1 Institut National de la Recherche Agronomique, Laboratoire de Nutrition Humaine et Physiologie Intestinale, Institut National Agronomique Paris-Grignon, 75231 Paris Cedex 05; and 2 Laboratoire d'Histologie-Embryologie, Faculté de Médecine Cochin, 75014 Paris, France
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ABSTRACT |
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Little is known
concerning the expression of amino acid transporters during intestinal
epithelial cell differentiation. The transport mechanism of
L-glutamate and its regulation during the differentiation
process were investigated using the human intestinal Caco-2 cell line.
Kinetic studies demonstrated the presence of a single, high-affinity,
D-aspartate-sensitive L-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This
transport was clearly Na+ dependent, with a Hill
coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1
glutamate stoichiometry and corresponding to the well-characterized XA,G system. The excitatory amino acid transporter
(EAAT)1 transcript was consistently expressed in the Caco-2 cell line,
whereas the epithelial and neuronal EAAT3 transporter was barely
detected. In contrast with systems B0 and y+,
which have previously been reported to be downregulated when Caco-2
cells stop proliferating, L-glutamate transport capacity was found to increase steadily between day 8 and day
17. This increase was correlated with the level of EAAT1 mRNA,
which might reflect an increase in EAAT1 gene transcription and/or
stabilization of the EAAT1 transcript.
glutamate transport; intestinal epithelium
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INTRODUCTION |
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ENTEROCYTE DIFFERENTIATION is associated with increased expression of the enzymes and transporters involved in the digestive and absorptive functions of the intestinal epithelium. Epithelial cell lines undergoing differentiation and mimicking enterocyte differentiation represent unique tools for the analysis of these functions during enterocyte differentiation (25, 30). The Caco-2 cell line, which undergoes spontaneous enterocytic differentiation, is well recognized as being the most relevant in vitro model to investigate the impact of differentiation on the absorptive functions of epithelial cells (30). In this regard, the uptake of a variety of ions and organic nutrients including hexoses, bile acids, dipeptides, inorganic phosphate, and vitamin B12 by Caco-2 cells resembled that which occurs in the small intestinal epithelium (3, 7, 11, 18, 34). This model and others have helped us to gain an understanding of the acquisition of hydrolase activities during epithelial cell differentiation (25). In contrast, few data are currently available regarding the expression of amino acid transporters, primarily because of the overlapping specificity of these transporters and a lack of molecular characterization (19).
The dicarboxylic amino acid L-glutamate is mainly involved
in energy metabolism and thus plays a key role in epithelial cell physiology. Five L-glutamate transporters mediating
L-glutamate uptake through an XA,G
transport system have recently been identified in the central nervous
system and cloned from a human cDNA library (1,
2, 9). These proteins exhibit similar
functional characteristics and constitute the five subtypes of the
excitatory amino acid transporter (EAAT) family. The expression of
EAAT2, EAAT4, and EAAT5 is mainly restricted to the brain and the
retina, whereas sites in which the expression of EAAT1 and EAAT3
transcripts has been reported include heart, lung, liver, kidney,
placenta, and small intestine (1, 12).
Whether the effects associated with the enterocyte differentiation
extend to the transport of L-glutamate has not so far been investigated.
In this study, we addressed the transport of L-glutamate in Caco-2 cells and its regulation during the enterocytic differentiation process. Our results show an increased transport of L-glutamate in differentiated Caco-2 cells. Furthermore, the cell line expresses the messenger for EAAT1, whereas EAAT3 mRNA are barely detectable. Interestingly, we have observed an increased expression of EAAT1 in differentiated Caco-2 cells, suggesting that the increased transport of L-glutamate in differentiated Caco-2 cells is related to increased expression of the EAAT1 transporter.
