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Am J Physiol Cell Physiol 292: C1263-C1271, 2007. First published November 8, 2006; doi:10.1152/ajpcell.00382.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells

Sheng Li and A. Richard Whorton

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

Submitted 12 July 2006 ; accepted in final form 6 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (L-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The Km for transport was 57 ± 8 µM for 4F2HC-LAT1 and 520 ± 52 µM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-{alpha}-mediated I{kappa}B degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with D-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin.

nitric oxide; nitrosothiols; system L transporter; endothelial cells; smooth muscle cells; red blood cells

NITRIC OXIDE (NO) has been implicated in numerous cellular functions, from vascular smooth muscle relaxation to regulation of gene expression (11, 19, 26). The short half-life of NO can be prolonged by reaction with sulfhydryl groups of proteins or low molecular thiols to form more stable, biologically active S-nitrosothiols (23, 32). Although the actual concentration in the circulation is debated, S-nitrosothiols are known to be formed in vivo (8, 10, 17, 31, 33, 39) and are proposed to mediate diverse biological responses (7, 9). Among the circulating S-nitrosothiols, S-nitrosoalbumin is an important species and may act as reservoir of NO (8, 31, 34, 35). The physiological roles for S-nitrosoalbumin or mechanisms for transfer of NO equivalents from this circulating S-nitrosoprotein to vascular tissues are not known. However, mobilization of NO bioactivity from S-nitrosoalbumin by low-molecular-weight thiols leads to vasorelaxation (13, 29), and low-molecular-weight thiols are involved in the ability of S-nitrosoalbumin to inhibit platelet function (4).

Recently, our group and others have suggested that system L transporters mediate S-nitroso-L-cysteine (L-CSNO) uptake (14, 15, 20, 41). These amino acid transporters are composed of a common heavy chain (4F2HC) and a variable light chain and preferentially mediate Na+-independent uptake of large neutral amino acids (22, 30, 36). Using molecular approaches, we (14) recently reported that stereoselective uptake of L-CSNO is mediated by two major members of the system L transporter family: L-type amino acid transporter 1 (LAT1) and LAT2. Neither D-CSNO, S-nitrosoglutathione (GSNO), nor S-nitroso-N-acetyl penicillamine is a substrate for uptake and does not lead to cellular responses unless these nitrosothiols are coincubated with L-cysteine (14–16, 25, 41). This has led to the suggestion that L-cysteine undergoes transnitrosylation reactions forming L-CSNO, which acts as a carrier for transport of NO equivalents into intact cells (14, 41). The role of system L in transfer of NO equivalents from S-nitrosoalbumin into cells has not been investigated. However, S-nitrosoalbumin is in equilibrium with circulating low-molecular-weight thiols, including L-cysteine, making it possible that transnitrosylation leads to L-CSNO formation and uptake by vascular cells.

In this study, we examined the role of system L transporters in transfer of NO equivalents from low-molecular-weight thiols and S-nitrosoalbumin into vascular cells. We show that red blood cells, vascular endothelial cells, and vascular smooth muscle cells express components of system L and that these transporters are responsible for uptake of NO bioactivity from S-nitrosothiols. Uptake leads to nitrosylation of specific proteins and cellular responses. Whereas L-CSNO is taken up directly, the mechanism for transfer of NO equivalents from S-nitrosoalbumin appears to involve transnitrosylation between S-nitrosoalbumin and L-cysteine, followed by system L-mediated transport of L-CSNO into the intracellular compartment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. All reagents were obtained from Sigma-Aldrich unless specified otherwise. GSNO, S-nitrosocysteine, and S-nitrosoalbumin were synthesized as previously described (14, 32). Yields were >90% for GSNO and S-nitrosocysteine. Synthesis of S-nitrosoalbumin yielded a stoichiometry of 0.86 ± 0.04 mol S-NO/mol BSA. [3H]leucine was from Amersham Pharmacia Biotech, [35S]cysteine was from New England Nuclear, and sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) was from Toronto Research Chemicals. 2-Aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and {alpha}-(methylamino)isobutyrate were obtained from Sigma-Aldrich. An antibody to LAT1 was obtained from Trans Genic.

