Up-regulation and Polarized Expression of the Sodium-Ascorbic
Acid Transporter SVCT1 in Post-confluent Differentiated CaCo-2
Cells*
Nancy P.
Maulén
§,
Esther A.
Henríquez
,
Sybille
Kempe
,
Juan G.
Cárcamo
¶,
Alexandra
Schmid-Kotsas
,
Max
Bachem
,
Adolph
Grünert
,
Marcelo E.
Bustamante§,
Francisco
Nualart**, and
Juan Carlos
Vera

From the
Departamento de
Fisiopatología, the ** Departamento de
Histología y Embriología, Facultad de Ciencias
Biológicas, Universidad de Concepción, Barrio Universitario
S/N, Concepción, Chile, the § Departamento de
Biología Molecular, Facultad de Medicina, Universidad
Católica de la Santísima Concepción, Alonso de
Ribera 2850, Concepción, Chile, ¶ Instituto de
Bioquímica, Facultad de Ciencias, Universidad Austral de Chile,
Campus Isla Teja, Valdivia, Chile, and
Institut of Clinical
Chemistry, Faculty of Medicine, University of Ulm, 8907 Ulm, Germany
Received for publication, May 24, 2002, and in revised form, October 10, 2002
 |
ABSTRACT |
Human cells acquire vitamin C using two different
transporter systems, the sodium-ascorbic acid co-transporters with
specificity for ascorbic acid, and the facilitative glucose
transporters with specificity for dehydroascorbic acid. There is no
information on the mechanism of vitamin C transport across the
intestinal barrier, a step that determines the bioavailability of
vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like
cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2
cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the
dehydroascorbic acid transporters GLUT1 and GLUT3, and a third
dehydroascorbic acid transporter with properties expected for GLUT2.
Analysis by real time quantitative PCR revealed that the post-confluent
differentiation of CaCo-2 cells was accompanied by a marked increase
(4-fold) in the steady-state level of SVCT1 mRNA, without changes
in SVCT2 mRNA levels. Functional studies revealed that the
differentiated cells expressed only one functional ascorbic acid
transporter having properties expected for SVCT1, and transported
ascorbic acid with a Vmax that was increased at least 2-fold compared with pre-confluent cells. Moreover,
post-confluent Caco-2 cells growing as monolayers in permeable filter
inserts showed selective sorting of SVCT1 to the apical membrane
compartment, without functional evidence for the expression of SVCT2.
The identification of SVCT1 as the transporter that allows vectorial
uptake of ascorbic acid in differentiated CaCo-2 cells has a direct
impact on our understanding of the mechanism for vitamin C transport
across the intestinal barrier.
 |
INTRODUCTION |
Vitamin C is an essential micronutrient required for the
maintenance of a normal human physiology. Vitamin C is required for the
synthesis of collagen and carnitine, as a cofactor in enzymes to
maintain metal ions in their reduced form, and to protect tissues from
oxidative damage by scavenging free radicals (1-6). Also, reduced
vitamin C (ascorbic acid) can recycle glutathione and vitamin E, two
important biologic antioxidants. Recent evidence (7) indicates that an
increased intake of vitamin C is associated with a reduced risk of
chronic diseases such as cancer or cardiovascular disease.
Vitamin C exists in two chemically distinct forms in human plasma, the
reduced ascorbate ion form (ascorbic acid
(AA)1 and the oxidized
non-ionic form (dehydroascorbic acid (DHA)). Human cells acquire both
chemical forms of vitamin C by transporting them across the cell
membrane with the participation of two different transporter systems
that show absolute specificity for one or the other vitamin form (2, 8,
9). One transporter system behaves as a low affinity, high capacity
sodium-independent system and includes several members of the
facilitative glucose transporter family (GLUTs) (10-13). These
transporters show a high specificity for oxidized vitamin C and
transport dehydroascorbic acid down a substrate concentration gradient
(14-17). Twelve glucose transporter isoforms have been molecularly
characterized, and there is evidence that the isoforms GLUT1, GLUT3,
and GLUT4 are efficient dehydroascorbic acid transporters. A second
transport system for vitamin C is a high affinity, low capacity
sodium-dependent system (SVCTs) that corresponds to a
recently described family of mammalian sodium-ascorbic acid
co-transporters composed of two members, SVCT1 and SVCT2. These
transporters display high affinity for reduced vitamin C (9,
18-21).
