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Structure, Function, and Regulation of Human Cystine/Glutamate Transporter in Retinal Pigment Epithelial Cells

¹ From the Departments of Cellular Biology and Anatomy, ² Biochemistry and Molecular Biology, ³ Obstetrics and Gynecology, and ⁴ Ophthalmology, Medical College of Georgia, Augusta.

	Abstract

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PURPOSE. The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x_c^-) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells.

METHODS. Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x_c^- in these cells was established by substrate specificity and Na⁺-independence of the uptake process. The human x_c^- light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N-acetylpenicillamine (SNAP) and the expression of x_c^- was studied at the functional and molecular levels.

RESULTS. ARPE-19 cells take up cystine as well as glutamate in the absence of Na⁺. Substrate specificity studies indicate that although the uptake of cystine in the absence of Na⁺ is mediated by multiple amino acid transport systems including x_c^-, the uptake of glutamate in the absence of Na⁺ occurs exclusively via x_c^-. The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x_c^-. The activity of x_c^- in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc.

CONCLUSIONS. ARPE-19 cells express the cystine/glutamate transporter x_c^- (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.

	Introduction

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Glutathione plays an important role in several physiologic processes including protection of cells against oxidative damage.^{1 2 3} Oxidative damage is thought to contribute to such diseases as age-related macular degeneration⁴ and diabetic retinopathy.⁵ Glutathione has been detected in high concentrations in retina and retinal pigment epithelium (RPE).^{6 7} The protective effects of exogenously administered glutathione against oxidative damage in cultured human RPE have been reported.⁸ We have shown recently that glutathione protects the reduced-folate transporter in RPE cells from nitric oxide (NO)-induced loss of transport function.⁹

Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine. Glutamate and glycine occur at relatively high intracellular concentrations; therefore, cysteine availability largely determines glutathione synthesis. The extracellular concentration of cysteine is quite low because this amino acid typically exists in the disulfide form, cystine.¹⁰ To date, two different transport systems have been described that mediate the uptake of cystine in mammalian cells. They are b^0,+ and x_c^-.^{11 12} The expression of b^0,+ is limited mostly to small intestine and kidney. b^0,+ is a Na⁺-independent transport system for cystine and also for a variety of neutral and cationic amino acids. This transport system exists as a heterodimer consisting of either rBAT (protein related to b^0,+ amino acid transport system) or 4F2hc (heavy chain of the 4F2 cell surface antigen) as the heavy chain and b^0,+ AT (b^0,+ amino acid transporter) as the light chain.^{13 14 15} In contrast to b^0,+, the system x_c^- is expressed ubiquitously, but at low levels, in mammalian tissues under normal conditions.¹⁶ The expression of this system is upregulated, however, by oxidative stress.^{17 18 19 20} Due to the widespread expression and regulation by oxidative stress, the system x_c^- is believed to be the primary transport system related to the uptake of cystine into cells for glutathione synthesis. x_c^- is Na⁺ - independent and mediates the entry of cystine into cells coupled to the efflux of glutamate.²¹ Because of this obligatory exchange function, x_c^- is called cystine/glutamate transporter. x_c^- is also a heterodimer, consisting of 4F2hc as the heavy chain and xCT (x_c^- transporter) as the light chain.²² 4F2hc is a subunit common to several amino acid transport systems including x_c^- and b^0,+, whereas xCT is unique to system x_c^-.¹¹ 4F2hc has been cloned from different animal species including humans.¹¹ In contrast, xCT has been characterized at the molecular and functional level only in mouse.²²

The purpose of the present investigation was threefold: to provide evidence from functional studies for the expression of the transporter x_c^- in the human retinal pigment epithelial cell line ARPE-19; to clone the light chain of the transporter, xCT, from an ARPE-19 cell cDNA library and study its function in an heterologous expression system; and, to investigate the regulation of x_c^- by NO in ARPE-19 cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
[³⁵S]-Cystine and [³H]-glutamate were purchased from New England Nuclear Corp. (Boston, MA). [³H]-Alanine and [³H]-arginine were purchased from Amersham Corp. (Piscataway, NJ). Cell culture supplies were purchased from Life Technologies (Gaithersburg, MD). Restriction enzymes were from New England Biolabs (Beverly, MA), 3-Nitroso-N-acetylpenicillamine (SNAP) was purchased from Research Biochemicals International (Natick, MA). Human retinal pigment epithelial cells (ARPE-19), a rapidly growing human RPE cell line²³ established in the laboratory of L. Hjelmeland (University of California, Davis), were provided by R. B. Caldwell (Medical College of Georgia, Augusta, GA). HeLa cells used for heterologous expression were purchased from American Type Culture Collection (Manassas, VA).