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EXPERIMENTAL PROCEDURES |
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Cell Culture
The Caco-2 cell line was kindly provided by Dr. G. Trugnan (INSERM U410, Paris, France) and was used between passages 50 and 79. Cells were seeded at 4 × 104 cells/cm and grown as monolayer cultures in high-glucose Dulbecco's modified Eagle's medium (Life Technologies, Cergy-Pontoise, France) supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 6 mM L-glutamine, and 200 µg/ml gentamicin. The medium was replaced every 2 days. Confluence was reached on days 7-8 after seeding. Cells were then subcultured using 0.05% trypsin in 0.02% EDTA.Transport Measurements
The uptake of L-glutamate was measured in 24-well plates for 2 min at 37°C using a cluster-tray technique (5). The culture medium was renewed to feed cells 3 h before the transport assays started. Cells were then prepared for transport experiments by incubation of the monolayer for 15 min in HEPES-saline buffer (HSB) containing (in mM) 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1 MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with Tris. This buffer was then discarded, and L-glutamate transport was initiated by adding 1 ml of HSB containing 0.01-2 mM L-glutamate trace-labeled with 9.25 kBq of L-[G-3H]glutamate (1.8 TBq/mmol; Amersham, Les Ulis, France). Uptake was terminated by removing the transport medium and washing the cells with 3 × 1 ml of ice-cold HSB containing an excess of unlabeled L-glutamate. The cells were harvested in 500 µl of 0.1 N NaOH, and the cell-associated radioactivity was determined by liquid scintillation counting. In selected experiments, Na+-independent L-glutamate transport was evaluated. For this purpose, NaH2PO4 was omitted from the HSB, and NaCl was replaced by choline chloride. Na+-dependent L-glutamate transport was calculated as the difference between the transport measured in Na+-containing and Na+-free buffers. For competition experiments, amino acids were added to the transport medium at a final concentration of 100 µM. In one experiment, L-glutamate accumulation was compared in (aminooxy)acetate-treated and nontreated Caco-2 cells to evaluate the contribution of transport and metabolism to L-glutamate accumulation. The transaminase inhibitor (aminooxy)acetate (final concentration 2.5 mM) was added in the culture medium 15 min before the beginning of the transport experiment. The uptake of L-glutamate was then measured after 5 min, as described above. All the results presented were corrected for the accumulation of L-glutamate in adherent extracellular fluids, as previously described (16). The protein contents of wells were determined using the method developed by Smith et al. (32), and results were expressed as picomoles of L-glutamate transported per milligram of cell proteins.RNA Extraction
Total RNA from Caco-2 cells seeded in six-well plates was extracted using TRI InstaPure reagent (Eurogentec, Seraing, Belgium). One milliliter of TRI InstaPure was added per ten square centimeters, and the cell lysates were homogenized by repeated pipetting. RNA extraction was performed according to the manufacturer's specifications, and the RNA was resuspended in RNase-free water. The control and normalization of RNA samples was achieved using densitometry analysis of the ribosomal 28S and 18S on an ethidium bromide-stained agarose gel and optical density quantification.RT-PCR
For RT-PCR assays, the EZ rTth RNA PCR kit (Perkin Elmer, Branchburg, NJ) was used with primers chosen from the cDNA sequence of the human EAAT1 and EAAT3 transporters identified by Arriza et al. (Ref. 1; Table 1). Before amplification, total RNA samples were treated with deoxyribonuclease I, amplification grade (Life Technologies, Cergy-Pontoise, France), at 1 U/µg RNA for 15 min at room temperature. Inactivation of the enzyme was achieved by the addition of 1 µl of 20 mM EDTA and heat treatment at 65°C for 10 min. Amplification products were analyzed on a 5% polyacrylamide nondenaturing gel stained with ethidium bromide and photographed under ultraviolet light.
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Riboprobe Synthesis
Two different approaches were used to design riboprobes for the ribonuclease protection assay (RPA). The EAAT3 probe was constructed using classical cloning in a plasmid, whereas the EAAT1 probe was directly engineered by RT-PCR using a modified primer containing the T7 promoter.EAAT1 riboprobe. A 5'-EAAT1 primer and a T7-modified 3'-EAAT1 primer (Table 1) were used for RT-PCR, with RNA from Caco-2 cells as a template. The PCR product (200 bp = 182 bp corresponding to the transcript + 18 bp corresponding to the T7 promoter) was used directly for the T7 RNA polymerase transcription to synthesize the biotin-labeled EAAT1 riboprobe using biotin-14-CTP/NTP and BrightStar BIOTINscript kit (Ambion, Austin, TX).
EAAT3 riboprobe.
RT-PCR was carried out using Caco-2 RNA and EAAT3 primers (Table 1).
The RT-PCR product was inserted in the pPCR-Script Amp vector (kit no.
