Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y+

Roberto Sala1, Bianca Maria Rotoli1, Emanuela Colla1, Rossana Visigalli1, Alessandro Parolari2, Ovidio Bussolati1, Gian C. Gazzola1, and Valeria Dall'Asta1

1 Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale e Clinica, Università degli Studi di Parma, 43100 Parma; and 2 Cattedra di Cardiochirurgia, Centro Cardiologico Monzino Istituto di Ricovero e Cura a Carattere Scientifico, Università degli Studi di Milano, 20122 Milan, Italy


    ABSTRACT
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Human umbilical vein endothelial cells transport arginine through two Na+-independent systems. System y+L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na+, and referable to the expression of SLC7A6/y+LAT2, SLC7A7/y+LAT1, and SLC3A2/4F2hc. System y+ is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha ) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y+. Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y+L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha , while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.

system y+L; cationic amino acid transporters; SLC7A genes; lipopolysaccharide; nitric oxide


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NITRIC OXIDE (NO) produced by endothelial cells is an important regulator of vascular tone, cell adhesion, and vascular permeability (2, 34). Depending on concentration, NO can have beneficial and protective effects or exert deleterious actions through enzyme inhibition, DNA damage promotion, induction of lipid peroxidation, or antioxidant depletion (21). Under physiological conditions, low levels of endothelial NO are synthesized from L-arginine through a calcium/calmodulin-dependent NO synthase [NO synthase type III (NOS3)].

The possible involvement of modifications of arginine transport in the regulation of endothelial NO synthesis has been investigated in short-term and chronic experiments. Short-term stimulation of arginine transport, obtained with shear stress (41), treatment with A2-purinoceptor agonists (43), or incubation in the presence of bradykinin (4), is followed by enhanced NO production. Less clear results have been obtained when arginine uptake is stimulated chronically. Several reports described a slowly ensuing stimulation of arginine transport by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha ) in porcine and human endothelium (7, 28, 38). Stimulation of arginine transport by proinflammatory cytokines has been confirmed in porcine endothelial cells by other authors (15), who, however, found no correlation between the increased uptake of the cationic amino acid and enhanced production of NO (16). In contrast, in a study performed in human umbilical vein endothelial cells (HUVECs), a correlation was found between NO production and arginine transport (23).

Most of those investigations were based on previous characterization studies that attributed endothelial transport of arginine to a single Na+-independent pathway identified with the ubiquitous system y+ (6, 31). However, it is now accepted that at least four distinct transport pathways, systems y+, y+L, b0,+, and B0,+, cooperate for arginine transport in mammalian cells (14, 37). Indeed, recent results from our group have demonstrated that, in human fibroblasts, arginine transport, once attributed solely to system y+, is due to the additive operation of systems y+ and y+L (11). Although both systems are Na+-independent exchange mechanisms for cationic amino acids, they present clear-cut differences (see Refs. 14 and 37 for review). System y+ is membrane potential dependent and interacts with neutral amino acids with a very low affinity. Its activity is referable to the cationic amino acid transporter (CAT) family of monomeric transporters, encoded by SLC7A genes. The ubiquitous CAT1, encoded by SLC7A1, and the two transporters CAT2A and CAT2B, derived from the alternative splicing of the SLC7A2 transcript, are the best characterized members of the family (8, 30). Conversely, system y+L is not sensitive to membrane potential and exhibits a high-affinity, strictly Na+-dependent interaction with neutral amino acids, such as leucine. System y+L is a member of the recently characterized group of the heterodimeric amino acid transporters (see Ref. 45 for review), formed by a light and a heavy subunit. In the case of system y+L, the heavy subunit is 4F2hc/CD98, the product of the SLC3A2 gene, while two alternative light chains have been characterized, y+LAT1 (encoded by SLC7A7) and y+LAT2 (encoded by SLC7A6).

In this report, we document the presence of system y+L in human endothelium and describe the effects of LPS and TNF-alpha on the discriminated transport mechanisms as well as on the expression of the respective genes. The results point to system y+ as the sole target of these proinflammatory compounds and exclude a direct correlation between transport changes and nitrite production.


    MATERIALS AND METHODS
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Cell culture and experimental treatment. Cultures of HUVECs were obtained from three distinct cords according to the method of Jaffe et al. (24), with minor modifications as described previously (6). Cells were routinely grown in collagen-coated 10-cm-diameter dishes in medium 199 (M199), with glutamine concentration raised to 2 mM. The culture medium was supplemented with 40% fetal bovine serum (FBS), 20 µg/ml endothelial cell growth supplement, and 15 U/ml heparin. The conditions of culture were as follows: pH 7.4, 5% CO2 in air, and 37°C. Cultures consisted of homogeneous endothelial populations, as determined by typical cobblestone morphology and positivity to von Willebrand's factor and CD31/platelet endothelial cell adhesion molecule-1 antigens (39). Culture medium was always renewed 24 h before the experiment.