Red blood cell preparation and cell culture. Human red blood cells from healthy volunteers were collected in heparinized tubes and washed three times by dilution with 10 vol of HBSS containing 25 mM HEPES, pH 7.4 (HBSH). The buffy coat was removed after each wash. Erythrocytes were resuspended with HBSH at a final 0.1 hematocrit and used within 24 h. Rat aortic smooth muscle cells, human aortic smooth muscle cells (Clonetics), and human umbilical vein endothelial cells (Clonetics) were isolated and cultured by established methods as previously described (14).

Expression of system L in oocytes. Plasmids containing 4F2HC, LAT1, or LAT2 cDNA were constructed as previously described (14). Adult female Xenopus laevis ovaries were obtained from NASCO and treated with collagenase A (2 mg/ml) in Ca2+-free medium (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) to remove the follicular layer. In vitro-synthesized cRNAs (73.6 nl, 200 ng/µl) were injected (14), and oocytes were maintained at 18°C in buffer containing 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and gentamicin (100 µg/ml), pH 7.6. L-CSNO uptake was determined 2 days after injection. Oocytes were washed in the uptake buffer (100 mM choline chloride, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 5 mM Tris, pH 7.4) and incubated in uptake buffer containing 35S-labeled L-CSNO (~6–30 µCi/µmol) and 1 mM MTSES for 15 min at room temperature. Oocytes were then washed five times with ice-cold uptake buffer and lysed in radioimmunoprecipitation assay buffer. Radioactivity was counted by liquid scintillation spectrometry.

RT-PCR. Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen) and reverse transcribed by oligo(dT) using an Omniscript RT kit (Qiagen) at 37°C for 60 min. Full-length 4F2HC or fragments of LAT1 and LAT2 were amplified with the use of primers reported previously (14). PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized under UV light in the presence of ethidium bromide. Fragments were excised from gels and isolated, and their identity was confirmed by sequence analysis.

Direct measurement of uptake. To measure amino acid uptake, cells were incubated with either 35S-L-CSNO or with [3H]leucine (25 µCi/µmol) in HBSH at 37°C, washed five times with ice-cold HBSH to remove extracellular radioactivity, and lysed into radioimmunoprecipitation assay buffer with 1% SDS. In these studies, uptake of intact L-CSNO was determined in the presence of 1 mM MTSES to prevent free labeled cysteine uptake as previously described (14). Radioactivity was determined by liquid scintillation spectrometry.

Determination of S-nitrosothiols by chemiluminescence. Intracellular S-nitrosothiols was determined by ozone-based chemiluminescence using either Cu(I)/cysteine (6) or acidic triiodide to release NO (40). Briefly, cells were treated with nitrosothiols in HBSH at 37°C, washed four times with ice-cold HBSH, and scraped into PBS. Cells were sonicated for 10 s and centrifuged at 14,000 rpm for 10 min. For the Cu(I)/cysteine method, extracts were added to a reflux apparatus containing a mixture of 1 mM cysteine saturated with Cu(I) chloride, pH 6.5. At this pH, nitrite and nitrate are not measured. For the triiodide method, extracts were collected in 40 mM N-ethylmaleimide. After 30 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 of 5% acidified sulfanilamide for 5 min to remove nitrite. Samples were then injected into a mixture of KI and I2 in glacial acetic acid (40) in a reflux apparatus. In both cases, the NO released was analyzed by chemiluminescence using an NO 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. Comparison of the two methods gave qualitatively identical results.

