Multiple pathways for cationic amino acid transport in rat thyroid epithelial cell line PC Cl3
Tiziano Verri,1
Cinzia Dimitri,1
Sonia Treglia,2
Fabio Storelli,2
Stefania De Micheli,2
Luca Ulianich,3,4
Pasquale Vito,3,5,6
Santo Marsigliante,1
Carlo Storelli,1 and
Bruno Di Jeso2
1Laboratory of General Physiology and 2Laboratory of General Pathology, Department of Biological and Environmental Sciences and Technologies, University of Lecce, Lecce; 3Department of Cellular and Molecular Biology and Pathology "L. Califano" and 5BioGeM Consortium, Federico II University of Naples, Naples; 4Institute of Endocrinology and Experimental Oncology "G. Salvatore," Italian National Research Council, Naples; and 6Laboratory of Molecular Genetics, Department of Biological and Environmental Sciences, University of Sannio, Benevento, Italy
Submitted 27 January 2004
; accepted in final form 10 October 2004
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ABSTRACT
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Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y+LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y+L, and b0,+ systems, as assessed by L-arginine uptake and kinetics, inhibition of L-arginine transport by N-ethylmaleimide and neutral amino acids, and L-cystine transport studies. y+L and y+ systems account for the highest transport rate (with y+L > y+) and b0,+ for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, and b0,+AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y+LAT1, and rBAT/b0,+AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term L-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.
cationic amino acid transporters; heteromeric amino acid transporters; system y+; system y+L; system b0,+; thyrotropin; L-arginine
IN MAMMALIAN CELLS, several carrier systems with distinct transport properties participate and/or cooperate in Na+-independent (b+, y+, y+L, and b0,+) and Na+-dependent (B0,+) transport of cationic amino acids across the plasma membrane, thus contributing to regulation of cationic amino acid availability within the cells, nitric oxide synthesis, and many other cell functions (see, e.g., Ref. 47). In recent years, the genes coding for most of the transport systems that mediate cellular transport of cationic amino acids in mammalian cells have been identified and characterized at the molecular level (Table 1; among many recent comprehensive reviews see Refs. 4, 6, 9, 13, 29, 4244). Na+-independent transport of cationic amino acids is mediated by several members of the solute carrier 7 (SLC7) gene family. Among these, the cationic amino acid transporters (members of the CAT subfamily; SLC7A1/CAT-1, SLC7A2/CAT-2A/2B, SLC7A3/CAT-3 and SLC7A4/CAT-4) essentially carry L-arginine, L-lysine, and L-histidine, possess much lower affinity for other amino acids, and virtually function as uniporters (system y+). In addition, certain glycoprotein-associated amino acid transporters (light chains belonging to the gpaAT subfamily: SLC7A6/y+LAT2, SLC7A7/y+LAT1, SLC7A9/b0,+AT) transport L-arginine, L-lysine, L-histidine, and many neutral amino acids, such as L-leucine, L-methionine, and L-glutamine, and function as obligatory antiporters exchanging cationic for neutral substrates and Na+ (system y+L) or neutral substrates alone (system b0,+). The glycoprotein-associated amino acid transporters require interaction via a disulfide bridge with the type II membrane glycoprotein members of the solute carrier 3 (SLC3) gene family, namely, the heavy chains SLC3A1/rBAT (which interacts with SLC7A9/b0,+AT) and SLC3A2/4F2hc (which interacts with SLC7A6/y+LAT2 and SLC7A7/y+LAT1). The glycoprotein does not play an active role in the actual amino acid transport function (32) but is necessary for trafficking of the glycoprotein-associated amino acid transporter to the plasma membrane. On the other side, Na+-dependent transport of cationic amino acids is ascribed to a member of the solute carrier 6 (SLC6) gene family, namely, SLC6A14/ATB0,+, that carries many neutral as well as cationic amino acids in cotransport with Na+ and Cl (system B0,+) (20, 37).
Because of the complexity of the organ and the difficulty of accessing the follicular epithelium by conventional membrane transport methodologies, information regarding amino acid transport systems in the thyroid is scanty. Nevertheless, the thyroid gland produces a large amount of thyroglobulin (Tg), the major soluble protein of the thyroid, which is extracellularly stored and concentrated (up to 750 mg/ml) in the follicular lumen for future liberation of thyroid hormones. To our knowledge, the specific information available on the presence of membrane transport systems that mediate the transport of cationic amino acids in the thyroid is limited to Northern blot detection of SLC7A7/y+LAT1 mRNA in mouse (31) and lack of detection of SLC6A14/ATB0,+ mRNA in human tissue (37). Therefore, as a contribution to understanding of cationic amino acid transport mechanisms in thyroid cells, we used L-arginine and L-cystine as substrates to study cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells) (16). This rat thyroid cell line represents a very popular model for in vitro studies on thyroid biology and, like other rat thyroid immortal untransformed cell lines (namely, FRTL-5 and WRT cells), it retains most of the typical features of a normal differentiated follicular thyroid cell, among which are thyrotropin (TSH) dependence for growth and functions and, at the molecular level, Na+-I symporter (NIS), Tg, and thyroperoxidase (TPO) expression (for recent reviews on in vitro rat thyroid cell systems see, e.g., Refs. 23 and 27).
In the present paper, we provide evidence that y+, y+L, and b0,+ transport activities and related SLC7A1/CAT-1, SLC7A2/CAT-2B, SLC3A2/4F2hc, SLC7A7/y+LAT1, SLC7A6/y+LAT2, SLC3A1/rBAT, and SLC7A9/b0,+AT mRNA transcripts are detectable in PC Cl3 cells, which represents the first description of an expression pattern of transport systems for cationic amino acids in a thyroid follicle-derived cell type, and evidence that such an expression pattern reflects that of the rat thyroid gland. In addition, as for many thyroid-specific activities required for thyroid hormone synthesis (including Tg synthesis, TPO expression, and I uptake), we show that L-arginine uptake is responsive to TSH as a consequence of differential expression of some of the detected transport systems under TSH stimulation. Finally, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that its long-term deprivation influences the expression pattern of the transport systems for cationic amino acids.
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MATERIALS AND METHODS
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Materials.
Calf serum, L-glutamine (100x solution), penicillin-streptomycin (100x solution), and Coons modified Hams F-12 culture medium were purchased from Labtek Eurobio (Milan, Italy). Hydrocortisone, transferrin, L-glycyl-L-histidyl-L-lysine acetate, and somatostatin were from ICN Biomedicals (Costa Mesa, CA). Insulin and TSH were obtained from Sigma (St. Louis, MO). L-[2,3,4-3H]arginine monohydrochloride (40 Ci/mmol; 1,480 GBq/mmol) and L-[14C]cystine (300 mCi/mmol; 11,100 MBq/mmol) were obtained from Perkin-Elmer Life Sciences (Boston, MA). Sigma was the source for all the other (reagent grade) chemicals.
Cell culture.