Cell Culture
ARPE-19 cells were cultured in 75 cm² flasks with Dulbecco’s modified Eagle’s medium: nutrient mixture F12 (DMEM:F12), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were passaged by dissociation in 0.05% (w/v) trypsin and seeded in 24-well culture plates (Fisher, Norcross, GA).

Uptake Measurements in ARPE-19 Cells
For uptake experiments, the culture medium was removed from RPE cells and the cells were subsequently washed twice with uptake buffer, which lacked Na⁺. The composition of the uptake buffer was 25 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES)/Tris, 140 mM N-methyl-D-glucamine chloride, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose, pH 7.5. Uptake was initiated by adding 250 µl of uptake buffer containing radiolabeled substrates. Uptake measurements were done with incubations for a desired time at 37°C. At the end of the incubation, uptake was terminated by removal of the medium by aspiration followed by three washes with ice-cold uptake buffer without the radiolabeled substrates. The cells were then solubilized in 0.5 ml of 1% sodium dodecylsulfate in 0.2 N NaOH and transferred to scintillation vials for quantitation of radioactivity. This protocol is the same as described previously from our laboratory for the measurement of amino acid uptake in mammalian cells expressing heterologously the cloned amino acid transport proteins b^0,+AT,^{13 15} LAT1,²⁴ and LAT2.²⁵

RT-PCR for 4F2hc mRNA and Restriction Analysis of the RT-PCR Product
The x_c system is a heterodimer consisting of 4F2hc as the heavy chain and xCT as the light chain. To determine whether ARPE-19 cells express 4F2hc mRNA, we used ARPE-19 cell mRNA for RT-PCR analysis with 4F2hc-specific primers. The sense primer was 5'-GCAGAAGTGGTGGCACAC-3'corresponding to the nucleotide positions 451–468 in human 4F2hc cDNA,²⁶ and the antisense primer was 5'-TTTTGTATGCTCCCCAGTAG AA-3'corresponding to the nucleotide positions 1033–1054 in human 4F2hc cDNA. The expected size of the RT-PCR product based on the nucleotide positions of the primer pair was 604 bp. The identity of the resulting product was established by restriction analysis. The product was gene cleaned and digested with BalI and PstI. The expected sizes of the digestion products were 335 and 269 for BalI and 412 and 192 for PstI.

Construction of cDNA Library
The SuperScript plasmid system (Life Technologies) was used to establish a unidirectional cDNA library with poly(A)⁺ mRNA isolated from ARPE-19 cells. Poly(A)⁺ mRNA was prepared by subjecting total RNA twice to oligo(dT)-cellulose affinity chromatography before use in library construction. The cDNA products with sizes greater than 1 kbp were separated by size-fractionation and used for ligation at SalI/NotI site in pSPORT1 vector.

cDNA Library Screening and DNA Sequencing
The ARPE-19 cell cDNA library was screened under low stringency conditions as described previously^{13 15 24 25} using a mouse xCT cDNA fragment as the probe. The probe was prepared by RT-PCR using mouse lung mRNA and primers specific for mouse xCT. The primers 5'-TACCTGCAGGGCAATATG-3' (sense) and 5'-GGGCGTTTGTATCGAAGA-3'(antisense), corresponded to the nucleotide positions 362–379 and 1546–1563 in mouse xCT cDNA²² and the expected size of the RT-PCR product was 1.2 kbp. The product was subcloned in pGEM-T vector and sequenced for establishment of its identity. After the screening of the ARPE-19 cell cDNA library with the mouse xCT cDNA probe, positive clones were identified, and the colonies were purified by secondary screening. Both sense and antisense strands of the cDNA were sequenced using an automated Perkin–Elmer Applied Biosystems (Framingham, MA) 377 Prism DNA sequencer. The sequence was analyzed using the GCG sequence analysis software package, GCG version 10 (Genetics Computer Group, Inc., Madison, WI).

