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Institute of Pharmacology & Therapeutics, Faculty of Medicine, 4200–319 Porto, Portugal
2To whom correspondence should be addressed. E-mail: patricio.soares{at}mail.telepac.pt.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15%) required extracellular sodium. [14C] -L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). Both L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited [14C]-L-leucine accumulation. The efflux of [14C]-L-leucine was markedly increased (P < 0.05) by L-leucine and BCH, but not by L-arginine. In IEC-6 cells, but not in Caco-2 cells, the uptake of [14C]-L-leucine at acidic pH (5.0 and 5.4) was greater (P < 0.05) than at pH 7.4. In conclusion, it is likely that system B0 might be responsible for the sodium-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine may include system LAT1, whose activation results in transstimulation of L-leucine outward transfer.
KEY WORDS: • L-system • leucine • Caco-2 cells • IEC-6 cells • amino acid exchanger
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), but mechanisms regulating its intestinal absorption have not yet been clearly defined. Recent studies from our laboratory have shown that L-DOPA uptake in intestinal epithelial cells may be promoted through the L-type amino acid transporter (2
,3
), as has been found in renal epithelial cells (4
) and at the level of brain capillary endothelium (4
–8
). However, the presence of the L-type, for leucine preferring, amino acid transporter (LAT) has not been detected in cultured intestinal epithelial cells currently employed in intestinal transport studies. The main reasons to use cultured epithelial intestinal cells in this type of study are concerned with the high cellular heterogeneity of the intestinal epithelium and the complex intestinal physiology and morphological structure.
The present work was designed to evaluate the presence and define the kinetic characteristics of the L-type amino acid transporter in cultured intestinal epithelial cell lines. For this purpose we measured the apical uptake of [14C]-L-leucine in monolayers of Caco-2 and IEC-6 cells. Caco-2 cells, an established epithelial cell line derived from a human colon adenocarcinoma, undergo enterocyte differentiation in culture (9
). This cell line also has been suggested to possess attributes that make it a suitable in vitro model system for the investigation of transport across the small intestinal epithelium (10
, 11
). IEC-6 cells are a rat epithelial cell line that in culture has features of small intestinal crypt cells (12
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Caco-2 cells (ATCC 37-HTB; passages 39–49) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Cells were grown in minimal essential medium (Sigma Chemical, St. Louis, MO) supplemented with 106 U/L penicillin G, 250 µg/L amphotericin B, 100 ng/L streptomycin (Sigma), 20% fetal bovine serum (FBS; Sigma) and 25 mmol/L HEPES (Sigma). IEC-6 cells were obtained from the "Deutsche Sammlung von Mikroorganismen und Zellkulturen" (ACC-111; Passages 3–14) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Cells were grown in Dulbecco’s modified Eagle’s medium (45%) and RPMI 1640 (45%) supplemented with 10% FBS, 100 U/L insulin and 106 U/L penicillin G, 250 µg/L amphotericin B and 100 ng/L streptomycin. For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:3 and subcultured in Costar petri dishes with 21 cm2 growth area (Costar, Badhoevedorp, The Netherlands). For uptake studies, the cells were seeded in collagen-treated 24-well plastic culture clusters (i.d. 16 mm, Costar) at a density of 40,000 cells per well (2.0 x 104 cells/cm2). The cell medium was changed every 2 d, and the cells reached confluence after 4 (IEC-6) or 7 (Caco-2) days of initial seeding. For 24 h before each experiment, the cell medium was free of FBS. Experiments with IEC-6 and Caco-2 cells were generally performed 2 and 5 d after cells reached confluency, usually 6 and 12 d after the initial seeding, respectively; each cm2 contained 40 and 100 µg of cell protein.
Transport of [14C]-L-leucine.