This study was performed with PC Cl3 cells, a rat thyroid epithelial (follicular) cell line derived from an 18-month-old normal Fischer rat (16). Cells were routinely grown on 35-mm-diameter dishes in Coons modified Hams F-12 medium supplemented with calf serum (5%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 ng/ml), and a six-growth factor mixture consisting of insulin (1 µg/ml), hydrocortisone (3.62 µg/ml), transferrin (5 µg/ml), L-glycyl-L-histidyl-L-lysine acetate (20 ng/ml), somatostatin (10 ng/ml), and TSH (1 mU/ml) (complete culture medium). The cells were maintained in a water-saturated atmosphere of 5% CO2 and 95% air at 37°C and transferred to new dishes every 35 days. The complete culture medium was routinely changed every 23 days and always renewed 24 h before an experiment. When required for TSH dependence studies, cells were grown in complete culture medium, with the sole exception of TSH varying between 0 and 10 mU/ml.
For L-arginine deprivation experiments, after complete cell attachment had been reached, cells were washed with PBS and incubated with either complete culture medium (normally containing 2.4 mM L-arginine) or medium lacking L-arginine. These media were prepared in accordance with published formulations, the appropriate amino acid being left out as required.
Uptake studies in PC Cl3 cells.
For transport studies, cells were used 23 days after plating (8090% confluence) on 35-mm-diameter dishes. Growth medium was removed, and dishes were washed three times in 3 ml of choline medium (in mM: 137 choline chloride, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, 10 HEPES; pH 7.4 with Tris) prewarmed to 37°C. Choline was replaced by 137 mM NaCl (sodium medium) in those experiments in which sodium dependence was studied. Uptake media were prepared by adding the labeled amino acid [L-[2,3,4-3H]arginine monohydrochloride (final concentration 0.51 µCi/ml) or L-[14C]cystine (final concentration 0.16 µCi/ml)] to choline or sodium medium. When L-cystine was present, the uptake medium contained 5 mM diamide as an oxidizing agent. Uptake was started by the addition of 1 ml of uptake medium (at 37°C) to the plate and terminated by removing uptake medium from the plate and washing it five times in cold stop solution (in mM: 132 NaCl, 14 Tris, 5 L-arginine; pH 7.4 with HCl at 4°C). Uptake periods had been assessed previously for all concentrations studied (see, e.g., Fig. 1A for 0.2 mM L-arginine and Fig. 5A for 0.050 mM L-cystine). Consequently, the incubation times used never exceeded 30 s. Nonspecific binding was assessed by measuring zero-time uptake, which was achieved by adding the uptake medium and immediately removing it and stopping the uptake. Cell lysates were obtained by adding 1 ml of 0.5% Triton X-100 per dish; 150 µl of this lysate were removed for scintillation counting in 4 ml of EcoLite+ scintillation fluid (ICN Biomedicals, High Wycombe, UK), and 25 µl were used for protein determination according to the method of Lowry et al. (25). The zero point was subtracted from the 30-s value, and uptake was expressed as picomoles per minute times milligram of protein.

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Fig. 1. A: Time course of L-arginine (substrate, S) uptake by PC Cl3 cells. Cells grown in complete culture medium were preliminarily washed 3 times in either sodium or choline medium (for details, see MATERIALS AND METHODS). L-Arginine uptake was then determined at the indicated times in sodium and choline medium supplemented with 0.2 mM L-arginine (and 1 µCi/ml L-[2,3,4-3H]arginine). Data points are means ± SE of 4 independent determinations within 1 representative experiment. B: kinetics of L-arginine uptake by PC Cl3 cells. Cells grown in complete culture medium were preliminarily washed 3 times in choline medium. L-Arginine uptake was then determined with 30-s incubations in choline medium supplemented with 0.0055 mM L-arginine (and 0.51.5 µCi/ml L-[2,3,4-3H]arginine; inset). The main body of B represents the Woolf-Augustinsson-Hofstee plot of the experimental data reported in the inset. Kinetic parameters in this experiment were as follows: Km,1 = 0.048 ± 0.002 mM, maximal velocity (Jmax,1) = 461.40 ± 11.49 pmol·min1·mg protein1, Km,2 = 0.261 ± 0.080 mM, Jmax,2 = 779.70 ± 71.04 pmol·min1·mg protein1, P = 440.30 ± 19.58 nl·min1·mg protein1. P, permeability. Note that when analyzed as a single component, the saturable transport of L-arginine displayed the following kinetic parameters: Km = 0.065 ± 0.006 mM, Jmax = 581.07 ± 29.56 pmol·min1·mg protein1.
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Fig. 5. A: time course of L-cystine uptake by PC Cl3 cells. Cells grown in complete culture medium were preliminarily washed 3 times in either sodium or choline medium. L-Cystine uptake was then determined at the indicated times in sodium and choline media supplemented with 0.05 mM L-cystine (and 0.16 µCi/ml L-[14C]cystine). Data points are means ± SE of 3 independent determinations within 1 representative experiment. B: effect of L-leucine, L-arginine, and L-glutamic acid on L-cystine influx in the absence of sodium. Cells grown in complete culture medium were preliminarily washed 3 times in choline medium. L-cystine uptake was then determined with 30-s incubations in choline medium supplemented with 0.05 mM L-cystine (and 0.16 µCi/ml L-[14C]cystine) in the presence or absence of 5 mM D-mannitol (control), L-arginine, L-leucine, or L-glutamic acid. Data points are means ± SE of 3 independent determinations within 1 representative experiment.
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Statistical analysis.
Each experiment was repeated at least three times. Data points reported in Figs. 15, 7, and 8 are given as means ± SE. Within a single experiment, each data point represents three to four replicate measurements; SE bars are shown wherever they exceed the size of the symbols. Differences between groups were tested with Students t-test.

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Fig. 8. A: growth curve of PC Cl3 cells in the presence and absence of L-arginine in the culture medium. Cells in exponential growth in complete culture medium (+L-arginine) were changed to a medium lacking L-arginine (L-arginine) at day 1 (arrow), and the no. of cells was counted at the indicated times. d, Days. B: semiquantitative (relative-quantitative) RT-PCR analysis of CAT-1, CAT-2A, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, b0,+AT, and ATB0,+ expression. RT-PCR was performed on equal amounts of total RNA (1 µg) isolated from PC Cl3 cells grown for 7 days either in complete culture medium containing 2.4 mM L-arginine (+) or in complete culture medium lacking L-arginine () in the presence of specific pairs of primers designed on the nucleotide sequences of rat transporters and a 1-to-9 ratio 18S primer-competimer mix (for details, see MATERIALS AND METHODS and Table 2).
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RT-PCR and Southern blotting.