Human bone marrow stromal cells (a gift of Dr. Nicola Giuliani, Dept. of Internal Medicine, University of Parma) were grown in M199 supplemented with 10% FBS.

For stimulation, TNF-alpha and LPS (from Escherichia coli, serotype O55:B5) were added (from 100× stocks in water) to complete growth medium for the times indicated for each experiment. As shown by cell protein content or endothelial cell population, treatment with the two compounds produced no significant cell loss up to 48 h.

Amino acid influx. The experiments were carried out on HUVEC subcultures resulting from 3.5 × 104 cells seeded into 2-cm2 wells of disposable Falcon 24-well trays (Becton Dickinson Labware Europe, Le Pont De Claix, France) in 1 ml of growth medium. Cells were employed after 2-3 days, when cultures were almost confluent (15 ± 2 µg protein/cm2). All the experiments were performed using the cluster-tray method for the measurement of solute fluxes in adherent cells (18) with appropriate modifications. Cell monolayers were washed twice in Earle's balanced salt solution (EBSS) containing (in mM) 117 NaCl, 26 NaHCO3, 5 KCl, 1.8 CaCl2, 1 NaH2PO4, 0.8 MgSO4, and 5.5 glucose. L-Arginine influx was assayed with a 30-s incubation of the cells in the same solution containing L-[14C]arginine. Preliminary experiments indicated that, in this interval, arginine influx approached linearity (results not shown). The experiments were terminated by three rapid washes (<10 s) in ice-cold 0.1 M MgCl2. Cell monolayers were extracted in 0.2 ml of ethanol, and the radioactivity of extracts was determined with a Wallac Microbeta Trilux. Extracted cell monolayers were then dissolved with 0.5% sodium deoxycholate in 1 M NaOH, and protein content was determined directly in the well using a modified Lowry procedure, as previously described (18).

In the experiments in which Na+-independent transport was to be measured, a modified bicarbonate-free EBSS buffered at pH 7.4 with 20 mM Tris · HCl (NMG-EBSS) was employed. In this solution, N-methyl-D-glucamine and choline salts replaced NaCl and NaH2PO4, respectively.

The osmolality of the solutions was routinely checked with a vapor pressure osmometer (model 5500, Wescor) and found to be 280 ± 10 mosmol/kg.

Expression of influx data. Amino acid influx is expressed as micromoles per milliliter of intracellular water per minute. The intracellular fluid volume was estimated by urea distribution space, as described previously (10), and found to be 6.88 ± 0.84 µl/mg protein. This value was not significantly changed by the experimental treatments (results not shown).

Kinetic parameters of arginine influx were determined by nonlinear regression analysis as follows
&ugr;=<FR><NU>V<SUB>max</SUB><IT>·</IT>[S]</NU><DE><IT>K</IT><SUB>m</SUB><IT>+</IT>[S]</DE></FR><IT>+K</IT><SUB>d</SUB><IT>·</IT>[S] (1)
for a saturable system plus diffusion, where v is the initial influx, Vmax is the maximal influx, Km is the Michaelis constant, Kd is the diffusion constant, and [S] is substrate concentration.

The following equation was employed to describe a competitive-type inhibition of amino acid transport
&ugr;=&ugr;<SUB>0</SUB>−<FR><NU>I<SUB>max</SUB><IT>·</IT>[I]</NU><DE>[I]<SUB>0.5</SUB><IT>+</IT>[I]</DE></FR> (2)
where v is the initial influx, v0 is the uptake in the absence of the inhibitor, Imax is the maximal inhibition, and [I]0.5 is the inhibitor concentration required for half-maximal inhibition.

Amino acid efflux. Arginine efflux was determined by sequentially removing aliquots of the extracellular medium and replacing them with fresh solution as described by Rotoli et al. (42) for the efflux of neutral amino acids. Briefly, cells were loaded with L-[14C]arginine (5 µCi/ml) for 5 min in M199, washed twice with EBSS in the presence or absence of NEM (0.5 mM), and incubated in 0.4 ml of the same solution. Escape of L-[14C]arginine from cells was measured at 10-s intervals up to 1 min, and the amino acid remaining in the cells was determined after extraction of cell monolayers in 0.2 ml of ethanol. Data are expressed as percentage of the total arginine accumulated before the efflux. Initial efflux was calculated from the derivative at time 0 of the single-exponential function
y=A·e<SUP>−Kx</SUP>+c (3)
where A is the amount of arginine at time 0, K is the efflux constant, and c is the residual intracellular amino acid (%/s).