On the day of the experiment, the growth medium was aspirated and the cell monolayers were preincubated for 30 min in Hanks’ medium at 37°C. The Hanks’ medium had the following composition (mmol/L): NaCl 137, KCl 5, MgSO4 0.8, Na2HPO4 0.33, KH2PO4 0.44, CaCl2 0.25, MgCl2 1.0, Tris HCl 0.15 and sodium butyrate 1.0, pH 7.4. Apical uptake was initiated by the addition of 1 mL Hanks’ medium with a given concentration of the substrate. Time-course studies were performed in experiments in which cells were incubated with 0.25 µmol/L [14C]-L-leucine for 1, 3, 6, 12, 30 and 60 min. Saturation experiments were performed in cells incubated for 6 min with 0.25 µmol/L [14C]-L-leucine in the absence and in the presence of increasing concentrations of the unlabeled substrate (0.3–30 µmol/L). In one set of experiments, [14C]-L-leucine accumulation was compared in (aminooxy)acetic acid (AOA)-treated and untreated cells to evaluate the contribution of transport and metabolism to [14C]-L-leucine accumulation (13
). The transaminase inhibitor AOA (2.5 mmol/L) was added to the culture medium 15 min before the beginning of the transport experiment. The uptake of [14C]-L-leucine was then measured after 6 min, as described above. In experiments performed in the presence of different concentrations of sodium, sodium chloride was replaced by an equimolar concentration of choline chloride. In inhibition studies, test substances were applied from the apical side and were present during the incubation period only. During preincubation and incubation, the cells were continuously shaken and maintained at 37°C. Uptake was terminated by the rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette followed by a rapid wash with cold Hanks’ medium and the addition of 250 µL of 0.1% v/v Triton X-100 (dissolved in 5 mmol/L Tris · HCl, pH 7.4). Radioactivity was measured by liquid scintillation counting.
Fractional outflow of intracellular [14C] -L-leucine was evaluated in cells loaded with 0.25 µmol/L [14C]-L-leucine for 6 min and then the corresponding efflux monitored over 24 min, in the absence and the presence of different amino acids. Fractional outflow was calculated using the following expression: [14C] -L-leucinefluid/([14C] -L-leucinefluid + [14C] -L-leucinecell), where [14C] -L-leucinefluid indicates the amount of [14C]-L-leucine (in pmol/mg protein) that reached the fluid bathing the apical cell side and [14C] -L-leucinecell (in pmol/mg protein) indicates the amount of [14C]-L-leucine accumulated in the cell monolayer.
Protein assay.
The protein content of cell monolayers was determined by the method of Bradford (14
), with human serum albumin as a standard.
Cell viability.
Cells were preincubated for 30 min at 37°C and then incubated in the absence or the presence of L-leucine and test compounds for a further 6 min. Subsequently, the cells were incubated at 37°C for 2 min with trypan blue (0.2% wt/v) in phosphate buffer. Incubation was stopped by rinsing the cells twice with Hanks’ medium and the cells were examined using a Leica microscope. Under these conditions, >95% of the cells excluded the dye.
Data analysis.
Km and Vmax values for the uptake of [14C]-L-leucine, as determined from a competitive uptake inhibition protocol (15
), were calculated from nonlinear regression analysis using the GraphPad Prism statistics software package (16
). Arithmetic means are given with SEM. Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test for multiple comparisons or Student’s t test for comparisons between Caco-2 and IEC-6 cells. A P-value < 0.05 was assumed to denote a significant difference.
Chemicals.
The L- and D-amino acids, 2-aminobicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), N-(methylamino)-isobutyric acid (MeAIB), AOA and trypan blue were purchased from Sigma Chemical, St. Louis, MO. [14C] -L-leucine, specific activity 11.2 GBq/mmol, was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). At 6 min, when uptake was linear and considering intracellular water as 7.0 ± 0.7 µL/mg protein (17
), the intracellular [14C]-L-leucine concentration was 5.3 ± 0.5 and 2.3 ± 0.1 µmol/L in IEC-6 and Caco-2 cells, respectively, at a medium concentration of 0.25 µmol/L. This represented a cell concentration of [14C]-L-leucine that was 20 and 8 times higher in IEC-6 and Caco-2 cells, respectively, than the corresponding medium concentration. The accumulation of [14C]-L-leucine [in pmol/(mg protein · 6min)] was not affected by pretreatment with the transamination inhibitor AOA (Caco-2 cells, 19.7 ± 0.6 vs. 18.1 ± 0.5; IEC-6 cells, 42.1 ± 1.2 vs. 44.8 ± 1.3). In a subsequent set of experiments designed to determine the kinetics of the L-type amino acid transporter, cells were incubated for 6 min with [14C]-L-leucine (0.25 µmol/L) in the absence or presence of increasing concentrations (0.3–30 µmol/L) of unlabeled L-leucine (Fig. 2
). Kinetic parameters of [14C] -L-leucine uptake (Km and Vmax) were determined by nonlinear analysis of the specific analysis of inhibition curve for L-leucine and are given in Table 1
. The affinity of the transporter for L-leucine in Caco-2 cells was similar to that in IEC-6 cells, as evidenced by similar Km.