Total RNA was extracted from PC Cl3 cells, as well as from rat thyroid and control tissues, with the TRIzol reagent (Invitrogen, Carlsbad, CA). RNA samples (0.5 µg) were subjected to RT-PCR with the GeneAmp RNA-PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturers protocol. Briefly, RT was performed for 12 min at 42°C in the presence of random hexamers, and the resulting cDNA was subjected to PCR with specific pairs of primers derived from rat or human sequences as reported in Table 2. PCR amplification was performed for cycles of denaturation, annealing, and extension, respectively, as follows: for CAT-1, 35 s at 95°C, 120 s at 58°C, 60 s at 72°C (35 cycles); for CAT-2A, 35 s at 95°C, 120 s at 58°C, 60 s at 72°C (39 cycles); for CAT-2B, 35 s at 95°C, 120 s at 60°C, 60 s at 72°C (39 cycles); for 4F2hc, 35 s at 95°C, 60 s at 52°C, 60 s at 72°C (35 cycles); for y+LAT1/2 (human-derived primers), 35 s at 95°C, 120 s at 55°C, 60 s at 72°C (35 cycles); for y+LAT1 (rat-derived primers), 35 s at 95°C, 60 s at 55°C, 60 s at 72°C (35 cycles); for y+LAT2 (rat-derived primers), 35 s at 95°C, 60 s at 55°C, 60 s at 72°C (35 cycles); for rBAT, 35 s at 95°C, 45 s at 60°C, 60 s at 72°C (35 cycles); for b0,+AT, 35 s at 35°C, 45 s at 60°C, 60 s at 72°C (35 cycles); for ATB0,+, 35 s at 95°C, 60 s at 55°C, 60 s at 72°C (39 cycles). In all cases, final synthesis was performed at 72°C for 7 min. RT-PCR products were separated on a 1% agarose gel and stained with ethidium bromide. To assess their identity, RT-PCR products were cloned in pCRII-TOPO vector (TOPO TA Cloning System, Invitrogen), and recombinant plasmids were transformed into Escherichia coli cells (TOP10F' strain). The identity of each insert was confirmed by direct sequencing on the plasmids with M13 reverse and/or M13 forward primer.
For Southern blotting of the amplification products derived from PC Cl3 cells and control tissues (see Fig. 6A), RT-PCR reaction products were subjected to electrophoresis in a 1% agarose gel and transferred by capillarity onto Hybond N+ nylon membranes (Amersham, Little Chalfont, UK) according to standard procedures (34). Probes to detect 4F2hc, rBAT, b0,+AT, y+LAT1, y+LAT2, and CAT-1 were prepared starting from rat intestinal RNA, whereas probes to detect CAT-2A, CAT-2B and ATB0,+ were prepared starting from rat liver, lung, and colon, respectively. In particular, for the preparation of probes that distinguished CAT-2A from CAT-2B, the following two pairs of rat-specific primers at the alternatively spliced region of CAT-2 were used: 5'-CCTTACCCCGCATTCTGTTTG-3' (forward primer) and 5'-AAATGACCCCTGCAGTCATCG-3' (reverse primer), which allowed amplification of a 115-bp CAT-2A-specific product, and 5'-CCCAATGCCTCGTGTAATCTA-3' (forward primer) and 5'-TGCCACTGCACCCGATGACAA-3' (reverse primer), which allowed amplification of a 121-bp CAT-2B-specific product. RT-PCR products were cloned in pCRII-TOPO vector and recombinant plasmids transformed as reported above. The identity of each insert was confirmed by sequencing. Routinely, inserts were excised by EcoRI digestion, separated from the vector on 1% agarose gel, and purified by Quantum Prep Freeze N Squeeze DNA gel extraction spin columns (Bio-Rad Laboratories, Hercules, CA). Inserts were labeled by random priming (Random Primers DNA Labeling System; Invitrogen) with [
-32P]dCTP (1 x 106 cpm/ml; Perkin-Elmer Life Sciences). Hybridization was visualized by autoradiography (Hyperfilm-
max films; Amersham) with standard protocols.

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Fig. 6. A: expression of amino acid transporters potentially involved in L-arginine uptake by PC Cl3 cells. RT-PCR was performed on equal amounts of total RNA (0.5 µg) isolated from PC Cl3 cells grown in complete culture medium and rat control tissues with specific pairs of primers designed on the nucleotide sequences of rat [except y+LAT (human)] transporters (for details, see MATERIALS AND METHODS and Table 2). H2O indicates no RNA in reverse transcription (negative control). Southern blot was resolved with probes specific for the single transporters. B: discrimination between y+LAT transporters in PC Cl3 cells. To ascertain the presence of y+LAT1 and/or y+LAT2 transporters, specific primers designed on the sequence of rat y+LAT1 and y+LAT2 were used. H2O indicates no RNA in reverse transcription (negative control). Markers are 1-kb DNA ladder (Invitrogen). C: expression of amino acid transporters in rat thyroid gland. RT-PCR was performed on equal amounts of total RNA (0.5 µg) isolated from rat thyroid and control tissues with specific pairs of primers designed on the nucleotide sequences of rat transporters. H2O indicates no RNA in reverse transcription (negative control). For details, see MATERIALS AND METHODS and Table 2.
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Semiquantitative (relative-quantitative) RT-PCR.
Total RNA was extracted from PC Cl3 cells grown under different experimental conditions [in the presence or absence of TSH (see Fig. 7C) or in the presence or absence of extracellular L-arginine (see Fig. 8B)] with TRIzol reagent. To obtain cDNA, 1 µg of total RNA was reverse transcribed for 1 h at 42°C with SuperScript II reverse transcriptase and random hexamers (Invitrogen) according to the manufacturers protocol. One microliter of RT product was used to perform PCR with specific pairs of primers derived from rat sequences as reported in Table 2. To perform amplification under relative-quantitative conditions, primers and competimers for 18S rRNA (QuantumRNA 18S Internal Standards, 324 bp; Ambion, Austin, TX) were added to the PCR mixture according to the manufacturers protocol. For all genes tested, a ratio of 1:9 for 18S primer to competimer was used. The optimal cycle number was determined by pilot experiments as that in which reactions for the specific gene and 18S in control and test samples were in the linear range. For a given gene tested, all samples were assayed concomitantly, using aliquots of the same PCR mixture and cDNA derived from individual samples; in addition, PCR products were loaded in the same agarose gel. Relative-quantitative PCR amplifications were performed as follows: for CAT-1, 40 s at 95°C, 60 s at 58°C, 70 s at 72°C (33 cycles); for CAT-2A, 40 s at 95°C, 60 s at 60°C, 70 s at 72°C (41 cycles); for CAT-2B, 40 s at 95°C, 60 s at 60°C, 70 s at 72°C (41 cycles); for 4F2hc, 40 s at 95°C, 60 s at 56°C, 70 s at 72°C (27 cycles); for y+LAT1, 40 s at 95°C, 60 s at 56°C, 70 s at 72°C (33 cycles); for y+LAT2, 40 s at 95°C, 60 s at 55°C, 70 s at 72°C (33 cycles); for rBAT, 40 s at 95°C, 60 s at 60°C, 70 s at 72°C (37 cycles); for b0,+AT, 40 s at 95°C, 60 s at 60°C, 70 s at 72°C (37 cycles); for ATB0,+, 40 s at 95°C, 60 s at 58°C, 70 s at 72°C (41 cycles). In all cases, final synthesis was performed at 72°C for 7 min. Each semiquantitative RT-PCR experiment was repeated at least three times. RT-PCR products were separated on a 2% agarose gel and visualized with ethidium bromide with a Gel-Doc 2000 gel documentation system equipped with a charge-coupled device camera for real-time image capture (Bio-Rad Laboratories). Band intensities were quantified with Quantity One 4.1.1 software (Bio-Rad Laboratories). The identity of each amplified RT-PCR product was verified by sequence analysis after cloning in pCRII-TOPO vector as described above.

