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Am J Physiol Cell Physiol 294: C1012-C1020, 2008. First published February 20, 2008; doi:10.1152/ajpcell.00411.2007
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VASCULAR BIOLOGY

A cystine-cysteine shuttle mediated by xCT facilitates cellular responses to S-nitrosoalbumin

Jun Zhu, Sheng Li, Zermeena M. Marshall, and A. R. Whorton

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina

Submitted 7 September 2007 ; accepted in final form 17 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

S-nitrosothiols; cysteine; cystine; amino acid transporters

NITRIC OXIDE (NO), following reaction with oxygen, can modify cysteine thiols leading to formation of S-nitrosothiols via a process termed S-nitrosation (7, 9, 12, 13). S-nitrosothiols have a much longer half-life than NO and exist at measurable levels in biological fluids. In the vasculature, both S-nitrosoalbumin and S-nitrosohemoglobin have been demonstrated and proposed to serve as pools of deliverable NO bioactivity (6, 12, 28). Whereas a role for circulating NO and its derivatives is well accepted, mechanisms for transfer of NO bioactivity into cells are only recently emerging. Diffusion of free NO from sites of synthesis or following release from circulating S-nitrosothiols may be limited because of rapid binding to hemoglobin in red blood cells (21, 22, 31). Low-molecular-weight thiols such as cysteine may circumvent this problem by functioning in movement of NO bioactivity within the vascular compartment. In fact both cysteine and glutathione have been suggested to facilitate biological responses to S-nitrosoalbumin (5) and S-nitrosohemoglobin (14). This concept is further supported by work from our group and others, which show that members of the system L amino acid transporter family act as stereoselective transporters for S-nitroso-L-cysteine (CSNO) (19, 20, 35). Their ability to take up CSNO has led to the suggestion that system L components are critical for cellular responses to circulating S-nitrosothiols, including S-nitrosoalbumin (19, 35). The mechanism involved would require transnitrosation of extracellular cysteine by S-nitrosoalbumin followed by direct transfer into the intracellular compartment without any requirement for extracellular nitrosothiol breakdown and release of free NO (19, 35, 36).

The source of extracellular cysteine to drive transfer is not known. However, it is known that cystine concentrations are relatively high in extracellular fluids including plasma and that cells are able to adjust extracellular redox status through mechanisms that produce an apparent reduction of cystine to cysteine (15, 17). One possible pathway for reduction of extracellular cystine would require cystine uptake followed by cysteine release (2). Such a mechanism has also been proposed by others to explain glutamate-inhibitable transfer of NO from S-nitrosoglutathione (GSNO) into cells (35). In many cells, cystine uptake occurs via the heterodimeric amino acid transport system xc– (27, 33). This transporter functions as a Na+-independent antiporter exchanging extracellular cystine for intracellular glutamate and is composed of the variable light chain xCT and a common heavy chain 4F2 (27, 33). Because cystine is readily reduced in the intracellular environment, cystine uptake by xCT increases intracellular cysteine levels and plays a vital role in both cellular growth and redox control by supplying intracellular cysteine necessary for glutathione and protein biosynthesis. As cysteine levels increase in cells, it is exported through bidirectional amino acid transporters (32, 33).

Thus it appears that xCT represents an important component of a cystine-cysteine shuttle bringing extracellular cystine into cells where it is reduced and subsequently exported via one of several neutral amino acid pathways. Outside cells, this cysteine is available to accept NO from S-nitrosothiols and serves as a carrier of NO bioactivity from the extracellular to intracellular compartment. To investigate this, we have manipulated the expression and activity of xCT in vascular smooth muscle cells and show that this amino acid transporter provides cystine uptake-dependent cysteine release. Furthermore, we show that cysteine released in response to xCT activity is required for cellular import of NO from extracellular S-nitrosothiols and for cellular responses to S-nitrosoalbumin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Unless otherwise indicated, all reagents were from Sigma-Aldrich. Thioglo was obtained from Covalent Associates, and [14C]cystine was from New England Nuclear. 3-Morpholino-sydnonimine (SIN-1) was obtained from Cayman Chemical. S-nitrosoalbumin was synthesized as previously described (19, 28). Antibodies (anti-phospho-Akt-Ser473, anti-phospho-Erk1/2-Thr202/Tyr204, and anti-GAPDH) were from Cell Signaling.