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). The [14C] -L-leucine uptake into Caco-2 was inhibited by glycine and L-isomers of the neutral amino acids and histidine and lysine. Proline, the acidic amino acids aspartate and glutamate, and the basic amino acids arginine and cystine did not inhibit [14C]-L-leucine uptake into Caco-2 cells. The profile of inhibition of [14C]-L-leucine uptake into IEC-6 cells was similar to that observed in Caco-2 cells, with the exception that glycine, L-alanine and L-lysine were ineffective in reducing substrate accumulation. In both Caco-2 and IEC-6 cells, L-aspartate produced a slight (P = 0.07) increase in [14C]-L-leucine accumulation. The inhibitory effect of D-amino acids on [14C] -L-leucine uptake was less marked than that obtained with L-isomers (Fig. 4
). Irrespective of their optical conformation, the most effective neutral amino acids in reducing the uptake of [14C] -L-leucine uptake into both Caco-2 and IEC-6 cells were isoleucine, phenylalanine, methionine, tyrosine and tryptophan.
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). Altogether, these results indicate that uptake of L-leucine in both types of cells may be promoted through the L-type amino acid transporter. To confirm this view, the next series of experiments studied the effect of MeAIB and 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH), inhibitors of the alanine preferring and L-type amino acid transporters, respectively. BCH, but not MeAIB markedly inhibited [14C]-L-leucine accumulation in both cell types (Fig. 6
). Taken together, these results suggest that the inward transfer of [14C] -L-leucine in both Caco-2 and IEC-6 cells may be promoted largely through the BCH-sensitive and sodium-independent L-type amino acid transporter.
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).
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25% of the amount of [14C]-L-leucine accumulated in the cells, i.e., both cell types were able to retain most of the substrate taken up (Fig. 8
). When the cells were incubated with 1 mmol/L L-leucine, the efflux of [14C]-L-leucine was markedly (P < 0.05) increased. This is in agreement with the view that the L-leucine transporter functions as an exchanger. In another series of experiments, the [14C]-L-leucine efflux was monitored for 24 min in the absence and the presence of 1 mmol/L BCH or 1 mmol/L L-arginine. BCH stimulated the efflux of [14C]-L-leucine in both Caco-2 and IEC-6 cells, with potency less (P < 0.05) than that of L-leucine (Fig. 8
). On the other hand, L-arginine did not stimulate the efflux of [14C]-L-leucine in either cell line. In contrast with that observed for the inward transport of [14C] -L-leucine, the spontaneous and L-leucine-stimulated outward transfer of [14C]-L-leucine was sodium independent in both Caco-2 and IEC-6 cells (Fig. 9
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although most of [14C]-L-leucine was entering the cells in a sodium-independent manner, a minor component of [14C]-L-leucine uptake (15%) required extracellular sodium. Apart the sodium-sensitive [14C]-L-leucine transporter, the major component of [14C]-L-leucine uptake into Caco-2 and IEC-6 cells was sodium independent, sensitive to BCH, but not to MeAIB, sensitive to neutral, but not acidic and basic amino acids and showed transstimulation by unlabeled leucine. Taken together, these results agree with the suggestion that the amino acid systems that may be responsible for [14C]-L-leucine transport include system B0 and system L. The sensitivity of [14C] -L-leucine uptake to BCH, but not to MeAIB, supports the view that [14C]-L-leucine inward transfer in Caco-2 and IEC-6 cells is not promoted by either the A- or the ASC-type amino acid transporter. System B0 is a broad-specificity amino acid transport system cotransporting neutral amino acids with sodium into cells that also accept BCH but not MeAIB (18
). System B0,+ is also a sodium-dependent transporter that has a broad-specificity for zwitterionic and basic amino acids and also accepts BCH but not MeAIB (18