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Fig. 7. A: effect of thyrotropin (TSH) on L-arginine uptake in PC Cl3 cells. Cells routinely grown in complete culture medium were maintained for 48 h in the presence of TSH concentrations varying between 0 and 10 mU/ml (in complete culture medium with the sole exception of TSH, which varied between 0 and 10 mU/ml). Before the uptake experiment, the cells were washed 3 times in either sodium or choline medium. L-Arginine uptake was then determined with 30-s incubations in sodium and choline media supplemented with 0.1 mM L-arginine (and 1 µCi/ml L-[2,3,4-3H]arginine). Data points are means ± SE of 4 independent determinations within 1 representative experiment. B: effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and forskolin on L-arginine uptake in PC Cl3 cells. Cells grown in complete culture medium but lacking TSH were maintained for 48 h in the absence (control) or presence of 1 mM 8-BrcAMP or 1 µM forskolin. Before the uptake experiment, part of the cells were preliminarily maintained for 5 min with or without 0.4 mM NEM. Cells were then washed 3 times in either sodium or choline medium. L-Arginine uptake was determined in NEM-treated and -untreated cells with 30-s incubations in sodium and choline medium supplemented with 0.01 mM L-arginine (and 0.5 µCi/ml L-[2,3,4-3H]arginine). Data points are means ± SE of 4 independent determinations within 1 representative experiment. C: semiquantitative (relative-quantitative) RT-PCR analysis of CAT-1, CAT-2A, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, b0,+AT, and ATB0,+ expression. RT-PCR was performed on equal amounts of total RNA (1 µg) isolated from PC Cl3 cells grown for 48 h either in complete culture medium containing 1 mU/ml TSH (+) or in complete culture medium lacking TSH () in the presence of specific pairs of primers designed on the nucleotide sequences of rat transporters and a 1-to-9 ratio 18S primer-competimer mix (for details, see MATERIALS AND METHODS and Table 2).
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RESULTS
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Basic transport of L-arginine in PC Cl3 cells.
The time course of L-arginine uptake by PC Cl3 cells is shown in Fig. 1A. Specific transport was recognizable immediately after the start of the incubation, and the amount of transported substrate was found to increase linearly at short incubation times. Equimolar substitution of sodium with choline in the uptake medium very slightly affected L-arginine uptake and only at longer incubation times, suggesting that L-arginine transport in PC Cl3 cells occurs virtually by sodium-independent mechanisms.
Kinetic analysis of L-arginine uptake, as measured in choline medium over a range of concentrations varying between 0.005 and 5 mM, is shown in Fig. 1B. L-Arginine transport was a complex phenomenon that could be described by the additive contributions of saturable process(es) and diffusion. The Woolf-Augustinsson-Hofstee transformation of the kinetic data in Fig. 1B is shown in the inset, where the saturable and diffusional components of the transport are graphically resolved. Together, these results suggest the occurrence at the plasma membranes of PC Cl3 cells of at least two saturable components as well as a diffusional component. To exclude the interference of the diffusional component, further kinetic experiments focusing on the saturable processes were always performed within a concentration range never exceeding 1 mM.
Competition assays were performed with a high concentration (5 mM) of selected amino acids as inhibitors of L-arginine uptake (Table 3). L-Arginine uptake, as measured at two L-arginine concentrations (0.01 and 0.1 mM), was strongly inhibited by excess of the cationic amino acid L-lysine in both choline and sodium media. The neutral amino acids L-leucine, L-glutamine, and L-methionine also inhibited L-arginine uptake (L-leucine and L-glutamine > L-methionine), although their inhibitory effect was more relevant in the presence than in the absence of sodium and their level of inhibition never reached that obtained by L-lysine. L-Alanine, L-phenylalanine, and L-tryptophan weakly inhibited or did not exert any significant inhibitory effect on 0.1 mM L-arginine uptake, whereas they consistently inhibited 0.01 mM L-arginine uptake (L-tryptophan and L-phenylalanine >> L-alanine), mostly in the presence of sodium. The acidic amino acid L-glutamic acid was also able to inhibit L-arginine uptake in both the absence and the presence of sodium.
Characterization of transport systems involved in L-arginine transport in PC Cl3 cells.
N-ethylmaleimide (NEM) selectively inhibits system y+ and thus can be used to discriminate the various mechanisms that mediate the uptake of cationic amino acids into cells (12). NEM was added to the culture medium 5 min before the transport assay at concentrations varying between 0 and 0.8 mM, and its effect on L-arginine transport was assessed in the presence and the absence of sodium (Fig. 2). L-Arginine uptake in PC Cl3 cells was only partially (<50%) inhibited by NEM in both experimental conditions. Inhibition by NEM was not further enhanced by prolongation of the treatment. Therefore, for further analyses throughout this study, NEM was always used for 5 min at a concentration of 0.4 mM.

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Fig. 2. Sensitivity to N-ethylmaleimide (NEM) of L-arginine transport in PC Cl3 cells. PC Cl3 cells grown in complete culture medium were treated for 5 min with the indicated concentrations of NEM by adding it to the culture medium from a 100x stock solution in water. After NEM incubation, cells were washed 3 times in either sodium or choline medium. L-Arginine uptake was then assayed with 30-s incubations in the same solutions supplemented with 0.1 mM L-arginine (and 1 µCi/ml L-[2,3,4-3H]arginine). Data points are means ± SE of 4 independent determinations within 1 representative experiment.
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Figure 3 shows the kinetic analysis of L-arginine transport in PC Cl3 cells in the selected concentration range of 0.010.75 mM, as performed in choline medium and in the absence or presence of 0.4 mM NEM. For the sake of simplicity, total L-arginine transport (transport in the absence of NEM) and the NEM-resistant component (transport in NEM-treated cells) were initially accounted for and therefore treated as single systems (Fig. 3A). Analogously, the NEM-sensitive component, obtained as the difference between total influx and NEM-resistant L-arginine uptake, was described as a single, saturable process (Fig. 3A). The Woolf-Augustinsson-Hofstee transformations of the saturable processes described in Fig. 3A are shown in Fig. 3B. With respect to the NEM-resistant component, the NEM-sensitive component exhibited a higher affinity (Km = 0.040 ± 0.011 vs. 0.084 ± 0.004 mM) and a lower maximal velocity (Jmax = 53.90 ± 6.50 vs. 144.90 ± 4.10 pmol·min1·mg protein1). A theoretical analysis was also performed with the a priori assumption that multiple transport systems are involved in total, NEM-resistant, and NEM-sensitive L-arginine transport (Fig. 3B). The theoretical kinetic parameters calculated under this assumption are reported in Fig. 3B for specific experiments, whereas overall Km calculations from three independent experiments are summarized in Table 4. Table 4 also provides Km values for total carrier-mediated L-arginine transport obtained from three independent experiments performed under the experimental scheme in Fig. 1B and a comparison with the Km values of various mammalian cloned and expressed transporters acting in a Na+-independent manner for the transport of cationic amino acids. Although highly limited by the low number of determinations (7 L-arginine concentrations ranging from 0.1 to 0.75 mM) and by the difficulty in discriminating and assigning Km and Jmax values to amino acid transport systems that operate with similar substrate affinities and/or maximal velocities (7, 8, 13), these results suggest that multiple high-affinity transport pathways for L-arginine transport, namely y+ system(s) on one side, and y+L and/or b0,+ system(s) on the other side, might operate in PC Cl3 cells.

