperatures and in the absence of glucose. Our results suggest that erythrocytes uptake cystine, intracellulary reduce it to cysteine and release it back to the environment when exposed to hyperosmolar conditions. Erythrocytes may have a role in the regulation of plasma cystine and cysteine concentrations and may contribute to the regulation of plasma redox status.

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Regulation of System x_c⁻Activity and Expression in Astrocytes by Interleukin‐1β

Jackman

Uliasz

Hewett

et al. 2010

Glia

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We recently demonstrated that interleukin-1β (IL-1β) increases system xc− (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes and microglia all express system xc− subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT— the light chain that confers substrate specificity— in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system xc− inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system xc− activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.

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Cerebral cystine uptake: a tale of two transporters

Cited by 146 publications

References 28 publications

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Hyperosmolarity Induced Cystine Transport and Cystine-cysteine Cycle between Erythrocytes and the Plasma

Regulation of System x_c⁻Activity and Expression in Astrocytes by Interleukin‐1β

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Cerebral cystine uptake: a tale of two transporters

Cited by 146 publications

References 28 publications

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Hyperosmolarity Induced Cystine Transport and Cystine-cysteine Cycle between Erythrocytes and the Plasma

Regulation of System xc−Activity and Expression in Astrocytes by Interleukin‐1β

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Regulation of System x_c⁻Activity and Expression in Astrocytes by Interleukin‐1β