Reverse transcription. Total RNA from subconfluent cultures of control and TNF-alpha - or LPS-stimulated HUVECs, seeded onto 10-cm2 wells, was isolated with Trizol (Life Technologies, Milan, Italy). RNA (2 µg), pretreated with RNase-free DNase, heated at 70°C for 10 min, and placed on ice for 1 min, was then incubated with a mixture containing 0.5 mM dNTPs mix, 25 ng/µl oligo(dT)15-18 (Life Technologies), 10 mM dithiothreitol, 1× first-strand buffer, 10 units of RNase inhibitor (Amersham Biotech, Milan, Italy), 200 units of SuperScript RT (Life Technologies), and water to a final volume of 20 µl for 1 h at 42°C. The reaction was stopped by heating at 70°C for 15 min. The duplex of RNA-DNA was treated with 5 units of RNase H (US Biochemicals, Cleveland, OH) at 37°C for 20 min, and the amount of single-strand cDNA was evaluated by fluorometry (Victor2 1420 Multilabel Counter, Wallac) with the fluorescent probe Oligreen (Molecular Probes, Eugene, OR) by using phage M13+ as single-strand DNA standard.

PCR and "semiquantitative PCR." For PCR, 200 ng of single-strand cDNA from each sample were amplified in a total volume of 50 µl with 1.25 U of Taq DNA polymerase (Qiagen, Milan, Italy), 1× PCR buffer, 0.2 mM each dNTPs, 1.5 mM MgCl2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proband sense and antisense primers (Table 1) at 0.4 µM each. The primers were designed according to the known sequences reported in GenBank with the help of the Primer3 program (42a). The sense primers for the transporter proteins hCAT2A and hCAT2B recognize different unique sequences resulting from alternative splicing of the SLC7A2 transcript. After an initial denaturation at 94°C for 2 min, samples were taken at 80°C to reduce formation of nonspecific amplification products. Taq polymerase (1.25 U) was then added, and the reaction continued with the annealing step at 60°C for 30 s and the extension step at 72°C for 1 min. Another 33 cycles were carried out, with the only difference being the 30-s denaturation step. A final extension of 5 min at 72°C was performed. These conditions were selected in preliminary experiments to verify that the PCR products were in the exponential phase of amplification for all the proband and GAPDH transcripts. Amplified mixtures (10 µl) were electrophoresed through a 2% agarose gel (NuSieve 3:1-FMC), stained with ethidium bromide (0.5 µg/ml) in 1× TBE buffer (0.1 M Tris, 90 mM boric acid, and 1 mM EDTA, pH 8.4), and visualized by ultraviolet light. Product size was established by comigration with a 100-bp ladder marker (Bio-Rad Laboratories, Milan, Italy). Pictures of the electrophoresed cDNAs were recorded with a digital DC 120 Kodak camera and quantified by ID Image Analysis Software (Kodak Digital Science).

                              
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Table 1.   Primers employed for RT-PCR

To confirm the identity with the reported human sequences, PCR products were ligated into pCRII-TOPO vector (TOPO TA cloning, Invitrogen, Groningen, The Netherlands) and used to transform CaCl2-competent E. coli. Plasmid DNA was extracted with the Jetquick plasmid miniprep spin kit (Genomed, Bad Oeynhausen, Germany); sense and antisense strands of the cDNA were sequenced (MWG Biotech, Ebersberg, Germany).

Northern blot. For Northern analysis, 15 µg of total RNA per lane were electrophoresed under denaturing conditions on 1% agarose gel containing 2.2 M formaldehyde. After staining with ethidium bromide to document equal sample loading and absence of degradation, RNA was transferred overnight to positively charged nylon membranes (Zeta Probe, Bio-Rad Laboratories) and then linked to the membrane by ultraviolet irradiation with 120 mJ in a Stratalinker UV cross-linker 1800 (Stratagene, La Jolla, CA). Filters were prehybridized for 10 min at 42°C and then hybridized overnight at 42°C in 50% formamide, 5% SDS, 4× sodium chloride-sodium phosphate-EDTA, 20 mM Na2HPO4, and 1× Denhardt's solution. Blots were washed according to the manufacturer's instructions and then exposed to Kodak XAR film at -70°C for the appropriate time. Filters were stripped before rehybridization in boiling 0.1× saline-sodium citrate-0.5% SDS for 10 min. Northern blots were hybridized to 32P-labeled cDNA probes (Ready-to-Go DNA Labeling Beads, Amersham Pharmacia Biotech) coding for human GAPDH (gift from Dr. R. Allen, American Red Cross Laboratories) and for human NOS3 (PCR generated). Densitometric analysis of the autoradiograms was performed at nonsaturating exposures with a Molecular Dynamics laser densitometer.

Immunoblotting. Cells, grown in 10-cm-diameter dishes, were washed twice with ice-cold PBS, scraped in the same solution, and collected by low-speed centrifugation. The pellet was suspended in 0.5 ml of lysis buffer containing 20 mM Tris · HCl (pH 7.4), 1 mM EDTA, 1% Triton, 1 mM Na3VO4, and a cocktail of protease inhibitors (Sigma, St. Louis, MO). Cell lysate, obtained by brief sonication (30 s) in an ice-cold bath, was centrifuged at 15,000 g for 20 min at 4°C. Protein concentration of the supernatant was determined with the Bio-Rad protein assay, with bovine serum albumin as standard. Sample aliquots of the supernatant, containing 30 µg of proteins, were employed immediately or stored lyophilized.