211188, Stratagene, La Jolla, CA). The cloned EAAT3 fragment (208 bp)
was sequenced with M13 and reverse-M13 primers and IsoBlue-stabilized
-35S-dATP (37 TBq/mmol; ICN, Costa Mesa, CA; Sequenase
7-deaza-dGTP DNA sequencing kit no. 70990, US Biochemical, Cleveland,
OH) to determine which of the T3 or T7 promoters of the plasmid was to be used for the antisense riboprobe (data not shown). The plasmid was
cut with Fok I (New England Biolabs, Beverly, MA), which
left a 179-bp antisense fragment plus 84 bp of the vector after the transcription reaction. After digestion with the restriction enzyme, the template was purified using the Magic DNA clean-up system (Promega,
Madison, WI). The antisense EAAT3 riboprobe was synthesized using the
same method as that used for the EAAT1 probe (BrightStar BIOTINscript
kit, Ambion).
Ribonuclease Protection Assay
RPA was performed as described in the RPA II kit (Ambion). Total Caco-2 RNA (25-100 µg) was hybridized with 1,250 pg of the biotinylated riboprobe (EAAT1 or EAAT3) at 45°C overnight, and digestion was ensured with the 1/1,000 diluted RNase A-RNase T1 mixture of the kit for 30 min at 37°C. The protected fragments were separated on a 5% denaturing polyacrylamide gel. After electroblotting on a nylon membrane (Ambion) the detection procedure was carried out using the BrightStar Biodetect kit (Ambion), and the membrane was exposed with both Kodak X-OMAT AR radiography film (1 h) and Ilford photographic paper (overnight). The scanning of areas was achieved using NIH Image 1.60.Enzymatic Activities
All enzyme activities were measured spectrophotometrically by recording the appearance of the reaction product as a function of time. Sucrase activity was determined according to Dahlqvist (4). Alkaline phosphatase activity was assessed according to the Eichholz technique (8) using p-nitrophenyl phosphate as substrate.Calculations and Statistics
All experiments were conducted in triplicate, and all experiments were confirmed using at least two independently seeded generations of cells. Results are expressed as means ± SE; statistical comparisons were made using the Tukey's studentized range test (GLM procedure, SAS 6.03, SAS Institute, Cary, NC). Transport kinetic parameters were obtained by fitting data to the Michaelis-Menten equation or to a linear model (NLIN and REG procedures, SAS 6.03). ![]() |
RESULTS |
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Characterization Of L-Glutamate Transport in Confluent Caco-2 Cells
The uptake of L-glutamate was measured on Caco-2 cells cultured in plastic dishes on day 8 after seeding. In the absence of extracellular Na+, there was no detectable uptake of L-glutamate after correction of the data for accumulation in extracellular adherent fluids (data not shown). In the presence of 137 mM NaCl, the rate of L-glutamate accumulation was almost constant over a 10-min period and slightly decreased thereafter (Fig. 1). The rate of glutamate accumulation for the first 5 min was not affected by cell pretreatment with the transamination inhibitor (aminooxy)acetate [uptake rate = 92 ± 6 and 87 ± 10 pmol · mg protein
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Transport of L-Glutamate During Caco-2 Cell Differentiation
Dramatic changes have been shown to occur during Caco-2 cell proliferation and differentiation. The rate of glutamate uptake was measured in Caco-2 cells between day 2 and day 22 after seeding (Fig. 4). On reaching confluence, Caco-2 cells underwent differentiation as assessed by the sharp increase in alkaline phosphatase and sucrase observed between day 8 and day 17 after seeding. During that period, a dramatic increase (+160%) was also seen in the rate of L-glutamate uptake. At any time after seeding, the inhibition of 10 µM L-glutamate uptake by 100 µM D-aspartate was similar to that measured on confluent cells (
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EAAT1 and EAAT3 Expression During Caco-2 Cell Culture
With RT-PCR, both EAAT1 and EAAT3 mRNA were seen to be present in the total RNA of day 17 Caco-2 cells (Fig. 6). The expression levels of EAAT1 and EAAT3 mRNA were then measured in Caco-2 cells at different ages (2, 6, 10, 14, 17, and 21 days after seeding) by RPA using 25 µg of total RNA. On day 2 and day 6 after seeding, the level of the EAAT1 transcript was below the detection limit. However, EAAT1 mRNA was clearly detected on day 10, and its level steadily increased between day 10 and day 17 (Fig. 7). Densitometric analysis indicated a fivefold increase in EAAT1 transcript abundance between day 10 and day 21. Under the same conditions, we failed to detect any EAAT3 transcript on any day after seeding (data not shown). With a larger amount of total RNA (100 µg) in the RPA, a faint signal from the protected fragment corresponding to the EAAT3 transcript was observed on day 17 after seeding (Fig. 8).