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Fig. 3. Kinetic analysis of NEM-sensitive and NEM-resistant components of L-arginine transport in PC Cl3 cells. A: cells grown in complete culture medium were preliminarily maintained for 5 min with or without 0.4 mM NEM to evaluate either NEM-resistant or total transport of L-arginine. Both treated and untreated (control) cells were then washed 3 times in choline medium, and L-arginine uptake was assayed with 30-s incubations in the same medium supplemented with increasing (0.010.75 mM) L-arginine concentrations (and 1 µCi/ml L-[2,3,4-3H]arginine). Data points are means ± SE of 3 independent determinations within 1 representative experiment. Data for the NEM-sensitive influx were calculated as the difference between total and NEM-resistant L-arginine influx at each indicated concentration of the amino acid. B: Woolf-Augustinsson-Hofstee graphic representation of total transport and NEM-resistant and -sensitive L-arginine uptake. In this experiment, kinetic parameters for total, NEM-resistant, and NEM-sensitive transport after 1-component analysis (dotted lines) were as follows: total transport, Km = 0.066 ± 0.006 mM, Jmax = 196.90 ± 9.59 pmol·min1·mg protein1; NEM-resistant component, Km = 0.084 ± 0.004 mM, Jmax = 144.90 ± 4.10 pmol·min1·mg protein1; NEM-sensitive component, Km = 0.040 ± 0.011 mM, Jmax = 53.90 ± 6.50 pmol·min1·mg protein1. Kinetic parameters for total, NEM-resistant, and NEM-sensitive transport after multiple-component analysis (solid lines) were as follows: total transport, Km,1 = 0.042 ± 0.017 mM, Jmax,1 = 142.70 ± 37.50 pmol·min1·mg protein1, Km,2 = 0.074 ± 0.010 mM, Jmax,2 = 191.60 ± 11.77 pmol·min1·mg protein1, Km,3 = 0.142 ± 0.026 mM, Jmax,3 = 231.10 ± 11.19 pmol·min1·mg protein1; NEM-resistant component, Km,1 = 0.064 ± 0.014 mM, Jmax,1 = 117.50 ± 18.86 pmol·min1·mg protein1, Km,2 = 0.088 ± 0.009 mM, Jmax,2 = 146.50 ± 5.99 pmol·min1·mg protein1; NEM-sensitive component, Km,1 = 0.023 ± 0.005 mM, Jmax,1 = 39.25 ± 3.77 pmol·min1·mg protein1, Km,2 = 0.443 ± 0.092 mM, Jmax,2 = 104.50 ± 10.95 pmol·min1·mg protein1.
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Table 4. Km of L-arginine transport in PC Cl3 cells and comparison with Km values from mammalian cloned and expressed transporters
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To further characterize the transport systems that mediate L-arginine transport in PC Cl3 cells, the effects of high concentrations (5 mM) of L-lysine and L-leucine on L-arginine uptake were evaluated in NEM-treated and untreated cells in both sodium and choline media (Fig. 4A). In both the presence and the absence of sodium, L-lysine completely inhibited L-arginine uptake in NEM-treated and untreated cells. In the presence of sodium, L-leucine strongly reduced L-arginine uptake in untreated cells while completely abolishing L-arginine uptake in NEM-treated cells, thus delineating the possible involvement of y+L in addition to y+ transport activity. To better define the effect of sodium on the inhibition of L-arginine transport by neutral amino acids, the uptake of L-arginine (0.01 mM) was measured in the presence or absence of sodium and in the presence of increasing concentrations (up to 2 mM) of L-leucine (Fig. 4B). L-Leucine inhibited L-arginine influx in both the absence and the presence of sodium, but its inhibitory effect was much stronger in its presence than in its absence, as assessed by the significant (P < 0.001) reduction of the L-leucine concentration required for half-maximal inhibition ([I]0.5) of L-arginine transport from 0.781 to 0.074 mM (Fig. 4B). Under our experimental conditions, i.e., L-arginine uptake concentration (0.01 mM) much lower (at least 1/10th) than the Km value of L-arginine carrier-mediated transport (
0.1 mM; see data relative to the NEM-resistant transport in Table 4), it can be assumed that the [I]0.5 of L-leucine equals the inhibition constant (Ki) (13). These results clearly suggest that sodium increases the affinity of L-leucine, which is consistent with the expression of y+L transport activity in PC Cl3 cells.

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Fig. 4. A: effect of L-leucine and L-lysine on L-arginine influx in NEM-treated and -untreated PC Cl3 cells in the presence and absence of sodium. Cells grown in complete culture medium were preliminarily maintained for 5 min with or without 0.4 mM NEM. Cells were then washed 3 times in either sodium or choline medium. L-Arginine uptake was determined with 30-s incubations in NEM-treated and -untreated cells using both sodium and choline medium supplemented with 0.1 mM L-arginine (and 1 µCi/ml L-[2,3,4-3H]arginine) in the presence of 5 mM D-mannitol (mann; control), L-lysine (Lys), or L-leucine (Leu). Data points are means ± SE of 4 independent determinations within 1 representative experiment. B: inhibitory effect of increasing concentrations of L-leucine on L-arginine influx in NEM-untreated PC Cl3 cells in the presence and absence of sodium. Cells grown in complete culture medium were preliminarily washed 3 times in either sodium or choline medium. L-Arginine uptake was determined with 30-s incubations of cells in both sodium and choline media supplemented with 0.01 mM L-arginine (and 0.5 µCi/ml L-[2,3,4-3H]arginine) in the absence or presence of increasing concentrations (0.252 mM) of L-leucine. The following equation was used to describe a competitive-type inhibition of amino acid transport: V = V0 [Imax x [I]/([I]0.5 + [I])], 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 [I] required for half-maximal inhibition (11, 13, 33). To compare the experimental data describing L-arginine entry in the absence and presence of sodium, uptake values measured in the presence of L-leucine (V) were normalized by the corresponding uptake value measured in the absence of L-leucine (V0), the above equation becoming V/V0 = 1 [Imax/V0 x [I]/([I]0.5 + [I])], where V/V0 is the relative L-arginine uptake and Imax/V0 is the relative Imax. In this experiment, regression parameters for L-arginine influx determined in the absence of sodium were [I]0.5 = 0.781 ± 0.469 mM and relative Imax = 0.73 ± 0.15, with V0 = 15.65 ± 1.39 pmol·min1·mg protein1, whereas regression parameters for L-arginine influx in the presence of sodium were [I]0.5 = 0.074 ± 0.018 mM and relative Imax = 0.83 ± 0.04, with V0 = 13.87 ± 0.34 pmol·min1·mg protein1. Data points are means ± SE of 4 independent determinations within 1 representative experiment.