Cell culture. Vascular smooth muscle cells were isolated from rat thoracic aortas using established methods and grown in a 1:1 mixture of DMEM and Ham's F12 (GIBCO) containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic (GIBCO) with insulin (10 mg/l), transferrin (5.5 mg/l), and selenium G (6.7 µg/l). A7r5 rat smooth muscle cells were obtained from American Type Culture Collection (ATCC) and grown in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic. Human umbilical endothelial cells were obtained from Clonetics and grown in EBM-2 media containing EGM-2 from Clonetics supplemented with 10% FBS.

Determination of cysteine release. For an experiment, cells were rinsed two times at 37°C with Hanks' balance salt solution containing 20 mM HEPES, pH 7.4 (HBSH). Cystine (100 µM) was added, and aliquots were taken with time to determine the amount of cysteine released. Cysteine was measured using a fluorescence assay based on Thioglo addition to the free thiol. Aliquots (250 µl) of cell medium were taken, centrifuged to remove any floating cells, and mixed with 5 µl of Thioglo (5 mM in acetonitrile) at room temperature. Five minutes after being mixed, 25 µl of 2 N acetic acid was added to stabilize the product and fluorescence determined ({lambda}ex = 378 nm; {lambda}em = 500 nm) using a fluorescence microplate reader (Tecan). Under these conditions the assay sensitivity was 5 pmoles of cysteine, and the fluorescence intensity was stable for at least 6 h when samples were kept in the dark.

High-pressure liquid chromatographic analysis of thiols released from cells. Small-molecular-weight thiols were separated and analyzed by HPLC following derivatization of reduced thiols using Thioglo. Fluorescent derivatives were separated on a C-18 column (25 cm, 5 µm Beckman Ultrasphere) under isocratic conditions (80% 20 mM sodium citrate, pH 5.0, 20% acetonitrile) using a flow rate of 0.75 ml/min and identified using a fluorescence detector. Coelution with authentic compounds was used to identify products.

RT-PCR analysis. Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen) and cDNA synthesized by reverse transcriptase reaction by using an Omniscript RT kit (Qiagen). cDNA sequences were used for amplification (35 cycles) using the PCR Master Kit (Promega). xCT primers sequences are given below. PCR products were separated by electrophoresis on 2% agarose gels and visualized under UV light using ethidium bromide.

Quantification of mRNA expression using quantitative real-time RT-PCR. cDNA sequences were amplified by using iQ-SYBR Green SuperMix (Bio-Rad). Quantitative real-time PCR reactions were performed using a Bio-Rad iCycler iQ system. PCR was carried out at 95°C for 3 min followed by 40 cycles at 95°C for 15 s, 54°C for 15 s, and 72°C for 15 s. Serial dilutions of xCT cDNA were used as a reference to develop a standard curve, and raw data were normalized to GAPDH. Primers used for xCT were 5'-CCTGGCATTTGGACGCTACAT-3' (forward) and 5'-TCAGAATTGCTGTGAGCTTGCA-3' (reverse) (29). Primers for GAPDH were 5'-CAAGAAGGTGGTGAAGCAGG-3' (forward) and 5'-CACCACCCTGTTGCTGTAGC-3' (reverse).

xCT expression. Human xCT cDNA (OpenbioSystem Collections, Clone-ID number 4562994) was amplified using the following primers: forward: 5'-TAAGATCTCGGAGGTATGGTCAGAAAGCCTG-3' and reverse: 5'-ATCAAGCTTTAACTTATCTTCTTC-3'. The PCR product was cloned into a pEGFP-C3 eukaryotic expression vector (Clontech) using appropriate restriction enzymes. The final plasmid was verified by DNA sequencing and used to transfect A7r5 rat vascular smooth muscle cells (ATCC) using Fugene6 (Roche). After 48 h, functional activity of xCT was determined as described.