Expression of the Cloned cDNA in HeLa Cells
The vaccinia virus expression system was used to functionally characterize the cloned cDNA.^{13 15 24 25} The human 4F2hc cDNA and the human xCT cDNA were cloned into pSPORT1 vector such that the sense transcription of the cDNA is under the control of T7 promoter. The human 4F2hc cDNA was isolated from a human placental choriocarcinoma cell (JAR) cDNA library and shown to be functional in heterologous expression systems.^{13 15 24 25} The cDNAs were transfected into HeLa cells grown in 24-well tissue culture plates using Lipofectin, and the functional expression of the cDNAs was analyzed 12 hours later by measuring radiolabeled amino acid uptake. Transfection was done with human 4F2hc cDNA, human xCT cDNA or human 4F2hc cDNA plus human xCT cDNA. The transport buffer was composed of 25 mM Hepes/Tris (pH 7.5), supplemented with 140 mM N-methyl-D-glucamine chloride, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. The incubation time for the transport measurements was 15 minutes at 37°C, after which the uptake medium containing the radioactive substrate was aspirated off, and the cells were washed with 2 x 2 ml of ice-cold transport buffer. The cells were then solubilized in 0.5% SDS in 0.2 N NaOH, transferred to vials, and radioactivity associated with the cells quantitated by liquid scintillation spectrometry. The experiments were repeated two to three times with independent transfections, each done in duplicate or triplicate. Data are presented as means ± SE of these replicate measurements.

Regulation Studies
To determine the effects of oxidative stress on the expression of the system x_c^- in ARPE-19 cells, confluent cell cultures were treated with 1 mM SNAP, a NO-donor, at 37°C for 2, 4, or 24 hours. Cells treated similarly but in the absence of SNAP served as controls. After the treatment, uptake of radiolabeled substrates (cystine, glutamate, alanine, and arginine) into these cells was measured as described previously.

Semiquantitative RT-PCR Analysis of the Steady-State Levels of mRNAs for xCT, 4F2hc and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
Confluent cultures of ARPE-19 cells were treated with or without 1 mM SNAP for 24 hours at 37°C and poly(A)⁺ mRNA was then prepared from these cells. RT-PCR was carried out using primer pairs specific for human xCT, human 4F2hc, and human GAPDH. The primers specific for human xCT were 5'-AGGGGAGTCTCCATTATCAT-3'(sense) and 5'-AAGTAGGC CACATTTGTCAG-3' (antisense) corresponding to nucleotide positions 373–392 and 1078–1097 of the xCT cDNA cloned from ARPE-19 cell cDNA library. The primers specific for human 4F2hc were the same as described previously for the analysis of 4F2hc mRNA in ARPE-19 cells. The primers specific for human GAPDH were 5'-AAGGCTGAGAACGGGAAGCTTGTCATCAAT-3' (sense) and 5'-TTCCCGTTCAGCTCAGGGATGACCTTGCCC-3' (antisense) corresponding to nucleotide positions 241–270 and 711–740 in human GAPDH cDNA.²⁷ Each of these RT-PCR products was subcloned in pGEM-T vector and sequenced to establish their identity. For semiquantitative RT-PCR, PCR following reverse transcription was carried out with varying numbers of cycles (range, 9 to 30). The products were size-fractionated on an agarose gel and subjected to Southern hybridization with probes specific for each of the three products. These probes were generated by labeling the respective subcloned RT-PCR products with [³²P]dCTP. The intensity of the hybridization signal was quantified using STORM phosphorimaging system (Molecular Dynamics, Sunnyvale, CA). The relationship between the intensity of the signal and the PCR cycle number was then analyzed to determine the linear range for the PCR product formation. The intensities of the signals within the linear range were used for data analysis.