Immunoblot and procedures. To measure ERK phosphorylation or I{kappa}B{alpha} levels, cells were incubated in HBSH and treated with either 100 ng/ml EGF (ERK phosphorylation) or 10 ng/ml human TNF-{alpha} (I{kappa}B levels), washed three times with ice-cold HBSH, scraped into lysis buffer (50 mM Tris·HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM beta-glycerolphosphate, 2.5 mM sodium pyrophosphate, and 1 mM PMSF), sonicated briefly, and centrifuged at 14,000 rpm for 10 min. Equivalent amounts of protein were separated by electrophoresis using 10% SDS-PAGE gels. After transfer to polyvinylidene difluoride membranes and blocking with casein, phosphorylation was detected with an antibody to phospho-ERK (Santa Cruz Biotechnology). I{kappa}B{alpha} protein levels were measured with an antibody to I{kappa}B{alpha} (Santa Cruz Biotechnology). A horseradish peroxidase-coupled secondary antibody was used for visualization by enhanced chemiluminescence. Blots were stripped using 100 mM beta-mercaptoethanol, 2% SDS, and 62.5 mM Tris·HCl, pH 6.7, at 50°C for 30 min and reprobed with an anti-ERK (Santa Cruz Biotechnology) antibody.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To begin our investigation of the role of system L in cellular uptake of S-nitrosothiols in the vasculature, we felt it was important to establish L-CSNO uptake parameters for both LAT1 and LAT2 and to demonstrate functional expression of these proteins in vascular tissues.

Characterization of L-CSNO transport by system L proteins. The saturation kinetics of L-CSNO uptake were determined in oocytes injected with cRNAs encoding either 4F2HC-LAT1 or 4F2HC-LAT2. As shown in Fig. 1A, L-CSNO uptake required coexpression of both 4F2HC and either LAT1 or LAT2. Uptake was rapid and linear for more than 15 min (Fig. 1B). Transport reconstituted by either 4F2HC-LAT1 or 4F2HC-LAT2 was saturable. Michaelis-Menten analysis of the data corrected for nonsystem L uptake gave apparent Km values of 57 ± 8 µM for 4F2HC-LAT1 and 520 ± 52 µM for 4F2HC-LAT2 (Fig. 1, C and D). Vmax values were 45 ± 2 and 97 ± 3 cpm/oocyte/min for 4F2HC-LAT1 and 4F2HC-LAT2, respectively, for the level of expression achieved. These values suggest that 4F2HC-LAT1 is a high-affinity transporter, whereas 4F2HC-LAT2 is a low-affinity transporter for L-CSNO.


Figure 1
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  		Fig. 1. Characterization of system L-reconstituted in oocytes: cRNAs corresponding to human LAT1, LAT2, or 4F2 heavy chain (4F2HC) were microinjected alone or in combination into freshly isolated Xenopus laevis oocytes. After 2 days, oocytes were rinsed and incubated with 35S-labeled S-nitroso-L-cysteine (L-CSNO) in uptake buffer containing 1 mM sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) for 15 min. Oocytes were collected, washed, and lysed in radioimmunoprecipitation assay buffer, and radioactivity taken up was determined. Data are means ± SE (n = 5–7). Brackets indicate concentration. A: uptake was measured at different concentrations of labeled L-CSNO in oocytes expressing various components of system L. Water was injected in oocytes as control. B: time course for uptake was determined by incubating oocytes with 25 µM labeled L-CSNO. C and D: data from A corrected for nonspecific uptake for oocytes expressing 4F2HC-LAT1 (C) or 4F2HC-LAT2 (D).

 
The substrate competition profiles for these two heterodimeric transporters for L-CSNO were examined by incubating labeled L-CSNO in the presence of various amino acids (Fig. 2). Uptake of L-CSNO mediated by either 4F2HC-LAT1 or 4F2HC-LAT2 was inhibited by BCH, leucine, isoleucine, phenylalanine, methionine, histidine, tryptophan, and valine but was not affected by glutamic acid or lysine. Alanine was a more effective competitive inhibitor for L-CSNO uptake mediated by 4F2HC-LAT1, whereas serine and threonine were more effective inhibitors of uptake mediated by 4F2HC-LAT2. These profiles are in general agreement with the competitive inhibition profiles for leucine and phenylalanine uptake mediated by system L transporters as published by others (22, 27, 30, 36) and confirm functional reconstitution of system L and its role in S-nitrosothiol uptake.


Figure 2
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  		Fig. 2. Amino acid competition profile for L-CSNO uptake in oocytes. Oocytes expressing either 4F2HC-LAT1 (A) or 4F2HC-LAT2 (B) were rinsed, and uptake was measured by incubating cells for 15 min with 25 µM 35S-labeled L-CSNO in uptake buffer containing 1 mM MTSES and 10 mM of the amino acids shown. Ctrl, control; BCH, 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid. Uptake of radiolabel was determined as in Fig. 1. Data are expressed as percent of control uptake and represent means ± SE (n = 3).