Protein samples were suspended in SDS-PAGE sample buffer, boiled at 100°C for 2 min, separated on a 12% SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in Tris-buffered saline (TBS) containing 1% casein, 0.33% gelatin, and 1% bovine serum albumin for 2 h at 30°C. Polyclonal antibody anti-NOS3 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:300, was added for 1 h at 30°C or overnight at 4°C. After four washes, the membrane was incubated with horseradish peroxidase-coupled anti-rabbit IgG antibody (Amersham Pharmacia Biotech), washed extensively, and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Nitrite production. Nitrite formation in the culture media of HUVECs was determined through a fluorometric approach. The method is based on the production of the fluorescent molecule 1H-naphthotriazole from 2,3-diaminonaphthalene (DAN) in an acid environment (33). For the experiment, cells were seeded into 2-cm2 wells of Falcon 24-well trays in 1 ml of complete growth medium (see Cell culture). After 2 days, medium was replaced with complete, phenol red-free medium (0.5 ml/well). TNF-alpha was added from a 100× stock in water, and nitrite was determined after 24 and 48 h. For nitrite determination, 100 µl of medium were put in wells of a black 96-well plate with a clear bottom (Corning, Cambridge, MA). DAN (20 µl of a solution of 0.025 mg/ml in 0.31 M HCl) was then added and, after 10 min at room temperature, the reaction was stopped with 20 µl of 0.7 M NaOH. Standards were performed in the same medium from a solution of 1 mM sodium nitrite. Fluorescence was determined with a Victor2 1420 Multilabel Counter.

Statistical analysis. Statistical analysis of transport and nitrite production data was performed with analysis of variance, unless otherwise stated. The comparison between densitometric results of RT-PCR in control and treated cells was performed with the Wilcoxon test for nonparametric data.

Materials. FBS was purchased from Euroclone (Milan, Italy) and M199 from Life Technology. L-[U-14C]arginine (313 mCi/mmol) was obtained from DuPont de Nemours (Bad-Homburg, Germany), DAN from Molecular Probes, TNF-alpha from Alexis (San Diego, CA), and ethanol from Carlo Erba (Milan, Italy). Sigma was the source of LPS as well as all other chemicals.


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Characterization of arginine influx in HUVECs. NEM inhibits selectively system y+ and, thus, can be employed to discriminate the various mechanisms operating the influx of cationic amino acids (13). To evaluate the effect of NEM on arginine transport in HUVECs, we performed a preliminary experiment in which the inhibitory effect of NEM, added to the culture medium 5 min before the transport assay at 0.1-2 mM, was assessed in the presence and absence of Na+ (Fig. 1). Arginine influx in HUVECs was completely Na+ independent and partially inhibited by NEM. In the presence and absence of Na+, Imax accounted for <50% of the initial influx. Preliminary experiments showed that inhibition of arginine influx by NEM was not further enhanced by prolongation of treatment (not shown). HUVEC cultures exhibited morphological alterations and a decrease in protein content when treated with >0.5 mM NEM (data not shown). For this reason, the inhibitor was employed at 0.5 mM throughout the study.


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Fig. 1.   L-Arginine influx (v) in human umbilical vein endothelial cells (HUVECs): Na+ dependence and sensitivity to N-ethylmaleimide (NEM). Cell monolayers maintained in complete growth medium were pretreated for 5 min with NEM. NEM was added to the incubation medium from a 100× stock solution in water. Cells were washed twice in Earle's balanced salt solution (EBSS, ) or in N-methyl-D-glucamine (NMG)-EBSS (open circle ), and L-arginine influx was assayed by 30-s incubations in the same solutions supplemented with 100 µM L-[14C]arginine (1 µCi/ml). Points are means of 4 independent determinations within 1 representative experiment. Error bars, SD. Experiment was repeated 3 times with similar results. Curves represent best fit of the data to Eq. 2. Regression parameters were as follows: inhibitor concentration required for half-maximal inhibition ([I]0.5) = 0.38 ± 0.13 mM and maximal inhibition (Imax) = 0.059 ± 0.006 µmol · ml cell water-1 · min-1 and [I]0.5 = 0.33 ± 0.12 mM and Imax = 0.078 ± 0.008 µmol · ml cell water-1 · min-1 in the presence and absence of Na+, respectively. [NEM], NEM concentration.

Figure 2 shows the kinetic analysis of L-arginine transport in HUVECs in the absence and presence of 0.5 mM NEM. Data in Fig. 2A indicate that, in the absence of NEM (total influx) and in NEM-treated cells (NEM-resistant influx), arginine transport can be described by the additive contributions of a single saturable system and a nonsaturable component, formally undistinguishable from diffusion in the concentration range adopted. The Hofstee plot of the saturable components of arginine influx is shown in Fig. 2B. For this representation, nonsaturable influx has been subtracted from the NEM-resistant component, while the NEM-sensitive fraction has been obtained from the difference between total influx and NEM-resistant arginine influx. Both components are accounted for by a single system, with a higher affinity of the NEM-resistant component (Km = 0.031 ± 0.002 vs. 0.102 ± 0.021 mM), while Vmax values were comparable (0.092 ± 0.005 and 0.110 ± 0.017 µmol · ml-1 · min-1 for NEM-resistant and NEM-sensitive components, respectively).