	Results

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Functional Evidence for the Expression of x_c^- System in ARPE-19 Cells
To provide evidence for the expression of system x_c^- in ARPE-19 cells, the uptake of cystine and glutamate into these cells was studied in a Na⁺-free medium and the substrate specificity of the uptake process assessed by competition experiments (Fig. 1) . The cells were found to take up [³⁵S]cystine and [³H]glutamate in the absence of Na⁺, suggesting the presence of Na⁺-independent transport mechanism(s) for these two amino acids in these cells. Competition experiments revealed that the Na⁺-independent uptake of [³⁵S]cystine was inhibitable by unlabeled cystine as well as by glutamate and a variety of neutral amino acids. In contrast, the Na⁺-independent uptake of [³H]glutamate was inhibitable only by three amino acids, namely cystine, cysteine, and unlabeled glutamate. The substrate specificity of the transport system mediating the Na⁺-independent uptake of glutamate in these cells matches with the known substrate specificity of system x_c^-. The inhibition of Na⁺-independent cystine uptake by glutamate is clear evidence for the involvement of system x_c^- in cystine uptake. However, the substrate specificity of the cystine uptake process indicates that system x_c^- may not be the only transporter mediating the uptake of cystine in these cells. These data show that ARPE-19 cells express system x_c^- and that measurement of Na⁺-independent glutamate uptake rather than cystine uptake identifies specifically the transport function of system x_c^- in these cells.

View larger version (23K): [in this window] [in a new window]   Figure 1. Inhibition of Na+-independent uptake of [35S]cystine (A) and [3H]glutamate (B) by various amino acids in cultured human ARPE-19 cells. Uptake of [35S]cystine (2.5 µM) and [3H]glutamate (2.5 µM) was measured in confluent ARPE-19 cells in a Na+-free medium for 15 minutes at 37°C in the absence or presence of 2 mM unlabeled amino acids (except for cystine, 0.5 mM). Results are presented as percent of control uptake measured in the absence of unlabeled amino acids. The values for control uptake (100%) were 46.8 ± 5.8 and 7.46 ± 0.27 pmol/106cells/15 minutes for [35S] cystine and [3H] glutamate, respectively. Values represent means ± SE for four determinations from two independent experiments.

View larger version (34K): [in this window] [in a new window]   Figure 2. RT-PCR and restriction analysis of 4F2hc mRNA in human ARPE-19 cells. Poly(A)+ RNA from ARPE-19 cells was used for RT-PCR with human 4F2hc-specific primers. The expected size of the RT-PCR product (uncut), predicted from the positions of the primers, is 604 bp. The RT-PCR product was gene-cleaned and then used for restriction analysis with BalI and PstI. DNA standards (100 bp–12.2 kbp) were run on the gel in parallel to determine the size of the RT-PCR product and restriction fragments. The expected sizes of the digestion products are 335 and 269 bp for BalI and 412 and 192 bp for PstI.

View larger version (91K): [in this window] [in a new window]   Figure 3. Amino acid sequence of human xCT cloned from ARPE-19 cells. The sequence of human xCT (hxCT) is aligned with the known sequence of mouse xCT (mxCT). Shaded boxes denote identical regions of the mouse and human xCT. Putative transmembrane domains are underlined and the N-linked glycosylation site in hxCT is indicated (Asn-314).

View larger version (13K): [in this window] [in a new window]   Figure 4. Transport function of the 4F2hc/xCT complex in HeLa cells. Cells were transfected with pSPORT vector (P + P), h4F2hc cDNA (P + F), hxCT cDNA (P + X) or hxCT cDNA plus h4F2hc cDNA (X + F). DNA content during transfection was kept constant by the addition of the pSPORT vector (P) as required. Expression of the transfected cDNAs was carried out by the vaccinia virus expression technique. Uptake of [35S]cystine (25 µM) and [3H]glutamate (25 µM) was measured in these cells in a Na+-free medium for 15 minutes at 37°C. Values are means ± SE for six determinations done with two independent transfections.

View larger version (21K): [in this window] [in a new window]   Figure 5. Substrate specificity (A) and saturation kinetics (B) of the transport function induced by the h4F2hc/hxCT complex in HeLa cells. Cells were transfected with either pSPORT alone or h4F2hc cDNA plus hxCT cDNA and the transfected cDNAs were expressed by the vaccinia virus expression technique. For the substrate specificity studies, uptake of [3H]glutamate (25 µM) in these cells was measured in a Na+-free medium for 15 minutes at 37°C in the absence or presence of 5 mM various amino acids. Data represent only the h4F2hc/hxCT complex-specific uptake (i.e., uptake in cells transfected with h4F2hc cDNA plus hxCT cDNA minus uptake in cells transfected with vector alone) and are given as percent of control uptake measured in the absence of unlabeled amino acids (100% = 11.0 ± 0.5 nmol/106 cells/15 min). Values are means ± SE for six determinations from two independent transfections. For saturation kinetics studies, uptake of glutamate was measured in a Na+-free medium for 15 minutes at 37°C over a glutamate concentration range of 0.025 to 2 mM. Data represent only the h4F2hc/hxCT complex-specific uptake. Values are means ± SE for six determinations from two separate transfections. Inset: Eadie–Hofstee plot.