 
Expression of system L components in vascular cells. System L uptake is widely expressed and is essential for neutral amino acid uptake (38). Less is known about system L expression in vascular endothelial or smooth muscle cells or about their role in uptake of NO equivalents by these cells. Because nitrosothiols have been proposed to function in regulation of cardiovascular physiology, we have used RT-PCR to investigate whether these cells contained system L components. As shown in Fig. 3A, both smooth muscle and endothelial cells express 4F2HC and both LAT1 and LAT2 transcripts. To confirm expression of LAT1 protein, we used a commercially available antibody and show (Fig. 3B) expression of the transporter in both cell types. Thus it is likely that both endothelial and smooth muscle cells have functional transporters that could mediate uptake of L-CSNO. In the following experiments, we examined mechanisms for L-CSNO uptake by cells and examined the role of transport in the biological activities of nitrosothiols.


Figure 3
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  		Fig. 3. A: expression of LAT1, LAT2, and 4F2HC transcripts in vascular cells. Total RNA was isolated from human umbilical vein endothelial cells (HUVEC) or human aortic smooth muscle cells (HAoSMC) and reverse transcribed and amplified with primers specific for human LAT1, LAT2, or 4F2HC. The PCR products were separated on 1.5% agarose gels. B: immunoblot analysis of LAT1 in vascular cells. Cells were grown to confluence, and total cell lysates were prepared, separated by PAGE, and probed using a LAT1-specific antibody.

 
L-CSNO uptake by vascular endothelial and smooth muscle cells. To measure uptake of L-CSNO, cells were incubated with increasing concentrations of 35S-L-CSNO. Both vascular endothelial and smooth muscle cells were found to take up L-CSNO in a concentration-dependent manner (Fig. 4A). The role of system L in uptake was confirmed by several approaches. First, uptake was shown to display an amino acid competition profile indicative of transport mediated by system L (Fig. 4C). Second, uptake was inhibited by BCH, a selective blocker of both LAT1 and LAT2 (Fig. 4B). Finally, uptake was shown to be Na+ independent (see GoFig. 6), an important property of system L transport. These data along with data showing expression of transcripts for both transporters (Fig. 3) demonstrate that vascular endothelial and smooth muscle cells express functional LAT, which participate in S-nitrosothiol uptake.


Figure 4
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  		Fig. 4. Uptake of L-CSNO by vascular endothelial and smooth muscle cells. A: HUVECs or rat aortic smooth muscle cells (RASMC) were incubated in HBSS containing 25 mM HEPES (HBSH) with increasing concentrations of 35S-L-CSNO for 15 min, and uptake was determined as in Fig. 1. B: effect of BCH on L-CSNO uptake was determined. C: competitive inhibition profile for L-CSNO uptake was also determined in the presence of amino acids (10 mM). Amino acids or BCH was dissolved in HBSH, and cells were preincubated for 5 min before addition of labeled L-CSNO and measurement of uptake. Data are means ± SE (n = 3).

 

Figure 5
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  		Fig. 5. Uptake of L-CSNO by human red blood cells (RBC). A and B: washed red blood cells (0.1 hematocrit) were incubated in HBSH with 2 µM 35S-L-CSNO for the times indicated or with increasing concentrations of labeled L-CSNO for 10 min at room temperature. Cells were washed with ice-cold HBSH and lysed in radioimmunoprecipitation assay buffer, and radioactivity taken up was determined. Data are means ± SE (n = 6). C and D: red blood cells were incubated in HBSH containing 25 µM 35S-L-CSNO and 10 mM amino acid as indicated or with increasing concentrations of BCH for 10 min, and uptake was determined as above. Data are expressed as percent uptake in the absence of inhibitors ± SE (n = 3). MeAIB, {alpha}-(methylamino)isobutyrate. E: red blood cells were incubated in HBSH containing 20 µM [3H]leucine or 25 µM 35S-L-CSNO for 10 min; 10 mM BCH, unlabeled leucine, or L-CSNO was added to determine competitive inhibition as indicated. Data are expressed as percentage of uptake in the absence of inhibitors ± SE (n = 3).