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To assess the existence of a b0,+ transport activity, we performed L-cystine uptake in both the presence and the absence of sodium (Fig. 5). The time course of 0.050 mM L-cystine uptake by PC Cl3 cells is shown in Fig. 5A. Substitution in the uptake medium of sodium with choline at equimolar concentration did not significantly affect L-cystine uptake, indicating that transport was independent of extracellular sodium. Furthermore, L-cystine uptake was inhibited by high concentrations (5 mM) of L-leucine and L-arginine, which strongly suggests the existence of a b0,+ system operating at the plasma membranes of PC Cl3 cells (Fig. 5B). Also, glutamic acid slightly, although not significantly (P > 0.05), inhibited L-cystine uptake, thus keeping open the hypothesis of the possible presence in PC Cl3 cells of cystine/glutamate exchange activity by the
system (see, e.g., Refs. 42 and 44). Together, these results suggest that y+, y+L, and b0,+ transport systems are present in the plasma membranes of PC Cl3 cells.
Gene expression analysis of transport systems potentially involved in L-arginine uptake in PC Cl3 cells and in rat thyroid gland.
The analysis of the expression of genes putatively involved in L-arginine transport in PC Cl3 cells was performed by RT-PCR and Southern blot assay. Results reported in Fig. 6A indicate that PC Cl3 cells express SLC7A1, which encodes for CAT-1, and the high-affinity splicing variant of SLC7A2, which encodes for CAT-2B. Furthermore, they express SLC7A9, which encodes for the b0,+-related light chain b0,+AT, as well as SLC3A1, which encodes for the b0,+ heavy chain rBAT. In addition, PC Cl3 cells expressed y+LAT transporters, as first assessed by using a pair of human y+LAT(1/2)-specific primers, and SLC3A2, which encodes for the y+L heavy chain 4F2hc. Further analysis performed by using rat y+LAT1- and y+LAT2-derived specific primers allowed identification of both SLC7A6, which encodes for the y+L-related light chain y+LAT2, and SLC7A7, which encodes for the y+L-related light chain y+LAT1 (Fig. 6B). All amplification products obtained from PC Cl3 cells were subcloned and sequenced to confirm their identity (data not shown). In our screening, we also tested PC Cl3 cell RNA for the low-affinity splicing variant of SLC7A2, which encodes for CAT-2A, and for SLC6A14, which encodes for ATB0,+, using rat-derived specific primers for each transporter. However, in both cases no amplification product was obtained (Fig. 6A). These results are in close agreement with the kinetic data and suggest that at least five transport entities, namely CAT1, CAT-2B, y+LAT1/4F2hc, y+LAT2/4F2hc, and b0,+AT/rBAT, may operate in PC Cl3 cells.
A similar expression analysis performed on rat thyroid gland by RT-PCR assay allowed detection of SLC7A1/CAT-1, SLC7A2/CAT-2A, SLC7A2/CAT-2B, SLC3A2/4F2hc, SLC7A7/y+LAT1, SLC7A6/y+LAT2, SLC3A1/rBAT, SLC7A9/b0,+AT, and SLC6A14/ATB0,+ (Fig. 6C). Here also, all amplification products obtained from the rat thyroid gland were subcloned and sequenced to confirm their identity (data not shown).
Effect of TSH on L-arginine uptake and expression of transport systems for cationic amino acids.
Like many thyroid-specific activities required for thyroid hormone synthesis (including Tg synthesis, TPO expression, and I uptake), L-arginine uptake in PC Cl3 cells was responsive to TSH. In fact, L-arginine uptake increased in a dose-dependent manner after 48-h incubation of the cells with increasing extracellular concentrations (010 mU/ml) of TSH (Fig. 7A). Equimolar substitution of sodium with choline in the uptake medium only very slightly (although not significantly; P > 0.05 for each tested TSH concentration) affected L-arginine uptake (Fig. 7A).
In the absence of TSH from the extracellular medium, the increase of L-arginine uptake was mimicked by 48-h incubation of the cells with the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 1 mM) or with the adenylate cyclase activator forskolin (1 µM) (Fig. 7B), thus suggesting the involvement of the cAMP-dependent pathway in the activation of L-arginine transport. Interestingly, L-arginine uptake increased in both NEM-treated and untreated cells, which is strongly indicative that both NEM-sensitive and NEM-resistant transport systems may be targets of the activation process observed (Fig. 7B). Again, substitution of sodium with choline in the uptake medium did not significantly (P > 0.05) affect L-arginine uptake (Fig. 7B).
Semiquantitative (relative-quantitative) RT-PCR analysis was performed to examine the expression of CAT-1, CAT-2A, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, b0,+AT, and ATB0,+ mRNA in PC Cl3 cells grown for 48 h either in the absence of TSH or in the presence of TSH 1 mU/ml (Fig. 7C). The QuantumRNA 18S internal standards were used as an internal control, resulting in a PCR product of 324 bp (Fig. 7C). A significant upregulation of the mRNA levels of CAT-1 [CAT-1-to-18S ratio (TSH) = 1.24 ± 0.11, CAT-1-to-18S ratio (+TSH) = 2.61 ± 0.15; no. of determinations (n) = 3; P < 0.01], CAT-2B [CAT-2B-to-18S ratio (TSH) = 0.25 ± 0.01, CAT-2B-to-18S ratio (+TSH) = 0.43 ± 0.02; n = 3; P < 0.05], 4F2hc [4F2hc-to-18S ratio (TSH) = 0.87 ± 0.09, 4F2hc-to-18S ratio (+TSH) = 1.63 ± 0.11; n = 3; P < 0.01], y+LAT1 [y+LAT1-to-18S ratio (TSH) = 0.61 ± 0.05, y+LAT1-to-18S ratio (+TSH) = 0.98 ± 0.07; n = 3; P < 0.05], rBAT [rBAT-to-18S ratio (TSH) = 1.26 ± 0.02, rBAT-to-18S ratio (+TSH) = 1.85 ± 0.12; n = 3; P < 0.05], and b0,+AT [b0,+AT-to-18S ratio (TSH) = 0.18 ± 0.03, b0,+AT-to-18S ratio (+TSH) = 0.35 ± 0.02; n = 3; P < 0.05] was observed passing from cells incubated in the absence of TSH to cells incubated with TSH 1 mU/ml, whereas y+LAT2 signal slightly (although not significantly) decreased [y+LAT2-to-18S (TSH) = 1.52 ± 0.05, y+LAT2-to-18S ratio (+TSH) = 1.40 ± 0.04; n = 3; P > 0.05]. On the other hand, CAT-2A and ATB0,+ mRNA signals were not detected in either the absence or the presence of TSH. Together, these results suggest that TSH differentially affects expression of the transport systems potentially involved in L-arginine transport in PC Cl3 cells.
Effect of long-term L-arginine deprivation on PC Cl3 cell growth and on expression of the transport systems for cationic amino acids.
The growth curve for PC Cl3 cells in the presence and absence of L-arginine is shown in Fig. 8A. At day 1, cells grown in normal culture medium (normally containing 2.4 mM L-arginine) were split into two 70,000-cell aliquots and grown for 7 days in normal culture medium or in a culture medium without 2.4 mM L-arginine. The absence of L-arginine from the culture medium resulted in a dramatic reduction of cell number (
6-fold at day 8), thus suggesting that extracellular supplementation of L-arginine is an important requirement for this thyroid cell line for normal growth, even in the presence of the six-hormone and/or growth factor mixture.