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Fig. 2.   Kinetic analysis of L-arginine transport in HUVECs. A: 30-s influx of increasing concentrations of L-[14C]arginine ([L-Arg], 0.001-0.73 mM, 4 µCi/ml) in cells pretreated with 0.5 mM NEM for 5 min (NEM-resistant influx, black-triangle) or in control cells (total influx, ). Points are means of 3 independent determinations within 1 representative experiment. Error bars, SD. Curves represent best fit of data to Eq. 1. B: Eadie-Hofstee graphical representation of NEM-resistant (black-triangle) and NEM-sensitive () arginine uptake. NEM-resistant arginine influx is obtained after subtraction of the nonsaturable component from influx data in A. Data of NEM-sensitive influx were calculated from the difference between total and NEM-resistant arginine influx at each indicated concentration of the amino acid. Lines are best-fit linear regressions.

To identify the NEM-resistant component, the effects of leucine on arginine influx were evaluated in the presence or absence of Na+. The results presented in Fig. 3A indicate that, in NEM-treated cells, leucine inhibited arginine influx only in the presence of Na+, with an Imax >80% of the NEM-resistant arginine influx. Conversely, no significant inhibition of arginine transport was observed in the absence of Na+. Leucine inhibition was restricted to the NEM-insensitive component of arginine influx, because the NEM-sensitive fraction was not significantly affected by <= 1 mM L-leucine (Fig. 3B).


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Fig. 3.   Effects of L-leucine on L-arginine influx in the presence and absence of Na+. HUVECs were washed twice in EBSS (filled symbols) or in NMG-EBSS (open symbols). Arginine influx was determined with 30-s incubations in the same solutions supplemented with 100 µM L-[14C]arginine (1 µCi/ml) in the presence of L-leucine. A: NEM-resistant component. Cells were pretreated with 0.5 mM NEM during the last 5 min of incubation in medium 199. Solid line, best fit of experimental data to Eq. 2. Regression parameters for arginine influx determined in the presence of Na+ were as follows: [I]0.5 = 0.671 ± 0.02 mM and Imax = 0.074 ± 0.008 µmol · ml cell water -1 · min-1. Data are means of 3 independent determinations. Error bars, SD (when greater than size of symbol). [Leu], leucine concentration. B: NEM-sensitive component. Data points represent the difference between arginine influx data (means of 3 independent determinations) in control and NEM-treated cells at each indicated concentration of leucine.

These results indicate that in HUVECs the NEM-resistant portion of arginine influx is mainly due to the activity of system y+L, while the NEM-sensitive, leucine-resistant fraction can be identified as system y+.

Effect of TNF-alpha and LPS on the bidirectional fluxes of arginine in HUVECs. In the experiment shown in Fig. 4, the effect of TNF-alpha and LPS on the discriminated influx of arginine was studied in HUVECs. For this purpose, cells were maintained in a complete growth medium in the presence of the two compounds for 3, 6, 9, 12, or 24 h, and arginine influx was determined in NEM-treated or untreated cells. TNF-alpha and LPS produced an increase of L-arginine transport through the sole NEM-sensitive component, i.e., system y+ (Fig. 4A), while no significant change was detected for the NEM-resistant component, i.e., for system y+L (Fig. 4B). System y+ stimulation required >= 6 h of treatment and was maximal after 9 h. At longer times of treatment, the stimulatory effect decreased and transport activity was not significantly different from untreated control after 24 h of incubation in the presence of TNF-alpha or LPS.


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Fig. 4.   Tumor necrosis factor-alpha (TNF-alpha ) and lipopolysaccharide (LPS) effect on endothelial pathways for L-arginine influx. Cells were incubated in the absence (control) and presence of 10 ng/ml TNF-alpha or 10 µg/ml LPS. After indicated times, cells were washed twice in EBSS, and L-arginine influx was assayed with 30-s incubations in the same solution in the presence of 100 µM L-[14C]arginine (1 µCi/ml) in untreated cells (total influx) and in cells pretreated for 5 min with 0.5 mM NEM (NEM-resistant influx). A: system y+. Data represent NEM-sensitive arginine influx calculated at each indicated time as the difference between total and NEM-resistant influx. B: system y+L. NEM-resistant arginine influx. Points are means of 4 independent determinations within 1 representative experiment. Error bars, SD. Experiment was repeated 4 times with similar results.

Table 2 shows the results of a kinetic analysis of arginine influx through systems y+ and y+L performed after 9 h of treatment with TNF-alpha . Data indicate that the stimulation of system y+ was accounted for by an increase in Vmax (0.323 vs. 0.130 µmol · ml-1 · min-1 obtained in control cells), while Km was not significantly modified. As expected, no significant changes of the kinetic parameters of system y+L were observed. Comparable results were obtained after LPS treatment (not shown).