 

Figure 6
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  		Fig. 6. Na+-independent uptake of L-CSNO by HUVEC, rat aortic smooth muscle cells (RASMC), and red blood cells. Cells were incubated in HBSH or with sodium-free HBSH in which 150 mM choline chloride was added. Uptake was determined by incubating cells for 10 min at 37°C with 2 µM 35S-L-CSNO as described in Fig. 4. Data are means ± SE (n = 3).

 
CSNO uptake by red blood cells. Previous work by others has suggested that red blood cells respond to low-molecular-weight thiols through selective uptake mechanisms. For example, incubation of red blood cells with L-CSNO but not GSNO was shown to lead to formation of S-nitrosohemoglobin (12). Moreover, previous studies have shown that incubation of red blood cells with L-CSNO but not GSNO led to formation of intracellular GSNO (28, 33). In addition, leucine uptake by red blood cells has been shown to display an amino acid competition profile indicative of system L transport (37).

To determine whether system L transport is involved in selective uptake in red blood cells, cells were incubated with labeled L-CSNO. As shown in Fig. 5, A and B, red blood cells take up labeled L-CSNO in a time- and concentration-dependent fashion. The amino acid competition profile for transport showed that uptake was inhibited by histidine, isoleucine, leucine, methionine, and phenylalanine but was not affected by arginine, asparagine, glutamic acid, lysine, proline, and {alpha}-(methylamino)isobutyrate, a system A transporter inhibitor (Fig. 5C). This pattern is similar to that seen in oocytes expressing system L components (Fig. 2) and patterns reported for system L in other mammalian cells (22, 27, 30, 36). Furthermore, as expected for system L-mediated transport, BCH competitively inhibited L-CSNO uptake by >70% (Fig. 5D). In other experiments, we found that red blood cells also take up labeled leucine, the prototypical substrate for system L, and found that BCH inhibits both leucine and L-CSNO uptake (Fig. 5E). Because leucine and L-CSNO are competitive substrates for the same transporter, we were also able to demonstrate that leucine and L-CSNO inhibit uptake of each other (Fig. 5E). In addition, CSNO uptake by red blood cells was found to be Na+ independent (Fig. 6). Together, these data show that system L is functionally present in mature red blood cells and is capable of mediating L-CSNO uptake.

Role of system L in transport of NO equivalents. We previously reported that L-CSNO is taken up intact by system L transport (14). In the following studies, we followed this observation and used a different approach to show that these transporters are involved in movement of NO equivalents from outside to inside the cell. To accomplish this, we incubated endothelial cells with S-nitrosothiols, washed to removed excess extracellular S-nitrosothiol, lysed the cells, and used the resulting lysate to measure total cell-associated nitrosothiol (small molecular weight and protein S-nitrosothiol) using ozone-based chemiluminescence detection assays. As shown in Fig. 7, A and B, L-CSNO but not D-CSNO or GSNO led to an increase in cell-associated nitrosothiols. Transport of NO equivalents was inhibited by BCH and displayed an amino acid competition profile consistent with system L transport of L-CSNO. These data confirm the stereospecificity and selectivity of transport when cell-associated NO equivalents are used as an index of transport and suggest that biological responses that depend on transfer of NO equivalents from S-nitrosothiols require system L-mediated uptake of L-CSNO.


Figure 7
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  		Fig. 7. Transfer of nitric oxide equivalents from L-CSNO into cells. A and B: HUVECs were incubated for 15 min at 37°C with the concentration of nitrosothiol given, rinsed 4 times with ice-cold HBSH, scraped, and sonicated in PBS. After centrifugation, nitrosothiol content of extracts was analyzed by chemiluminescence using the Cu(I)/cysteine method. A represents output from chemiluminescence detector, whereas B represents means ± SE (n = 6). Each peak represents the nitrosothiol content of a single sample. GSNO, S-nitroso-glutathione; Cont, control. C and D: cells were incubated at 37°C with 100 µM L-CSNO plus 10 mM of the amino acids given. After 30 min, cells were rinsed 4 times in ice-cold HBSH, scraped, and sonicated in 40 mM N-ethylmaleimide. Nitrosothiol context of the extracts was analyzed by chemiluminescence using the acidic triiodide method. C represents output from the detector, whereas D represents means ± SE (n = 3). Each peak represents the nitrosothiol content of a single sample.