Construction of GFP-tagged xCT adenovirus (ad-xCT). The following primers: 5'-TTTAGGTACCGCCATGGTGAGCAAGGG-3' (forward) and 5'-ATCAAGCTTTCATAACTTATCTTCTTC-3' (reverse) were used to amplify the NH2-terminal enhanced green fluorescent protein (EGFP) tagged xCT from the pEGFP-xCT plasmid. The PCR fragment containing an NH2-terminal EGFP-tagged sequence was cloned into the pAd-Shuttle CMV adenovirus vector (ATCC). Correct insertion was confirmed by DNA sequencing. This construct was used to transform BJ5183-AD-1 cells (Stratagene). Positive adenoviral recombinants were transfected to Ad-293 cells (Stratagene). Recombinant adenoviruses were generated and titrated (11, 20). Functional xCT activity was determined in smooth muscle and endothelial cells following exposure to ad-xCT by measuring cysteine release. Recombinant adenoviruses containing EGFP (ad-EGFP) alone were prepared by the same approach and used for infection controls.

Determination of S-nitrosothiols by chemiluminescence. Intracellular S-nitrosothiols were determined by ozone-based chemiluminescence using acidic triiodide to release NO (34). Briefly, cells in six-well plates were treated with nitrosothiols in phosphate-buffered saline (PBS) at 37°C, washed five times with ice-cold PBS, and then scraped into 0.5 ml of PBS containing 40 mM N-ethylmaleimide. After 15 min on ice, cells were sonicated for 10 s and centrifuged at 14,000 rpm for 10 min. The supernatant was treated with 1/10 vol 5% acidified sulfanilamide for 5 min to remove nitrite. Samples were then injected into a mixture of KI and I2 in glacial acetic acid (34) in a reflux apparatus. The NO released was analyzed by chemiluminescence using a Nitric Oxide Analyzer (Sievers). In control studies, pretreatment of samples with 5 mM HgCl2 completely abrogated the signal suggesting that S-nitrosothiols were being measured. Sulfanilamide completely prevented responses to nitrite when the triiodide method was used. Standard curves were prepared using GSNO.

Immunoblot. EGFP or EGFP-tagged xCT-expressing cells were lysed on ice in 150 µl RIPA buffer containing 6 M urea. To measure phosphorylation of ERK1/2 and AKT, modified RIPA buffer containing 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma) was used. Lysates were sonicated and centrifuged at 12,000 g. After separation by SDS-PAGE, proteins were transferred to polyvinyldifluoride membranes, blocked with casein and proteins were identified using specific primary antibodies. Visualization was done using ECL (Amersham).

Cystine uptake. Cystine uptake was measured as previously reported (4).

Statistical analysis. Multiple comparisons were made by ANOVA. Comparisons of paired samples were made by Student's t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Others have previously shown that Caco2 cells reduce extracellularly added cystine to cysteine and adjust the CSH/CSSC ratio in culture medium to about –80 mV (16, 17). In the present study, we have found that rat vascular smooth muscle cells behave in a similar manner. Incubation of these cells in HBSH containing 100 µM cystine led to a time-dependent reduction of cystine and accumulation of cysteine in the medium (Fig. 1A). After 3 h, the net amount of cysteine in the medium reached ~7 nmol/mg cell protein. When aliquots of medium were analyzed by HPLC for small-molecular-weight thiols, only cysteine was identified (Fig. 1B). The mechanism involved is not known. Of several possibilities, cells may transfer reducing equivalents to directly reduce cystine to cysteine outside cells. It is also possible that cells take up cystine, reduce it intracellularly, and release cysteine back to the extracellular space. Since we have previously shown that smooth muscle cells express system xc– activity and that this transporter is effectively inhibited when the intracellular/extracellular glutamate gradient is reduced by addition of extracellular glutamate (4), we began to investigate possible mechanisms by using glutamate to block cystine uptake. When cells were incubated with 5 mM glutamate, we saw complete inhibition of cysteine accumulation in the medium (Fig. 1A). Inhibition by glutamate is important since it shows that cystine uptake is required for appearance of cysteine outside cells and because glutamate inhibition of system xc– suggests that xCT mediates uptake.