View larger version (34K): [in this window] [in a new window]   Figure 6. Upregulation of the Na+-independent uptake of cystine (A) and glutamate (B) in ARPE-19 cells by SNAP. Confluent cells were treated in the absence or presence of 1 mM SNAP at 37°C for 1, 6, or 24 hours. Uptake of [35S]cystine (2.5 µM) and [3H]glutamate (0.2 µM) was measured in these cells for 30 minutes at 37°C in a Na+-free medium. Values are means ± SE for six determinations from two independent experiments.

View larger version (12K): [in this window] [in a new window]   Figure 7. Kinetics of glutamate uptake in control () and SNAP-treated (•) ARPE-19 cells. Confluent cells were treated with or without 1 mM SNAP for 24 hours at 37°C. Uptake of glutamate was measured in these cells for 30 minutes at 37°C in a Na+-free medium over a glutamate concentration range of 25 µM to 1000 µM. Values are means ± SE for six determinations from two independent experiments. Results are presented as Eadie–Hofstee plots (V, glutamate uptake in nmol/mg of protein/30 min; S, glutamate concentrationin µM).

The influence of the SNAP treatment was studied on the steady-state levels of mRNA transcripts specific for the two protein components of system x_c^-, namely 4F2hc and xCT. mRNA samples isolated from control and SNAP-treated ARPE-19 cells were used for semiquantitative RT-PCR for the determination of the levels of mRNA transcripts. As an internal control, the steady-state levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined in the mRNA samples in parallel. The results of these experiments showed that treatment with SNAP did not alter the steady-state levels of 4F2hc mRNA (Fig. 8) . The small increase in 4F2hc mRNA seen in SNAP-treated cells versus control cells was similar to the increase in the steady-state levels of GAPDH mRNA. In contrast, the steady-state levels of xCT mRNA increased markedly in SNAP-treated cells compared to control cells and this increase was much higher than observed in the case of 4F2hc mRNA and GAPDH mRNA. These results demonstrate that the SNAP-induced increase in the transport activity of system x_c^- is associated with an increase in the expression of xCT with no significant change in the expression of 4F2hc.

View larger version (29K): [in this window] [in a new window]   Figure 8. Analysis of steady-state levels of mRNA for xCT, 4F2hc, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in control (- SNAP) and SNAP-treated (+ SNAP) ARPE-19 cells. Confluent cells were treated with or without 1 mM SNAP for 24 hours at 37°C. Poly(A)+ RNA was then isolated from these cells and used for semiquantitative RT-PCR. Primer pairs specific for human xCT mRNA, human 4F2hc mRNA and human GAPDH mRNA were used. RT-PCR was done with a wide range of PCR cycles (9–30). The resultant products were run on a gel and then subjected to Southern hybridization with 32P-labeled cDNA probes specific for xCT, 4F2hc, and GAPDH. The hybridization signals were quantified using the STORM Phosphorimaging System, and the intensities that were in the linear range with the PCR cycle number were used for analysis. Left: representative Southern hybridization signal. Right: band intensity in SNAP-treated cells relative to band intensity in control cells.

	Discussion

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System x_c^- is a Na⁺-independent exchanger for cystine and glutamate and couples the influx of cystine to the efflux of glutamate in cells. This system can be studied by monitoring the Na⁺-independent uptake of radiolabeled cystine or glutamate, which occurs in exchange with intracellular glutamate. Functional studies in ARPE-19 cells using this approach demonstrate that system x_c^- is expressed in these cells. In addition to system x_c^-, other transport systems such as b^0,+ may also be present in these cells that contribute to the influx of cystine. However, when glutamate is used as the substrate instead of cystine, the uptake appears to be mediated exclusively by system x_c^-.