 
Role of L-cysteine in cellular responses to S-nitrosoalbumin. In the following experiments, we investigated mechanisms for transport of NO equivalents from physiologically relevant NO carriers such as S-nitrosoalbumin into cells. Because it is known that S-nitrosoalbumin can transnitrosylate L-cysteine, leading to formation of L-CSNO (33), it seems reasonable to propose that transnitrosylation and formation of L-CSNO followed by system L uptake plays an important role in cellular responses to S-nitrosoalbumin. To examine this possibility, we compared cellular responses to S-nitrosoalbumin in the presence and absence of L-cysteine. Two approaches were used. In the first, we measured effects on cellular nitrosothiols as described above. In the second, we measured NO bioactivity resulting from transfer of NO equivalents by examining changes in activity of selected signaling pathways.

As shown in Fig. 8A, when vascular endothelial cells were incubated with S-nitrosoalbumin alone, we were unable to detect any increase in cell-associated S-nitrosothiols. In contrast, coincubation of S-nitrosoalbumin with L-cysteine led to a dramatic increase. Thiols whose S-nitroso derivatives are not transported by system L (D-cysteine, L-cysteinylglycine, and glutathione) were unable to function as carriers of NO equivalents from S-nitrosoalbumin into cells (Fig. 8, A and B). The potentiating effect of L-cysteine on NO transfer was blocked by BCH in a concentration-dependent manner (Fig. 8C). These findings demonstrate that L-cysteine and system L transporters are absolutely required for transfer of NO equivalents from S-nitrosoalbumin into cells and suggest that NO equivalents are rapidly transferred from S-nitrosoalbumin to L-cysteine to form L-CSNO. The combined activities of transnitrosylation and system L uptake provide a novel pathway for movement of NO equivalents from albumin into cells.


Figure 8
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  		Fig. 8. Transfer of nitric oxide equivalents from S-nitrosoalbumin into cells: A: HUVECs were incubated in HBSH for 15 min at 37°C with 100 µM of S-nitrosoalbumin (SNOAlb) in the presence of 100 µM of the thiols given. CysGly, L-cysteinylglycine; CSH, cysteine. B: cells were incubated with S-nitrosoalbumin in combination with equimolar concentrations of either D- or L-cysteine. C: cells were incubated with 100 µM of S-nitrosoalbumin plus 100 µM L-cysteine in the presence of increasing concentrations of BCH. In each experiment, nitrosothiols were extracted as in Fig. 6 and analyzed by chemiluminescence using the Cu(I)/cysteine method. Data are representative of 3 similar experiments.

 
To determine NO bioactivity resulting from transfer of NO equivalents, we next examined the role of L-cysteine in S-nitrosoalbumin-mediated regulation of MAPK and NF-{kappa}B signaling pathways. In initial studies, we observed that L-CSNO blocked EGF-stimulated ERK phosphorylation in vascular smooth muscle cells (Fig. 9A). We then investigated the role of system L in this process and the requirement for cysteine in nitrosothiol-mediated inhibition of ERK phosphorylation in cells treated with S-nitrosoalbumin. As shown in Fig. 9B, L-CSNO inhibited EGF-stimulated ERK phosphorylation. The effect was blocked by BCH, indicating that system L is involved. Unmodified albumin, unmodified albumin plus L-cysteine, and S-nitrosoalbumin in the absence of L-cysteine did not affect ERK phosphorylation. In contrast, S-nitrosoalbumin plus L-cysteine inhibited this effect. System L is required because BCH blocked the inhibitory effect of S-nitrosoalbumin plus L-cysteine.