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DISCUSSION |
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Although several studies have focused on the uptake of dipolar and
cationic amino acids in the Caco-2 cell line and suggested a decrease
in the transport capacity through systems B0 and
y+ during enterocytic differentiation (22,
23), the transport of anionic amino acids and its
evolution during epithelial cell maturation have received little
attention. Our results demonstrate that 1)
L-glutamate uptake occurs through a single, high-affinity Na+-dependent component that exhibits the functional
characteristics of the XA,G system; 2)
expression of the XA,G
-like system is modulated
during epithelial cell differentiation, with a steep increase in
Vmax occurring when cells cease to proliferate and undergo enterocytic differentiation; 3)
L-glutamate uptake in the Caco-2 cell line is associated
with expression of the EAAT1 transporter subtype; and 4)
enhanced L-glutamate transport in differentiated Caco-2
cells is correlated with increased levels of EAAT1 transcripts.
Our results indicate that the uptake of L-glutamate in the Caco-2 cell line involved a single, high-affinity Na+-dependent transport system. Assuming a cell volume of 3.7 µl/mg protein (3, 6), a 12-fold ratio between the intracellular and extracellular glutamate concentrations was achieved after 5 min. This high ratio did not reflect glutamate metabolism but was the consequence of a true accumulative transport process, as reflected by comparison of the rate of uptake measured in (aminooxy)acetate-treated and nontreated Caco-2 cells. In contrast to our previous observation in the rat IEC-17 intestinal cell line (17), we have found no evidence for the existence of Na+-independent L-glutamate transport or for a low-affinity Na+-dependent transport component. Striking differences have already been reported between these two intestinal epithelial cell lines concerning amino acid transport: the Caco-2 cell line was shown to exhibit a high transport capacity for dipolar amino acids through the B0 transport system soon after passaging, whereas we found no evidence for the presence of this epithelium-specific transport system in the IEC-17 cell line (16). The IEC-17 intestinal cell line exhibits many of the characteristics of undifferentiated intestinal crypt cells (26). It may represent a much earlier stage in the differentiation process than preconfluent Caco-2 cells and thus may express transport proteins that are lost during the first steps of enterocytic transformation. Alternatively, interspecies differences may account for these discrepancies, because the presence of low-affinity Na+-dependent glutamate transport has also been reported in rat small intestine (24), whereas the high-affinity, Na+-dependent component accounts for all glutamate uptake in the human jejunal brush-border membrane (27).
We characterized the specificity, kinetic properties, and stoichiometry
of L-glutamate uptake in the Caco-2 cell line. In agreement
with the results reported by Nicklin et al. (21), we
observed that L-glutamate uptake in the Caco-2 cell line
was inhibited by L- as well as D-aspartate and
was unaffected by D-glutamate and neutral and cationic
amino acids. The Km of the transport reaction in
our study was similar to that determined in human brush-border membrane
vesicles (90 µM) (27), being of the same magnitude as
that reported by Nicklin et al. (21) for transepithelial fluxes across Caco-2 cell monolayers (65.0 µM). The Hill coefficient of 2.9 calculated from the measurement of glutamate uptake in the
presence of Na+ concentrations ranging from 0 to 140 mM
suggests a Na+-glutamate stoichiometry of 3:1, this being
consistent with the stoichiometry reported for EAAT1 and EAAT3
transporters responsible for the expression of the
XA,G transport system (14,
15). Together, these characteristics indicate that
glutamate transport occurs through the widespread XA,G
system.