Figure 1
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  		Fig. 1. A: reduction of extracellular cystine by vascular smooth muscle cells. Smooth muscle cells were grown to confluence in six-well plates, rinsed with Hanks' balance salt solution containing 20 mM HEPES, pH 7.4 (HBSH), and incubated at 37°C in HBSH in the presence of 100 µM cystine (CSSC) with or without 5 mM glutamate (Glu). Samples were collected at times indicated and analyzed for thiol content using a fluorescence plate reader. Data represent total nmoles of cysteine in the buffer per milligram cell protein (means ± SE, n = 7, *P < 0.01 and **P < 0.05 compared with corresponding control samples). B: high-performance liquid chromatography (HPLC) analysis of thiols present in buffer bathing cells. Aliquots of buffer were taken from cells incubated for 1 or 2 h with or without CSSC. After addition of Thioglo, small-molecular-weight thiols were analyzed by HPLC using a fluorescence detector. The fluorescent peak identified comigrated with authentic, Thioglo-derivatized cysteine.

 
We have previously shown system xc– expression in vascular smooth muscle cells by functional demonstration of inducible cystine uptake (4) and more recently induction of xCT expression by electrophiles in smooth muscle cells has been reported (25). In the following experiments we wanted to verify that our smooth muscle cell cultures express xCT and that expression was inducible. To accomplish this, we used RT-PCR to demonstrate transporter mRNA in these cells. As shown in Fig. 2A, the xCT transcript is present. An important property of system xc– is that it is inducible by a variety of electrophilic agents, which are known to activate the transcription factor nrf2 (26). As shown in Fig. 2B, treatment of smooth muscle cells with 0.2 mM diethyl maleate (DEM) led to an ~14-fold increase in xCT expression as determined by real-time PCR. This increase in expression correlated with a threefold increase in [14C]cystine uptake (Fig. 2C), which was blocked by inclusion of 5 mM glutamate in the medium bathing cells. Together, these studies confirm that these smooth muscle cells express xCT constitutively and that DEM effectively induces both xCT expression and glutamate-inhibitable cystine uptake.


Figure 2
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  		Fig. 2. xCT expression in basal and diethyl maleate (DEM)-treated vascular smooth muscle cells (SMC). A: RT-PCR of RNA isolated from cells. Rat aortic smooth muscle cells (SMCs) were grown to confluence in T-25 flasks in media with or without 0.2 mM DEM and used to isolate total RNA. After synthesis of cDNA from equivalent amounts of extracted RNA, transcripts corresponding to xCT were amplified using specific primers (35 cycles). B: smooth muscle cells were treated with or without DEM and used for RNA extraction. Quantitative real-time RT-PCR was done using primers corresponding to xCT and GAPDH and calibrated against a standard curve developed using xCT cDNA. Raw data were normalized to GAPDH. Data represent means ± SE, n = 3, *P < 0.01. C: CSSC uptake in untreated and DEM-treated cells. Confluent monolayers of smooth muscle cells were incubated overnight with or without 0.2 mM DEM. Cells were rinsed and incubated in HBSH containing 1 µCi [14C]cystine (60 µM) in the presence or absence of 5 mM Glu. After 10 min at 37°C, cells were rinsed four times with ice-cold HBSH containing 0.6 mM unlabeled CSSC and lysed in HBSH containing 1% Triton X-100. CSSC uptake was determined by measuring 14C content of the cell lysate. Data given as mean counts per minute (CPM) taken up ± SE, n = 6, and normalized to total cell protein (*P < 0.01 and **P < 0.05).

 
If cysteine accumulation requires cystine uptake by xCT, then induction of xCT expression should lead to a more rapid and enhanced release. Data in the literature (4, 25) and data presented above show that DEM and SIN-1 effectively increase xCT expression in vascular smooth muscle cells. The effect of these inducers on cysteine release was tested in the following experiments. When smooth muscle cells were treated overnight with 0.2 mM DEM and then incubated with 100 µM cystine, we observed a linear increase in cysteine release (Fig. 3A). When compared with nontreated cells (Fig. 1), the net amount of cysteine released from DEM-treated cells after 3 h was ~18-fold higher reaching 130 nmol/mg cell protein. As expected if xCT transport is involved, 5 mM glutamate blocked cysteine accumulation outside DEM-treated cells (Fig. 3A). Again, high-performance liquid chromatography analysis showed that the only detectable reactive small-molecular-weight thiol appearing in the media bathing cells was cysteine (Fig. 3B). Similar results were seen when smooth muscle cells were pretreated overnight with 0.5 mM SIN-1 (Fig. 3C).