Figure 9
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  		Fig. 9. Role of L-cysteine in transfer of NO bioactivity from S-nitrosoalbumin. A and B: human aortic smooth muscle cells were incubated at 37°C in HBSH and stimulated with 100 ng/ml of EGF. A: L-CSNO was added 10 min before addition of EGF, and the incubation was continued for an additional 5 min. Cells were rinsed twice with ice-cold HBSH and lysed, and lysates were analyzed by immunoblot using an anti-phospho-ERK (pERK) antibody. B: cells were treated 10 min before addition of EGF with combinations of L-CSNO (200 µM), S-nitrosoalbumin (200 µM), an equivalent amount of unmodified albumin, L-cysteine (200 µM), or BCH (10 mM) as indicated. Immunoblots were done as above. Blots were stripped and reprobed with anti-ERK to demonstrate equal protein loading. C and D: HUVECs were incubated in HBSH at 37°C and treated with 10 ng/ml TNF-{alpha}. Five minutes before addition of TNF-{alpha}, combinations of L-CSNO (200 µM), S-nitrosoalbumin (200 µM), L-cysteine (200 µM), BCH (10 mM), or leucine (10 mM) were added as indicated. Immunoblots were prepared and probed with anti-I{kappa}B{alpha}. Data are representative of 3 similar experiments.

 
In similar studies, we also investigated the regulation of TNF-{alpha}-mediated activation of the NF-{kappa}B pathway in endothelial cells by nitrosothiols. Previous studies by others have shown that L-CSNO nitrosylates and inhibits IKK (25). This effect prevents phosphorylation of I{kappa}B, thereby inhibiting NF-{kappa}B activation by agents such as TNF-{alpha}. As shown in Fig. 9, C and D, TNF-{alpha} stimulated rapid degradation of I{kappa}B, which was completely prevented by coincubation with L-CSNO (9D). S-nitrosoalbumin was also able to block TNF-{alpha}-stimulated I{kappa}B degradation but only when coincubated in the presence of L-cysteine. The role of system L in the activity of these nitrosothiols was investigated using either leucine or BCH to competitively inhibit uptake of NO equivalents. As shown in Fig. 9D, both BCH and leucine blocked the ability of L-CSNO and S-nitrosoalbumin plus L-cysteine to reverse the actions of TNF-{alpha} on I{kappa}B levels. These data show for the first time that S-nitrosoalbumin regulates cell signaling pathways through mechanisms requiring L-cysteine and uptake of L-CSNO by system L transporters. Moreover, these findings suggest that S-nitrosoalbumin can donate nitrosylating equivalents directly to cells using L-cysteine as a carrier.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we extend our earlier work and show that coexpression of system L components (4F2HC and LAT1 or LAT2) is required for L-CSNO uptake. This ability of system L proteins to transport L-CSNO is likely important for the biological actions of NO and, because transport is specific and selective, explains why responses to L-CSNO are unique and distinguishable from other S-nitrosothiols (GSNO or S-nitroso-N-acetyl-penicillamine) or from NO. For example L-CSNO but not GSNO or S-nitroso-N-acetyl-penicillamine has been shown to modify protein tyrosine phosphatase 1B (14) and GAPDH (21) and inhibit IKK (25) in intact cells. Similarly, L-CSNO but not GSNO is able to transfer NO equivalents across red cell membranes, leading to formation of intracellular GSNO and S-nitrosohemoglobin (12). Stereoselective actions of nitrosothiols have been reported in the circulation, where L-CSNO is a potent vasodilator but D-CSNO is not (5), and in the central nervous system, where L-CSNO but not D-CSNO is important in regulation of ventilatory signaling (16).