Semiquantitative (relative-quantitative) RT-PCR was used to examine the expression of CAT-1, CAT-2A, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, b0,+AT, and ATB0,+ mRNA in PC Cl3 cells grown for 7 days in the presence or absence of extracellular L-arginine (Fig. 8B). Comparable mRNA levels were observed in the presence and absence of L-arginine for CAT-1 [CAT-1-to-18S ratio (+L-arginine) = 1.35 ± 0.06, CAT-1-to-18S ratio (L-arginine) = 1.18 ± 0.04; n = 3; P > 0.05], 4F2hc [4F2hc-to-18S ratio (+L-arginine) = 0.69 ± 0.05, 4F2hc-to-18S ratio (L-arginine) = 0.73 ± 0.03; n = 3; P > 0.05], y+LAT1 [y+LAT1-to-18S ratio (+L-arginine) = 0.74 ± 0.06, y+LAT1-to-18S ratio (L-arginine) = 0.83 ± 0.05; n = 3; P > 0.05], y+LAT2 [y+LAT2-to-18S ratio (+L-arginine) = 0.98 ± 0.04, y+LAT2-to-18S ratio (L-arginine) = 1.25 ± 0.11; n = 3; P > 0.05], rBAT [rBAT-to-18S ratio (+L-arginine) = 2.41 ± 0.08, rBAT-to-18S ratio (L-arginine) = 2.73 ± 0.09; n = 3; P > 0.05], and b0,+AT [b0,+AT-to-18S ratio (+L-arginine) = 0.80 ± 0.03, b0,+AT-to-18S ratio (L-arginine) = 0.96 ± 0.05; n = 3; P > 0.05], whereas CAT-2A and ATB0,+ were not detected in either experimental condition. On the other side, CAT-2B mRNA expression was highly reduced [CAT-2B-to-18S ratio (+L-arginine) = 0.62 ± 0.04, CAT-2B-to-18S ratio (L-arginine) = 0.28 ± 0.06; n = 3; P < 0.01] by long-term L-arginine deprivation.
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DISCUSSION
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In the present study we report uptake, RT-PCR, and Southern blot analysis data supporting the concept that multiple transport systems for cationic amino acids are present at the plasma membranes of rat thyroid PC Cl3 cells. In particular, we demonstrate that cationic amino acid transport occurs by activity of y+, y+L, and b0,+ systems (Figs. 15, Tables 3 and 4) and is possibly supported by expression of the genes encoding for CAT-1 (SLC7A1), CAT-2B (SLC7A2), 4F2hc (SLC3A2), y+LAT1 (SLC7A7), y+LAT2 (SLC7A6), rBAT (SLC3A1), and b0,+AT (SLC7A9) (Figs. 6, A and B, 7C, and 8B). PC Cl3 are highly differentiated thyroid cells that express Tg, TPO, NIS, and TSH receptors (23, 27). They are considered unpolarized cells in that they have lost the capacity to form monolayers and/or to develop tight junctions in culture. However, our results indicate that both "basolateral" (CAT-1, CAT-2B, 4F2hc/y+LAT1, 4F2hc/y+LAT2) and "apical" (rBAT/b0,+AT) transporters may coexist in PC Cl3 plasma membranes. Similarly, FRTL-5 cells (also unpolarized but highly differentiated rat thyroid epithelial cells; for details see Ref. 23) seem to express the same pattern of cationic amino acid transporters as PC Cl3 cells (C. Dimitri, unpublished observations), which suggests that such equipment might be common to thyroid follicule-derived cells. Results obtained in the rat thyroid epithelial cell model are confirmed and corroborated by the RT-PCR analysis performed on total RNA isolated from the thyroid gland, which gave for the whole organ in the rat an expression profile similar to that described in PC Cl3 cells (Fig. 6C). However, in addition to CAT-1, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, and b0,+AT, the rat thyroid gland expresses both CAT-2A and ATB0,+ transcripts. This finding is not surprising if we consider the complexity of the thyroid gland (with its epithelial follicles, blood vessels, lymphatics, innervation, C cells, accessory cells, etc.) in combination with the relatively small size of the organ in the rat (
5 mm) and the contiguity of other satellite tissues and organs, such as the parathyroid glands (which in the rat are embedded in the dorsal surface of the thyroid gland), the salivary glands, and the trachea. For instance, CAT-2A is expressed in many different tissues and cell types (13), including fibroblasts (11), whereas ATB0,+ is expressed in the trachea and salivary glands (37) as well as in endothelial cells (although to a low extent; Refs. 24, 26). Therefore, heterogeneity in the composition and possible contamination of the rat thyroid total RNA preparation might explain the differences between epithelial cells in culture and in the whole organ on one side and the discrepancy with respect to the lack of detection of ATB0,+ by Northern blotting in human thyroid (37) on the other. At the moment, it is difficult to address the physiological relevance of the transport systems found in both PC Cl3 cells and thyroid gland in the context of epithelial thyroid cell functions. However, the fact that epithelial thyroid cells are endowed with uptake and release systems for cationic amino acids resembling those that allow epithelial cells of kidney proximal tubule and small intestine to perform both basic cellular functions and transepithelial (vectorial) transport of cationic and neutral amino acids (see, e.g., Refs. 42 and 44) makes it possible to hypothesize the involvement of such transporters in the blood-to-lumen and/or lumen-to-blood movement of amino acids at the thyroid follicular level, possibly functional in Tg (colloid) synthesis and/or degradation. The identification in thyroid follicule-derived cells as well as in the thyroid gland of membrane transporters that are involved in type I (rBAT) and non-type I (b0,+AT) cystinuria and lysinuric protein intolerance (LPI; y+LAT1) (see, e.g., Ref. 6) also raises an intriguing question regarding the possible effects of such genetic defects at the thyroid level. To our knowledge, no information is available in the literature regarding the status of thyroid in cystinuric and LPI patients. However, the recent availability of mouse models for type I (SLC3A1-deficient mice; Ref. 30) and nontype I (SLC7A9-deficient mice; Ref. 15) cystinuria might aid careful examination of this pathophysiological question.