Figure 3
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  		Fig. 3. Effect of DEM and 3-morpholino-sydnonimine (SIN-1) on CSSC uptake and cysteine release in vascular smooth muscle cells. A: smooth muscle cells were grown to confluence in six-well plates and incubated overnight in the presence of 0.2 mM DEM. Cells were rinsed 2 times with HBSH and incubated at 37°C in HBSH in the presence of 100 µM CSSC with or without 5 mM Glu. Samples were collected at times indicated and analyzed for thiol content using a fluorescence plate reader. Data represent total nmoles of cysteine in the buffer (means ± SE, n = 7, *P < 0.01 compared with controls at each point). B: HPLC analysis of thiols present in buffer bathing cells after pretreatment with DEM. Aliquots of buffer were taken from cells incubated for 1 or 2 h with or without CSSC. After addition of Thioglo, small-molecular-weight thiols were analyzed by HPLC using a fluorescence detector. The fluorescent peak identified comigrated with authentic, Thioglo-derivatized cysteine. C: cells were incubated overnight with 0.5 mM SIN-1, rinsed twice with HBSH, and incubated in HBSH containing 100 µM CSSC with or without 5 mM Glu. Data given as means ± SE (n = 3).

 
Further evidence for xCT involvement in cysteine release was obtained in experiments in which we overexpressed an xCT fusion protein in A7r5 rat smooth muscle cells. For these studies, we constructed an EGFP fusion protein that we used to visualize xCT expression in cells using fluorescence microscopy. When cells were transfected with an EGFP-xCT plasmid, ~2–5% of cells showed evidence of GFP fluorescence (data not shown). As seen in Fig. 4B, cells transfected with EGFP-xCT also exhibited a protein at the expected size for the fusion protein, suggesting expression of xCT. Cells transfected with EGFP alone served as controls and exhibited similar expression of fluorescent protein. When these cells were incubated with 100 µM cystine or cystine plus 5 mM glutamate, we saw enhancement of glutamate-inhibitable cysteine accumulation only in cells overexpressing EGFP-xCT (Fig. 4A). Cysteine release in EGFP-xCT expressing cells was time dependent and 10-fold higher after 3 h when compared with cells expressing EGFP alone. It should be pointed out that 4F2 heavy chain expression is generally not limiting so that expression of light chain (xCT) leads to functional reconstitution of system xc–. Thus, as we found when we induced xCT using DEM, overexpression of EGFP-xCT leads to enhanced cysteine accumulation in the medium.


Figure 4
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  		Fig. 4. Role of xCT in cysteine release from vascular smooth muscle cells. Cells were transfected with constructs encoding enhanced green fluorescent protein (EGFP) or EGFP-xCT fusion protein. Cysteine release was determined at 40, 80, and 180 min as described in Fig. 1 (A), and cell lysate was examined by immunoblot for expression of the constructs using anti-GFP antisera (B). Cysteine release data given as means ± SE, n = 3.

 
In subsequent studies, to achieve more uniform expression of xCT in primary rat smooth muscle cells, we used an adenoviral delivery system. As shown in Fig. 5B, EGFP-xCT expression in cultured primary rat smooth muscle cells increased with the amount of ad-xCT used. Expression was relatively uniformly distributed among all cells in a given culture dish (data not shown). Increased expression of xCT in these cells led to increased glutamate-inhibitable cysteine release. Although human endothelial cells appear to be more sensitive to ad-xCT infection (Fig. 5, C and D), similar xCT-dependent cysteine release was seen. These data confirm our findings presented above and further demonstrate that xCT overexpression can reconstitute a cystine-cysteine shuttle and drive cysteine release from cells.


Figure 5
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  		Fig. 5. xCT transporter-dependent cysteine-release in adenovirus-infected rat vascular smooth muscle cells (RASMC). RASMC cells (A and B) or human umbilical vein cells (C and D) were grown to ~70% confluence in six-well plates. Cells were then exposed to EGFP-tagged xCT adenovirus for 48 h. A and C: cells were rinsed and incubated for 1 h with 100 µM CSSC with or without 5 mM Glu to determine cysteine release as described in Fig. 1. B and D: cells were lysed using RIPA buffer containing 6 M urea, and the lysates were used to demonstrate EGFP-xCT expression by immunoblot using a GFP antibody (top). Blots were stripped and reprobed using a GAPDH antibody to demonstrate equal loading (bottom). Data are from representative experiments.