The term "transport system" is used to designate a physiological
membrane functional process that may be catalyzed by different polypeptides or "transporters." Five different genes encoding five
polypeptides that could mediate glutamate uptake through an
XA,G transport system have been cloned, and two of
them, EAAT1 and EAAT3, are expressed in peripheral organs. With the use
of transient expression in COS cells, it has been shown that EAAT1- and
EAAT3-mediated L-glutamate uptake exhibit similar
inhibition profiles and kinetic characteristics (1). The
Km calculated for the uptake of
L-glutamate by either of these two proteins was similar to
that determined in our experiments (48 and 62 µM for EAAT1 and EAAT3,
respectively), and the Km calculated in the
D-aspartate transport was similar for the two proteins
(Km = 60 and 47 µM for EAAT1 and EAAT3,
respectively). It is therefore difficult to determine which of these
two proteins is responsible for glutamate transport solely on the basis
of functional experiments. Using RT-PCR, we detected the presence of
both EAAT1 and EAAT3 transcripts in the Caco-2 cell line. However, the
EAAT3 transcript was barely detectable when we used the highly specific
and sensitive ribonuclease protection assay, whereas EAAT1 was still
clearly expressed in this cell line. The presence of the EAAT1
transcript and the weak expression of EAAT3 in Caco-2 cells were quite
unexpected. The latter protein has been cloned in humans by Arriza et
al. (1) and Kanai and Hediger (12) (hEAAC1
protein), and the expression of hEAAC1 mRNA has been demonstrated in
human intestine (13). In contrast, our results
constitute the first report of the presence of the EAAT1 glutamate
transporter in an intestinal epithelial cell line. The reasons for
these unexpected features may arise from the karyotypic specificity of
the Caco-2 cell line. This cell line was established from a
colon carcinoma and has been shown to be hypertetraploid, with the loss
of normal chromosomes 9 and 21 (10). The chromosomal loci
for the EAAT1 and EAAT3 genes have recently been established as
5p11-p12 and 9p24, respectively (31,
33). Reassignment of the EAAT3 gene to a poorly
transcripted region during chromosomal alteration may result in a
drastic reduction of EAAT3 expression. On the other hand, it should be
borne in mind that Caco-2 cells have a colonic origin and may exhibit
certain differences with enterocytes, including the expression of
different transporter subtypes.
The third important result of our study was the demonstration of a
2.5-fold increase in the glutamate transport rate during Caco-2 cell
differentiation. The increase in L-glutamate transport capacity (Vmax) probably results from an
increase in the amount of active transporter in the brush-border
membrane rather than from the expression of a different transporter
subtype with a different affinity, because Km
did not vary during differentiation. The magnitude of
L-glutamate uptake was correlated to the amount of EAAT1
transcripts in postconfluent cells, suggesting that glutamate transport upregulation may result from an increase in gene
transcription or the stabilization of EAAT1 mRNA during cell
differentiation. Contrasting with our results concerning glutamate, the
uptake of cationic and neutral amino acids through systems
y+ and B0, respectively, was previously shown
to be downregulated when Caco-2 cells cease to proliferate and undergo
differentiation (22, 23). Both systems serve
the uptake of essential amino acids, the requirement for which depends
on protein synthesis rate and may thus decrease as cells cease to
proliferate. In contrast, the physiological meaning of upregulation of
system XA,G after confluence is more speculative.
L-Glutamine and L-glutamate are major metabolic
fuels of the enterocyte (20, 28), and the
increase in L-glutamate transport may compensate for
decreased L-glutamine uptake through system B0.
The rise in glutamate uptake may also aid postmitotic Caco-2 cells in
maintaining adequate glutathione concentrations and thus resist
oxidative aggressions, because it has recently been demonstrated that
dietary glutamate (but not de novo synthesized glutamate) is used for
glutathione synthesis in the intestinal epithelium (29).
In conclusion, this study provides the first demonstration of the involvement of EAAT1 in L-glutamate transport in the Caco-2 intestinal epithelial cell line and its upregulation during the enterocytic differentiation process. This change may reflect the gradual settling of transporters involved in the vectorial transport of amino acids across the intestinal mucosa as well as modifications to epithelial cell amino acid requirements in relation to their differentiation. Further investigations are needed to explore these hypotheses.
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ACKNOWLEDGEMENTS |
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Critical reading of the manuscript by P. N. Boyaka is gratefully acknowledged.
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FOOTNOTES |
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A. Mordrelle was supported by Grant no. 94231 from the French Department of Higher Education and Research.
Address for reprint requests and other correspondence: J.-F. Huneau, INRA, Laboratoire de Nutrition Humaine et Physiologie Intestinale, Institut National Agronomique Paris-Grignon, 16 rue Claude Bernard, 75231 Paris Cedex 05, France (E-mail: huneau{at}inapg.inra.fr).
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. §1734 solely to indicate this fact.
Received 9 September 1999; accepted in final form 24 March 2000.
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