). The uptake of [14C]-L-leucine was inhibited by neutral amino acids such as phenylalanine, leucine and tyrosine and blocked by BCH, but not by MeAIB and the acidic and basic amino acids. For this reason, it is likely that system B0 rather than system B0,+ might be responsible for the sodium-dependent uptake of [14C]-L-leucine into Caco-2 and IEC-6 cells. Because these particular experiments were performed in the presence of sodium, it is likely that system B0,+ and/or system L might be responsible for this BCH-sensitive component of L-leucine uptake. System L transports neutral amino acids with high affinity (Km in the micromolar range) with no need for sodium in the extracellular medium and also shows very high capacity for transstimulation (18
). The finding that accumulation of [14C]-L-leucine in Caco-2 and IEC-6 cells was insensitive to pH variations fits well with the view that L-leucine uptake in these cells is promoted through system L, namely, the L-type amino acid transporter (LAT)1. In fact, the activity of the mammalian system L in various cell types and tissues has been reported to be independent of extracellular pH (19
).
The results of [14C]-L-leucine efflux studies in Caco-2 and IEC-6 cells are also quite valuable to define the nature of transporters involved in the handling of L-leucine. In fact, system L has been divided into subtypes LAT1 and LAT2, and both transporters induce transstimulable transport of neutral amino acids in a sodium-independent manner. This agrees with the finding that spontaneous and L-leucine–stimulated outward transport of [14C]-L-leucine in both Caco-2 and IEC-6 cells was similar in the presence and absence of extracellular sodium. Systems LAT1 and LAT2 function as exchangers (20
–22
) and leucine-induced efflux of [14C]-L-leucine agrees with the view that either transporter may participate in the exchange of [14C]-L-leucine. Because most of the [14C]-L-leucine did not leak out of the cells during the 24-min incubation in amino acid–free buffer and measurements of efflux in the absence of extracellular amino acids did not show a consistent efflux, the results suggest that [14C]-L-leucine transporters function as exchangers. LAT2-mediated leucine transport is accelerated by lowering the pH (22
), whereas LAT1-mediated transport is reported not to be influenced by pH (19
). Although IEC-6 cells showed some pH dependency in [14C]-L-leucine accumulation, that occurred only at acidic pH. Accordingly, Caco-2 and IEC-6 cells may transport [14C]-L-leucine through the pH-independent LAT1 transporter. Another result that agrees with this suggestion is that some substrates, particularly leucine, show lower affinity for LAT2 than for LAT1 (23
), which is consistent with the low Km values observed for L-leucine in both cell lines. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine (19
). This conflicts with the view that the sodium- and pH-independent L-type amino acid transporter in Caco-2 and IEC-6 cells may correspond to LAT1. On the other hand, the mRNA corresponding to LAT2 examined by Northern blot analysis was strongly expressed in the small intestine (21
, 22
).
We conclude that L-leucine in Caco-2 and IEC-6 cells is transported quite efficiently through the apical cell border, and several findings indicate that this uptake process is a facilitated mechanism involving sodium-dependent and sodium-independent transporters. It is likely that system B0 might be responsible for the sodium-dependent uptake of L-leucine. Transporters involved in sodium-independent uptake of L-leucine may include system LAT1, which also functions as an exchanger whose activation results in transstimulation of L-leucine outward transfer.
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FOOTNOTES |
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3 Abbreviations used: AOA, (aminooxy)acetic acid; BCH, 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid; FBS, fetal bovine serum; LAT, L-type amino acid transporter; L-DOPA, L-3,4-dihydroxyphenylalanine; MeAIB, N-(methylamino)-isobutyric acid.
Manuscript received 22 August 2001. Initial review completed 21 October 2001. Revision accepted 12 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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