 
In the following studies we determined the role of xCT in transfer of NO from extracellular S-nitrosothiols into cells leading to nitrosation of cellular thiols. To accomplish this, we overexpressed xCT in smooth muscle cells using ad-xCT, incubated these cells with S-nitrosoalbumin in the presence and absence of cystine, and measured total cellular S-nitrosothiol content using established methods (34). As shown in Fig. 6A, when xCT-overexpressing cells are incubated with S-nitrosoalbumin in the presence of cystine, S-nitrosothiol content was increased threefold (bar 4) compared with controls (bar 3). This increase was blocked by glutamate (bar 5) and by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH), a competitive inhibitor of system L transporters (18) (bar 6). These data support the hypothesis that cysteine provided by xCT accepts NO from S-nitrosoalbumin via transnitrosation and that the resulting CSNO enters cells through system L transporters.


Figure 6
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  		Fig. 6. Role of xCT in nitrosation of cellular thiols in smooth muscle cells by extracellular S-nitrosothiols. A: RASMCs were exposed to EGFP-tagged xCT adenovirus for 48 h. Cells were rinsed and incubated for 2 h at 37°C with 200 µM S-nitrosoalbumin (SNO-Albumin) and 200 µM CSSC with or without 5 mM Glu or 20 mM 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) as indicated. B: smooth muscle cells expressing endogenous levels of xCT were incubated as above with 200 µM CSSC with or without 100 µM S-nitroso-N-acetylpenacillamine (SNAP) in the presence or absence or 5 mM Glu or 20 mM BCH. C: xCT overexpressing cells were incubated with 100 µM CSSC plus 100 µM SNAP for 10–40 min at 37°C. In each case cells were rinsed 5 times with ice-cold phosphate-buffered saline, and cell extracts were prepared in the presence of 40 mM N-ethylmaleimide (NEM) as described. Total cellular S-nitrosothiol content was determined by chemiluminescence and expressed as pmoles of S-nitrosothiol per milligram cell protein calculated based on standard curves constructed using S-nitrosoglutathione (GSNO). Data given as means ± SD, n = 4 (*P < 0.01 compared with all other data for A and B and in C, *P < 0.01 and **P < 0.05 for comparison of xCT overexpressing to controls at each time point).

 
In other studies we investigated the role of xCT in cellular responses to S-nitroso-N-acetylpenacillamine (SNAP). We have previously shown that extracellular SNAP does not alter intracellular proteins unless it is coincubated with L-cysteine and that this effect is blocked by inhibitors of system L (20). To demonstrate the role of xCT in providing cysteine necessary for this pathway, we incubated muscle cells with SNAP with or without cystine for 2 h (Fig. 6B). Nitrosation of cellular thiols was dramatically increased when cells were incubated with SNAP plus cystine (bar 4 compared with bar 3). This effect was inhibited by glutamate and by BCH suggesting a requirement for both xCT and system L transport. Importantly, the effect of cystine in facilitating the ability of SNAP to nitrosate cellular thiols was seen in cells expressing endogenous levels of xCT expression and basal activity of the cystine-cysteine shuttle demonstrated in Fig. 1. Further proof of this concept was seen in experiments in which SNAP-mediated nitrosation of cellular thiols was determined in xCT-overexpressing cells. Coincubation of SNAP plus cystine led to a greater extent of nitrosation in xCT-expressing cells compared with that of control cells (Fig. 6C). Thus continuous cysteine release leads to sustained uptake of NO from extracellular S-nitrosothiols and nitrosation of cellular thiols.