                              
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Table 2.   Kinetic constants of discriminated L-arginine influx in HUVECs in the absence and presence of TNF-alpha

To assess the effect of TNF-alpha on two-way fluxes of arginine, the efflux of the cationic amino acid was compared in HUVECs treated for 9 h with the cytokine and in control cells (Fig. 5). Total arginine efflux was significantly faster from cells incubated for 9 h with TNF-alpha (initial efflux = 1.98 ± 0.05%/s vs. 1.71 ± 0.06%/s, Fig. 5A). In cells pretreated with NEM, the efflux of the cationic amino acid was significantly slowed and no effect of TNF-alpha was detectable (Fig. 5B). TNF-alpha stimulation of arginine efflux was therefore referable exclusively to a change of the NEM-sensitive component, which corresponds to the outward activity of system y+ (Fig. 5C), calculated by subtracting the NEM-resistant component from total efflux.


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Fig. 5.   Arginine efflux in HUVECs: effect of treatment with TNF-alpha . Cells were incubated for 9 h in medium 199 in the absence (control) and presence of TNF-alpha (10 ng/ml). Culture medium (0.33 mM arginine) was supplemented for 3 min with L-[14C]arginine (10 µCi/ml, carrier free), and cells were washed in EBSS in the absence (A) and presence (B) of 0.5 mM NEM and incubated in 0.4 ml of the same solution. C: NEM-sensitive component of arginine efflux, calculated by subtraction of data in B from data in A. Arginine efflux was calculated as described in MATERIALS AND METHODS. Lines represent best fit of data to Eq. 3. Data are means of 3 independent determinations. Error bars, SD (when greater than size of symbol).

Effects of TNF-alpha and LPS on the expression of cationic amino acid transporter genes in HUVECs. A preliminary analysis of the expression of genes involved in cationic amino acid transport in HUVECs was performed with RT-PCR. The results indicated that HUVECs express SLC7A1, which encodes for CAT1, SLC7A2, which is restricted to the CAT2B transcript, SLC7A6, for the y+L-related light chain y+LAT2, SLC7A7, for y+LAT1, and SLC3A2, which encodes for the y+L heavy chain 4F2hc/CD98.

Figures 6 and 7 report the results of an RT-PCR analysis of the expression of genes for cationic amino acid transporters in TNF-alpha -treated HUVECs. The cytokine caused a decrease in SLC7A1 expression after 6 h of treatment (Fig. 6A), whereas a significant increase in CAT2B expression was detected after 3 h of incubation in the presence of TNF-alpha (Fig. 6B). The densitometric analysis (Fig. 6C) showed that the relative density of SLC7A1/CAT1 to GAPDH RT-PCR product remained constant until 6 h of treatment and then decreased throughout the rest of the incubation. Conversely, the relative density of SLC7A2/CAT2B to GAPDH RT-PCR products increased progressively up to 6 h of incubation, remained high up to 12 h, and returned to basal values at 24 h. The Wilcoxon test with all the RT-PCR analyses performed on different cell strains revealed a highly significant increment (P < 0.01) of CAT2B expression at all times of treatment except 24 h and a significant decrease (P < 0.05) of SLC7A1 expression at >9 h of treatment. Similar experiments performed with LPS yielded comparable results (not shown).


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Fig. 6.   Effect of TNF-alpha on expression of genes related to system y+ in HUVECs. Cultures were incubated for the indicated times in the presence of TNF-alpha (10 ng/ml) before RNA extraction and reverse transcription. cDNA was employed as template for PCR coamplification reactions in which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were employed together with primers for SLC7A1/CAT1 (A) or SLC7A2/CAT2B (B). After electrophoresis of RT-PCR products, gels were stained with ethidium bromide and photographed. C: densitometric analysis of gels obtained in representative experiments in A and B. Relative intensity of SLC7A1 and CAT2B amplification products was normalized to that of the coamplified GAPDH product. The analysis, repeated twice in 3 different cell strains, yielded comparable results.



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Fig. 7.   TNF-alpha effect on expression of genes related to system y+L in HUVECs. Cultures were incubated in the presence of TNF-alpha (10 ng/ml) before RNA extraction and reverse transcription. DNA (200 ng) was employed as template for PCR coamplification reactions in which GAPDH primers were employed together with primers for 4F2hc, SLC7A6, or SLC7A7. After electrophoresis of RT-PCR products, gels were stained with ethidium bromide and photographed. Gels were obtained from a single representative experiment repeated in 3 different cell strains with comparable results.

RT-PCR products of the y+L-related genes SLC7A6 (y+LAT2), SLC7A7 (y+LAT1), and SLC3A2 (4F2hc) did not show any significant variation at any time of treatment with TNF-alpha (Fig. 7) or LPS (not shown).