System L transporters are known to be widely expressed, representing the major mechanism for large neutral amino acid uptake in most cells (for review, see Ref. 38). Expression in blood-brain barrier endothelium has also been demonstrated, and system L is highly expressed in tumors, tumor cell lines, and rapidly growing tissues. Data from the present study show that both LAT1 and LAT2 are expressed in vascular endothelium and smooth muscle cells. Transport data and amino acid competition profiles confirm the presence of functional transporters in these tissues. This is important because these vascular cells are likely to be primary targets for circulating nitrosothiols. In this compartment, S-nitrosoalbumin and S-nitrosohemoglobin are believed to be the principal carriers of NO bioactivity (7). Mechanisms through which these nitrosoproteins deliver their NO equivalents are not known. However, biological responses to both S-nitrosoalbumin and S-nitrosohemoglobin are known to be enhanced by low-molecular-weight thiols (12). In the case of S-nitrosoalbumin, L-cysteine has been shown to facilitate vasodilation (13, 29) and inhibit platelet function (4). Our present data also show that L-cysteine is required for both transfer of NO equivalents into cells and for cellular responses to S-nitrosoalbumin. The ability of S-nitrosoalbumin to inhibit EGF-stimulated ERK phosphorylation or TNF-{alpha} activation of I{kappa}B degradation is seen only when cells are incubated in the presence of both S-nitrosoalbumin and L-cysteine. Because D-cysteine and other low-molecular-weight thiols are not active and the effect of S-nitrosoalbumin plus L-cysteine is inhibited by either BCH or leucine, it appears that system L transport of L-CSNO is involved. Importantly, others have shown that IKK nitrosylation and inhibition by L-CSNO accounts for inhibition of TNF-{alpha}-induced I{kappa}B degradation and that this effect is specific for L-CSNO (25). Thus transnitrosylation, formation of L-CSNO, and uptake of L-CSNO by system L transporters are necessary for transfer of NO equivalents from S-nitrosoalbumin into cells. The initial step in this mechanism is transnitrosylation between S-nitrosoalbumin and L-cysteine. Although the equilibrium constant for transnitrosylation between glutathione or cysteine and S-nitrosoalbumin favors formation of S-nitrosoalbumin (33), the reaction can be quantitatively shifted toward formation of CSNO because of cellular uptake of this nitrosothiol by LAT proteins. Furthermore, the concentration of L-cysteine in plasma is in the range of 10 µM (1). This is 10- to 100-fold higher than the reported concentrations for S-nitrosoalbumin and would favor formation of L-CSNO. Furthermore, because the Km that we determined for transport mediated by both LAT1 and LAT2 is above expected extracellular nitrosothiol concentrations in the blood, uptake of L-CSNO is efficient and unlikely to be affected by physiological extracellular concentrations of amino acids. Regulation of this process by changes in plasma cysteine levels or by patterns and levels of LAT protein expression has not been studied.

Other mechanisms have also been proposed for transfer of NO equivalents from S-nitrosoalbumin. For example, plasma ascorbate can act as reductant for albumin-bound Cu2+ and thus promote Cu+-mediated release of NO from S-nitrosoalbumin (8). However, such a mechanism may be limited by rapid uptake and binding of NO to heme iron in red blood cells. Impairment of transfer mechanisms and disruption of movement of NO equivalents from nitrosoproteins may be important in cardiovascular responses to NO (7, 8) in certain diseases. Elevated levels of S-nitrosoalbumin have been resported in preeclampsia (8, 35) and have been suggested to be associated with hypertension and adverse outcomes in patients with end-stage renal disease (18). In preeclampsia, impairment may result from plasma ascorbate deficiency (8), although the effect of ascorbate supplementation on blood pressure and S-nitrosoalbumin levels in pregnant women has not been studied. Because oxidative stress is an important component of preeclampsia, plasma thiols are also affected such that the ratio of reduced to oxidized cysteine is lower (24). Whether this affects transfer of NO equivalents from S-nitrosoalbumin is not known. However, administration of N-acetylcysteine prevents hypertension in a preeclampsia animal model (3) and restores NO-mediated placental perfusion in women with preeclampsia (2).

In summary, the present study demonstrates that functional system L components are present in vascular endothelium, smooth muscle, and red blood cells. These cells respond to relevant circulating nitrosothiols such as S-nitrosoalbumin through a novel mechanism that requires transfer of NO equivalents from albumin to L-cysteine before uptake by system L. Importantly, the existence of these mechanisms implies that circulating L-cysteine functions as a carrier of NO activity in the cardiovascular system and suggests L-cysteine levels may regulate responses to NO.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. R. Whorton, C138B LSRC, Box 3813, Dept. of Pharmacology and Cancer Biology, Duke Univ. Med. 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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