System x_c^- functions as a heterodimer consisting of 4F2hc and xCT. Evidence is shown here for the expression of both of these components in ARPE-19 cells. The presence of 4F2hc mRNA in these cells was established by RT-PCR. The presence of xCT mRNA was shown by isolation of a full-length xCT cDNA from an ARPE-19 cell cDNA library. Even though the nucleotide sequence of human xCT cDNA has been deposited in the GenBank database by other investigators (accession number AB026891), no information is available on the functional identity of the clone. In this article, evidence is provided for the first time for the transport function of the cloned human xCT. This was accomplished by using an heterologous expression system in which human 4F2hc and human xCT were coexpressed. If expressed independently, neither of these proteins exhibits detectable transport function. However, when coexpressed, the 4F2hc/xCT heteromeric complex mediates the transport of cystine and glutamate. The functional characteristics of the transport process associated with the heteromeric complex are similar to those of system x_c^-. It is interesting that the substrate affinity of the heterologously expressed system x_c^- is significantly different from that of the constitutively expressed system x_c^- (k_t value: 48 ± 4 vs. 221 ± 18 µM). This difference is likely due to the different cell types used in the determination of the substrate affinity. The heterologously expressed system x_c^- was studied in HeLa cells, whereas the constitutively expressed system x_c^- was studied in ARPE-19 cells. The kinetic parameters of a transport system may differ significantly in different cell types, influenced by the cell type-specific differences in posttranslational modification such as glycosylation and in the microenvironment of the transport protein in the membrane.

After establishing the expression of system x_c^- in ARPE-19 cells at the functional and molecular level, we investigated the regulation of the expression of this transport system by NO. Exposure of the cells to SNAP, an NO donor, for 24 hours leads to an increase in the Na⁺-independent uptake of cystine and glutamate in these cells. This stimulatory effect is not seen if the exposure time is less than 6 hours. The increase in system x_c^- activity resulting from NO-exposure is accompanied by an increase in the maximal velocity of the transport process. The substrate affinity of the process is not altered. These results, together with the need for a long treatment period to elicit the observed effect, suggest that the increase in transport function is most likely due to an increase in the transporter density in the cell plasma membrane. This is supported by the findings that cycloheximide (an inhibitor of translation) and actinomycin D (an inhibitor of transcription) attenuate the stimulatory effect of SNAP to a significant extent. As system x_c^- is a heterodimer consisting of 4F2hc as the heavy chain and xCT as the light chain, the question arises as to which of these two proteins is expressed at higher levels as a result of exposure to NO. We addressed this question by quantifying the steady-state levels of 4F2hc mRNA and xCT mRNA in control and NO-treated cells. These studies have shown that NO increases only the steady-state levels of xCT mRNA. The levels of 4F2hc mRNA are not influenced by NO. The specific stimulatory effect of NO on xCT expression suggests that the levels of xCT constitute the rate-limiting factor in the formation of the transport-competent 4F2hc/xCT heteromeric complex. Furthermore, 4F2hc is not unique to system x_c^- because this protein is also a component of several other amino acid transport systems such as systems L, y⁺L, and b^0,+. Therefore, the stimulatory influence of NO on the expression of only xCT confers the specificity of NO action on system x_c^-.

The stimulation of x_c^- expression by NO has physiological relevance. Oxidative stress is expected to deplete the cellular levels of antioxidants such as glutathione. An increase in the expression of system x_c^- following exposure to NO may be a physiologic response of the cell to oxidative stress as an attempt to increase the cellular levels of cysteine for glutathione synthesis. Although studies from other laboratories have demonstrated the stimulatory effect of oxidative stress on system x_c^- in different cell types,^{17 18 19 20} for the first time here, the basis for the stimulatory effect at the molecular level is described. Several diseases in humans are associated with oxidative stress. These include diabetes, bacterial infection, AIDS, and several neurodegenerative disorders. It is speculated that the expression of system x_c^- may be significantly upregulated under these pathologic conditions in an attempt to increase glutathione levels as an antioxidant protective mechanism against the oxidative stress.

	Footnotes

 
Supported by funding from Research to Prevent Blindness, Fight for Sight Division of Prevent Blindness America, and National Institutes of Health Grants EY13089 and EY12830.

Submitted for publication May 26, 2000; revised September 18, 2000; accepted September 29, 2000.

Commercial relationships policy: N.

Corresponding author: Vadivel Ganapathy, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912. vganapat{at}mail.mcg.edu

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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