Expression and sensitivity to TNF-alpha of NOS isozymes in HUVECs. Figure 8A shows the results of a representative Northern blot experiment in which the abundance of NOS3 mRNA was determined during incubation of HUVECs in the presence of TNF-alpha (10 ng/ml). A decrease in the expression of NOS3 was detectable after 3 h of incubation in the presence of the cytokine. Quantification of the effect, performed through determination of the ratio of NOS3 to GAPDH (Fig. 8B), indicates that NOS3 mRNA expression was inhibited by >80% after 24 h of exposure to the cytokine. Western blot analysis (Fig. 8, C and D) showed that the decrease in NOS3 protein was less pronounced, with a decrease of 44% and 40% detected after 24 and 48 h, respectively, of treatment with TNF-alpha .


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Fig. 8.   Nitric oxide synthase type III (NOS3) expression in HUVECs: effect of TNF-alpha . Endothelial cultures were incubated for indicated times in the presence of TNF-alpha (10 ng/ml) before RNA extraction and Northern blot analysis with NOS3 and GAPDH probes (A). For densitometric analysis (B), relative intensity of NOS3 mRNA was normalized to that of GAPDH mRNA. Experiment was repeated twice in 3 different HUVEC strains with comparable results. C: representative Western blot analysis of NOS3 protein in control HUVECs and in cells incubated for 24 or 48 h in the presence of TNF-alpha (10 ng/ml). Experiment was repeated 3 times with comparable results. D: densitometric analysis of Western blot analysis in C. C, control; AU, arbitrary unit.

RT-PCR analysis indicated that no expression of NOS2 was detected in HUVECs in the absence of TNF-alpha or in the presence of the cytokine at 10 ng/ml for various times of incubation (Fig. 9). Under the same conditions of amplification, the expression of NOS2 was readily detected in human bone marrow stromal cells stimulated for 9 h with the cytokine.


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Fig. 9.   Lack of nitric oxide synthase type II (NOS2) expression in HUVECs. Cultures were incubated in the presence of TNF-alpha (10 ng/ml) before RNA extraction, reverse transcription, and coamplification with NOS2 and GAPDH primers. After electrophoresis of RT-PCR products, gels were stained with ethidium bromide and photographed. BMSC, RT-PCR products obtained with bone marrow stromal cells, incubated for 9 h with TNF-alpha (10 ng/ml), employed as a positive control. Experiment was repeated twice in 3 different HUVEC strains with comparable results.

Effect of TNF-alpha on nitrite production of HUVECs. The accumulation of nitrites in the extracellular medium of HUVECs treated with TNF-alpha for 24 and 48 h and in control cells was determined with a fluorometric method (Fig. 10). No significant difference was detected between TNF-alpha -treated and control cells at either time.


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Fig. 10.   Nitrite production ([nitrite]out) in HUVECs after treatment with TNF-alpha . Cell monolayers, grown in phenol red-free growth medium, were incubated for 24 or 48 h in the absence (control) and presence of TNF-alpha (10 ng/ml). Fluorometry was used to determine nitrite production in the culture medium. Values are means ± SD of 12 independent determinations in a representative experiment. Experiment was repeated 3 times with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The characterization of endothelial arginine transport in the present study has demonstrated a thus far overlooked complexity. The cationic amino acid enters endothelial cells through at least two high-affinity components. One component corresponds to system y+ and is due to the activities of CAT1 and CAT2B. The other component corresponds to the leucine-accepting system y+L and is also due to at least two transporters: the heterodimers 4F2hc/y+LAT1 and 4F2hc/y+LAT2. The activity of system y+L has been recently demonstrated in other human mesenchymal cells (11) where it depends on the expression of the genes SLC3A2, SLC7A6, and SLC7A7, which also are expressed in HUVECs. However, at variance with human fibroblasts, where SLC7A6, encoding for y+LAT2, seems to be predominant (11), SLC7A7 and SLC7A6 appear to be expressed at comparable levels in HUVECs. The physiological relevance of the contributions of systems y+ and y+L to arginine transport is incompletely understood. Under physiological conditions, the contribution of system y+ to arginine influx should prevail, since this system, in contrast to system y+L, is poorly sensitive to competition by neutral amino acids. In epithelial models, it is generally accepted that system y+L provides an efflux route for cationic amino acids in exchange for neutral amino acids (5, 45). However, the operation of system y+ also is bidirectional, a characteristic clearly documented here for endothelial cells.