We next used xCT-overexpressing cells to investigate whether this cystine-cysteine shuttle could promote cellular responses to extracellular S-nitrosothiols. As shown in Fig. 7A, whereas pretreatment with L-CSNO effectively inhibits EGF-stimulated ERK1/2 phosphorylation, as we have previously reported (19), S-nitrosoalbumin was not effective unless cells were incubated in the combined presence of S-nitrosoalbumin plus cysteine. The implication of this result is that cysteine is required for transnitrosation and movement of NO bioactivity from S-nitrosoalbumin to cysteine followed by L-type amino acid transporter (LAT)-mediated uptake of the newly formed L-CSNO. Given that expression of xCT can promote cysteine release, we proposed that xCT expression would facilitate cellular responses to S-nitrosoalbumin. Data in Fig. 7B show that this is the case. Pretreatment of xCT-overexpressing cells with S-nitrosoalbumin in the presence of cystine was found to block EGF-stimulated ERK1/2 and Akt phosphorylation in a concentration-dependent fashion with partial effects seen at concentrations in the range of 25 µM. This inhibitory effect was dependent on xCT expression since cells treated with ad-EGFP did not show measurable responses to S-nitrosoalbumin (see Fig. 7C, lane 5 vs. lanes 1 and 2), and cells that were not exposed to adenovirus showed minimal responses (Fig. 7D, lane 5 vs. lane 3). Inhibition was also found to be sensitive to extracellular glutamate (Fig. 7C, lanes 1 and 2 vs. lanes 3 and 4 and Fig. 7D, lane 3 vs. lane 4) further confirming a requirement for xCT and cystine uptake. Finally, the effect of cystine plus xCT could be mimicked by addition of exogenous cysteine (Fig. 7D, lane 6), and incubation with nonnitrosated albumin had no effect on phosphorylation (Fig. 7D, lanes 1 and 2). Taken together, data in Fig. 7 show that expression of xCT constitutes a cystine-dependent, glutamate-inhibitable system that facilitates cellular responses to S-nitrosoalbumin.


Figure 7
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  		Fig. 7. Role of xCT in cellular responses to SNO-Albumin. A: RASMCs were treated by CSNO (400 µM) for 15 min, or combinations of SNO-Albumin (200 µM) with or without cysteine (400 µM) for 120 min in PBS at 37°C. EGF (100 ng/ml) was added for an additional 5 min to stimulate mitogen-activated protein kinase and AKT signaling pathways. Cells were rinsed four times with PBS buffer and lysate prepared in RIPA buffer. ERK1/2 phosphorylation was determined by using a phospho-ERK1/2 antibody (top). Blots were stripped and reprobed with a GAPDH antibody (bottom) to demonstrate loading. B: RASMCs were exposed to ad-xCT for 48 h. Cells were treated with combinations of CSSC (200 µM) or SNO-Albumin (10–200 µM) for 120 min, and then stimulated with EGF (100 ng/ml) for an additional 5 min at 37°C. Cells were rinsed and lysates were prepared for immunoblot and probed with a phospho-ERK1/2 antibody (top). Blots were stipped and reprobed with anti-GAPDH (bottom). C: ad-xCT-treated cells or cells treated with ad-EGFP were incubated with combinations of CSSC (200 µM), SNO-Albumin (200 µM), with or without Glut (5 mM) for 120 min, after which, they were stimulated by addition of EGF (100 ng/ml) for 5 min as indicated. Cells were rinsed and lysates used to determine ERK1/2 phosphorylation. Blots were stripped and reprobed for phospho-AKT and stripped and reprobed a second time using a GAPDH antibody (bottom). D: control cells and ad-xCT-treated cells were incubated with albumin or SNO-Albumin as indicated. After rinsing was completed, cell lysates were prepared and analyzed by immunoblot for phosphor-ERK1/2 and phosphor-AKT. In each case data shown represent results for at least duplicate determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In these studies we show that system xc–-mediated cystine uptake is required for cysteine release. Although others have suggested extracellular delivery of reducing equivalents by dihydrolipoic acid for reduction of cystine to cysteine (10), we found that cysteine release by smooth muscle cells is dependent on cystine uptake. These findings suggest that cystine is taken up from the extracellular space, reduced intracellularly by abundant redox mechanisms, and released from cells as cysteine. This is in agreement with others who have also suggested a role for system xc– in the appearance of cysteine outside cells (2, 3, 35). Mechanisms for cysteine release from cells are not well understood. However, neutral amino acid transporters are known to have symmetric specificities for both intracellular and extracellular amino acids (23, 32) with transport occurring down gradients. As cysteine levels inside a cell increase due to cystine uptake, these transporters may export excess cysteine so that the combined activities of system xc– and a neutral amino acid transporter may comprise a cystine-cysteine shuttle. Amino acid transporters are known to function in a similar way in epithelial cells. For example, system b0,+ is a Na+-independent heterodimeric transporter that plays an important role in cystine uptake from both the intestinal lumen and especially the renal tubular lumen (24) and contributes to the total cysteine pool in plasma.