L-Arginine transport in PC Cl3 cells occurs by Na+-independent mechanisms. The absence of Na+-dependent transport systems for L-arginine is suggested by the negligible effect of Na+ on L-arginine uptake (Figs. 1A, 2, 4A, and 7A and Table 3) and lack of RT-PCR amplification products (even up to 50 cycles of amplification) using ATB0,+-specific primers (see Figs. 6A, 7C, and 8B). On the other hand, the notion that y+, y+L, and b0,+ transport systems may jointly operate in PC Cl3 cells is mainly supported by 1) the inhibitory effect of NEM on L-arginine transport and kinetics (Figs. 2 and 3), which allowed discrimination between cationic amino acid uniporters and cationic/neutral amino acid antiporters; 2) the inhibitory effect of L-leucine and other neutral amino acids (such as L-glutamine and L-methionine) on L-arginine transport in the presence of sodium (Fig. 4 and Table 3), which suggested y+L transport activity; and 3) the analysis of L-cystine uptake and inhibition pattern (Fig. 5), which allowed discrimination of b0,+ with respect to y+L transport activity, L-cystine being a specific substrate for the b0,+ system only. As assessed by molecular analysis (Figs. 6A, 7C, and 8B) and observed multiplicity of the NEM-sensitive component of L-arginine transport (Fig. 3B and Table 4), y+ transport activity should be sustained by CAT-1 and CAT-2B. Furthermore, y+L transport activity might be sustained by both y+LAT1 and y+LAT2 transporters, as supported by both molecular analysis (Figs. 6B, 7C, and 8B) and study of the inhibitory effect of neutral and acidic amino acids on L-arginine transport (Fig. 4 and Table 3). In particular, we observe that certain neutral amino acids, including L-leucine, L-glutamine and L-methionine, largely inhibit L-arginine transport in the presence of sodium, although their inhibitory effect is also exerted in the absence of sodium, but at a lower extent. In addition, we show that L-glutamic acid significantly inhibits L-arginine transport in the presence of sodium, although to a lower extent with respect to the above-mentioned neutral amino acids, and that a similar inhibition is also provided in the absence of sodium (Table 3). Because inhibition of L-arginine transport by L-glutamic acid in both the presence and the absence of sodium is a hallmark feature of the y+LAT2 transporter (3, 44), our results suggest that in addition to y+LAT1, y+L transport activity in PC Cl3 cells is due in part to y+LAT2. That this holds true is further supported by the finding that L-tryptophan also exerts inhibition on L-arginine transport in both the presence and the absence of sodium (with a very strong effect observed on 0.01 mM L-arginine uptake; Table 3), another peculiarity described for y+LAT2 (3). Interestingly, y+LAT1 mRNA has also been reported to be present in mouse thyroid tissue, although at low levels with respect to small intestinal and kidney tissues (31). Finally, b0,+ transport activity should be sustained by the b0,+AT transporter, as shown by molecular analysis (Figs. 6, 7C, and Fig. 8B) and L-cystine transport studies. Among the different expressed systems, it appears that in PC Cl3 cells y+L and y+ activities account for the highest transport rate (with y+L > y+) and b0,+ activity for a residual fraction of the transport (Figs. 25). This concept correlates well with the systematic observation from the semiquantitative RT-PCR experiments that, starting from the same amount of total RNA and under relative-quantitative conditions, an increasing cycle number is required to reach the lower third of the linear range of amplification of 4F2hc (
27 cycles), y+LAT1, y+LAT2, and CAT-1 (
33 cycles), rBAT and b0,+AT (
37 cycles), and CAT-2B (
41 cycles), the last invariably being the least-represented transcript amplified from the PC Cl3 total RNA pool.
TSH plays an important role in thyroid growth and differentiation, and several thyroid-specific genes and differentiation markers (such as Tg, TPO, NIS, and TSH receptor) and thyroid-specific transcription factors [such as thyroid transcription factor (TTF)-1, TTF-2, and Pax-8] are regulated by TSH and/or cAMP via PKA, the most important signaling pathway in the control of thyroid growth and function (23, 27). In the present study, we showed that in PC Cl3 cells, increasing TSH concentration in the extracellular medium results in increasing L-arginine transport (Fig. 7A) and that this effect is mimicked by 8-BrcAMP and forskolin (Fig. 7B). Interestingly, as found by semiquantitative RT-PCR analysis, TSH stimulation results in a significant upregulation of the mRNA levels of CAT-1, CAT-2B, 4F2hc/y+LAT1, and rBAT/b0,+AT but does not involve y+LAT2, CAT-2A, and ATB0,+, which supports the notion that only some of the transport proteins that mediate the movement of cationic amino acids across the plasma membranes of thyroid cells share the same TSH and/or cAMP hormonal control and/or regulation with thyroid-specific proteins.
To our knowledge, the present study represents the first attempt to characterize the pattern of L-arginine transporters in a thyroid follicular cell. Identification of such a pattern is an important step in assessing the status of L-arginine transport in the context of many physiological functions that are exerted by follicular cells and that involve L-arginine itself or L-arginine-derived products. In this respect, we assessed the effect of long-term L-arginine deprivation on PC Cl3 cell growth. As shown in Fig. 8A, PC Cl3 cells dramatically reduce their growth in the absence of external L-arginine. Interestingly, a significant reduction of CAT-2B transcripts is concomitantly observed in the total RNA pool of the cells deprived of L-arginine for 7 days (Fig. 8B), which suggests a role of CAT-2B in the cellular adaptive response to long-term amino acid starvation. That L-arginine supplementation is crucial for normal growth of PC Cl3 cells is in agreement with studies conducted on many "normal" cell lines (such as fibroblast, kidney epithelial, and lung cell lines) (35, 46) that have shown how these cells reach and maintain a condition of quasi-quiescence for several days in the absence of L-arginine supplementation. Under the same experimental treatment, HeLa cells and many other "transformed" or "malignant" cultured cell lines die within 13 days (35, 46), suggesting L-arginine deprivation as a nongenotoxic method for selective treatment of cancer cells. In this context, it must be emphasized that the "normal and differentiated" PC Cl3 cells have been transformed by various oncogenes to obtain "undifferentiated" (PC E1A, PC v-raf) and "transformed" (PC Py, PC E1A v-raf, PC E1A Py) thyroid cell lines, which together represent a well-validated in vitro model of multistep thyroid tumorigenesis (2, 14, 16, 41). This model might be very useful for assessing the effect of L-arginine on cell growth and function during the various phases of thyroid tumor progression. In addition, the positive correlation existing between extracellular L-arginine levels and CAT-2B expression suggests that L-arginine supplementation via CAT-2B might play a relevant role in regulating NO biosynthesis in thyroid cells, thus regulating many thyrocyte functions. In fact, at the thyroid level, NO produced by follicular cells seems to exert autocrine or paracrine actions in follicular cell proliferation and expression of growth and vasoactive factors and to play a highly relevant role in regulating the dynamics of the microvascular bed around active follicles, in both physiological and pathological conditions (see, e.g., Refs. 10, 1719). Also, proinflammatory cytokines, such as interleukin-1 and interferon-
, have been demonstrated to increase NO production in thyrocytes (22), suggesting a role for L-arginine availability in the inflammatory pathology of the thyroid.
In summary, we have shown that y+, y+L, and b0,+ transport activities and related SLC7A1/CAT-1, SLC7A2/CAT-2B, SLC3A2/4F2hc, SLC7A7/y+LAT1, SLC7A6/y+LAT2, SLC3A1/rBAT, and SLC7A9/b0,+AT mRNA transcripts are present in rat thyroid PC Cl3 cells and that such a profile can be extended to the rat thyroid gland. In addition, we have provided evidence that L-arginine uptake is controlled by TSH as upregulation of CAT-1, CAT-2B, 4F2hc/y+LAT1, and rBAT/b0,+AT occurs at the mRNA level under TSH stimulation. Finally, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term L-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.
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GRANTS
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This investigation was supported by PRIN2001, a grant from the Italian Ministry of Education, University and Research, and by grants from the University of Lecce (Fondi ex-60%, 20012003).
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FOOTNOTES
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Address for reprint requests and other correspondence: T. Verri, Laboratory of General Physiology, Dept. of Biological and Environmental Sciences and Technologies, Univ. of Lecce, Strada Provinciale Lecce-Monteroni, I-73100 Lecce, Italy (E-mail: physiol{at}ultra5.unile.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.
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