The overexpression of CAT2B associated with an increase of arginine transport has been repeatedly observed in endothelial models under nondiscriminating conditions (7, 15, 23, 28, 38). With the use of discriminating conditions, it is possible to ascertain that system y+L activity is not affected by treatment with TNF-alpha or LPS and that, consistently, the expression of SLC3A2, SLC7A7, or SLC7A6 does not exhibit any significant change. Only changes in the expression of CAT genes, and hence in system y+ activity, underlie the modifications in arginine transport induced by treatments with TNF-alpha or LPS. However, the regulatory mechanisms involved appear to be more complex than expected. Indeed, both compounds induce a clear-cut increase in CAT2B expression that foreruns the stimulation of arginine transport, although, as described for different examples of regulation of CAT genes (1), the increase in mRNA level appears to be greater than the transport change. In addition, TNF-alpha and LPS also affect the level of CAT1 mRNA, which is significantly decreased at later times of incubation (>9 h). Also in other cell models, such as macrophages (25, 35) and smooth muscle cells (19), TNF-alpha lowers CAT1 expression. In HUVECs, this effect may account for the rapid decline in the stimulation of arginine transport, detected after 12 h of incubation in the presence of TNF-alpha or LPS when CAT2B expression appears to be stimulated. The inability to discriminate between the activities of the two transporters on operational grounds prevents the validation of this hypothesis. However, the opposite effects observed on CAT1 and CAT2B on treatment with TNF-alpha and LPS suggest different functional roles of the two transporters. Moreover, the different time courses of the effects point to the involvement of different signals and/or transduction mechanisms.

In other cell models, high-output NO production is strictly linked to arginine transport through the coordinated induction of CAT2B and NOS2. In particular, this phenomenon has been thoroughly characterized in macrophages (22, 26, 35), vascular smooth muscle cells (16, 17, 19), and astrocytes (44). The situation is much less clear in endothelial cells. Under conditions of acute stimulation of arginine transport (4, 41, 43), an increased production of NO is observed, although the carrier involved in these effects has not been identified. On the other hand, it is known that chronic exposure to cytokines or LPS causes an overexpression of CAT2B and an increase of arginine transport (7, 15, 23, 28, 38), but data on possible consequences in terms of changes in NO synthesis are contrasting (16, 23). Under the conditions adopted here, the extracellular concentration of nitrites did not increase on treatment with TNF-alpha or LPS, notwithstanding the clear-cut stimulation of arginine transport. Although the results presented here do not exclude minor, transient changes in NO production on treatment of HUVECs with LPS or TNF-alpha , they exclude the occurrence of a sustained increase of NO production. On the other hand, under the same conditions, no basal or induced expression of NOS2 is detected at the mRNA (Fig. 9) or the protein level (results not shown). The lack of expression of NOS2 could possibly be attributed to the lack of interferon-gamma and interleukin-1, which have been employed in a recent study for the maximal expression of NOS2 associated with a high production of nitrites/nitrates in HUVECs (47). Whatever the reason for lack of NOS2 expression, this report suggests that, in the absence of NOS2 induction, stimulation of arginine transport through CAT2B by TNF-alpha is not associated with detectable changes in NO production in HUVECs.

Under the conditions employed here, NOS3 is the only source for the low, basal NO production, which appears unaffected by TNF-alpha or LPS treatment (Fig. 10). Because the Km of arginine for NOS3 is in the micromolar range (40), arginine, the intracellular concentration of which is in the millimolar range (3, 9), should not be a limiting factor for NO production. It is known that NOS3 activity is unaffected even by marked changes in the intracellular concentration of arginine (9); therefore, it is hardly surprising that the transient transport change described here can affect NO production through this NOS isozyme. Moreover, expression of this NOS isoform in HUVECs has been found to be inhibited by chronic exposure to TNF-alpha (Fig. 8), an effect consistent with the results obtained in other endothelial cell models (12, 27, 46). A structural association between NOS3 and CAT1 has been proposed that could imply a "channeling" of transported arginine toward NO synthesis (32). Although the data presented here do not support this hypothesis directly, they document a parallel decrease in the expression of NOS3 and CAT1, suggesting a coordinated regulation compatible with a functional link between the two proteins.

In nonendothelial cell models, the stimulation of system y+ raises arginine influx and efflux (29). Similar results obtained here for HUVECs indicate that TNF-alpha - or LPS-treated endothelial cells are endowed with an enhanced capability for arginine extrusion via system y+, suggesting that treatment with these compounds could promote the transendothelial flux of the cationic amino acid. In this case, stimulation of arginine transport would ensure an increased availability of the cationic amino acid to other cells of the vessel wall, such as smooth muscle cells, in which high extracellular levels of arginine are required to maintain high-output NO production through the functional axis CAT2B/NOS2 (17, 19). The validation of this hypothesis will be matter of further investigations.


    ACKNOWLEDGEMENTS

This study was partially supported by Istituto di Ricovero e Cura a Carattere Scientifico Centro Cardiologico Fondazione Monzino (Milan, Italy), Consiglio Nazionale delle Ricerche, Target Project "Biotechnology," and Ministero dell' Università e della Ricerca Scientifica e Tecnologica Progetto di Ricerca di Interesse Nationale "Coordinated Regulation of NO Production and Arginine Transport."


    FOOTNOTES

Address for reprint requests and other correspondence: V. Dall'Asta, Sezione di Patologia Generale e Clinica, Dipartimento di Medicina Sperimentale, Università di Parma, Via Volturno, 39, 43100 Parma, Italy (E-mail: valeria.dallasta{at}unipr.it).

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.

Received 5 July 2001; accepted in final form 4 September 2001.


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