The ability of cells to regulate extracellular cysteine levels is significant for several reasons. Cysteine is required by cells and must be available as either extracellular cystine or cysteine for normal growth. Since system xc– mediates cystine uptake in many cases, cells that lack this transporter require cysteine for growth. In this regard it has been shown that release of cysteine from dendritic cells is necessary for growth of T-lymphocytes (1) because these cells lack xCT. Furthermore, cell growth is regulated by the extracellular CSH/CSSC ratio, and cells appear to be capable of adjusting the redox potential to an optimal range (15, 16). Certain vascular diseases may also depend on CSH/CSSC ratios. Recent data suggest that more oxidized ratios may be proathrogenic perhaps by leading to increased expression of adhesion molecules in vascular endothelial cells (8). How system xc– participates in this scenario is not known, although it is clear that signals generated by oxidant stress activate nrf2 and enhance expression of xCT (26). Recently, the concept of a cystine-cystiene shuttle and cysteine release has also been reported to explain increased survival in cells overexpressing xCT (2).

Our recent work (19) and work by others (35) suggests that extracellular cysteine may also be necessary for intracellular importation of NO bioactivity. This may be especially critical for large-molecular-weight S-nitrosothiols such as S-nitrosoalbumin, which may serve as a circulating reservoir of NO (26). Because of the nature of these large molecules, they do not have direct access to the intracellular compartment and must deliver their NO by other means. For example, large-molecular-weight S-nitrosothiols may interact with cysteine and transfer their NO through transnitrosation to this low-molecular-weight thiol. After transnitrosation by S-nitrosoalbumin, CSNO can be taken up efficiently by stereoselective system L transporters (20, 35). Although transnitrosation in a static mixture of S-nitrosoalbumin and cysteine favors formation of S-nitrosoalbumin (30), transfer of NO bioactivity from S-nitrosoalbumin to the intracellular compartment is enhanced by the relatively higher concentrations of cysteine in plasma and by efficient removal (uptake) of newly formed CSNO mediated by system L uptake. Because cysteine release occurs continuously, this pathway may provide a mechanism for sustained cellular responses to a reservoir of S-nitrosothiols outside cells. Indeed, we have shown that the activity of the cystine-cysteine shuttle is required for nitrosation of cellular thiols in response to excellular S-nitrosothiols such that enhanced expression of xCT leads to increased delivery and that the extent of nitrosation increases with time. Furthermore, because nitrosation of critical components in cell signaling responses represents an important signaling modality for S-nitrosothiols, mechanisms such as xCT expression, which facilitate cystine uptake, may be critical for cellular responses. This conclusion is strongly supported by the current data which show that the ability of S-nitrosoalbumin to inhibit EGF-stimulated ERK1/2 phosphorylation is cystine driven and dependent on xCT expression. These mechanisms may also be involved in cellular responses to other cell-impermeant S-nitrosothiols, including S-nitrosoglutathione and SNAP. For example, Zhang et al. (35) have suggested that a similar xCT-dependent mechanism is responsible for glutamate-inhibitable NO transfer from GSNO into cells. In addition, our previous data (20) and data in this study show that xCT expression facilitates transfer of NO from SNAP into cells. Given these findings, it appears that such a shuttle mechanism provides a general mechanism for cellular responses to S-nitrosothiols dependent on both xCT and system L expression. It should also be emphasized that these mechanisms provide important alternative pathways for cellular responses to NO derivatives, which are different from and perhaps complimentary to those mediated by NO-activation of guanylyl cyclase.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-42444 and HL-61377 (to A.R. Whorton).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. R. Whorton, C138B Levine Sci. Res. Center, Dept. of Pharmacol. & Cancer Biol, Duke Univ. Medical Center, Durham, NC 27710 (e-mail: awho{at}duke.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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