Because humans are not capable of synthesizing vitamin C, it must be
obtained from the diet and then acquired by the different body cells.
Although we know the molecular identities of the vitamin C transporters
expressed in human cells (9, 13, 16, 17, 20-22), there is no
information regarding the identity and functional properties of the
cellular mechanisms that control vitamin C transfer across the
intestinal barrier and the bioavailability of vitamin C in humans.
Supplementation studies in normal volunteers have determined that the
step limiting vitamin C bioavailability in humans lies at the
transcellular transport within the intestine (23). However, we know
very little regarding the most basic aspects of this process, including
how vitamin C enters the apical membrane of the intestinal epithelium,
whether it is processed intracellularly, or how it exits the
basolateral membrane (1, 2, 24-29).
We used the human colon carcinoma cell line CaCo-2 as an in
vitro model for vitamin C transport in enterocyte-like cells. CaCo-2 cells have been extensively used to study the transcellular movement of nutrients, drugs, or metal ions in vitro because
they differentiate in culture, both structurally and functionally, resembling mature enterocytes (30-34). These cells have been
successfully used to study glucose transport regulation and to identify
the molecular components by which glucose crosses the intestinal
barrier. These studies revealed that CaCo-2 cells express several
glucose transporters such as the Na+-glucose co-transporter
SGLT, facilitative glucose transporters GLUT1, GLUT2, and GLUT3, and
the fructose transporter GLUT5. It was also shown that the polarized
distribution of the different transporters at the apical or basolateral
membrane compartments is central to the capacity of these cells to
transport glucose in a vectorial manner, i.e. apical to
basolateral direction (24, 26, 35, 36).
We present data indicating that cultured CaCo-2 cells transport both
reduced and oxidized vitamin C. Transport analysis, together with
competition and inhibition studies and RT-PCR analysis, revealed that
vitamin C uptake by CaCo-2 cells is mediated by two transporter families. We conclude that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with
properties expected for GLUT2. Moreover, our data indicate that SVCT
transporter expression is regulated during in vitro differentiation of CaCo-2 cells. Post-confluent differentiated CaCo-2
cells showed increased expression of SVCT1 at RNA and protein levels,
and this increased expression was accompanied by selective sorting of
SVCT1 to the apical membrane compartment. Our data have a direct impact
on our understanding of the mechanism for vitamin C transport at the
intestinal level, as well as on the vectorial transport of the vitamin
across the intestinal barrier.
 |
EXPERIMENTAL PROCEDURES |
CaCo-2 cells were used at passages 20-60 from stock cells grown
in T75 plastic flasks. They were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1%
nonessential amino acids, 1% L-glutamine,
penicillin/streptomycin, and fungizone. Cells were dispersed by
treatment with 0.25% trypsin, 5 mM EDTA at 80% confluence
(31, 34). Cell viability was always greater than 95% as assessed by
trypan blue exclusion. Tissue culture media, supplements, and tissue
culture reagents were obtained from Invitrogen.
Vitamin C uptake experiments were performed in 6-well plates containing
1 × 106 cells/well. For uptake assays, the cells were
deprived of fetal bovine serum, growth factors, and other media
supplements by replacing the culture media with incubation buffer (15 mM Hepes (pH 7.4), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, and 0.8 mM MgCl2) for 1 h before performing the
assays. Uptake was done in incubation buffer containing labeled
ascorbic acid or dehydroascorbic acid. Ascorbic acid transport assays
were performed in 0.6 ml of incubation buffer containing 0.1-0.4 µCi
of L-[14C]ascorbic acid (specific activity
8.2 mCi/mmol, PerkinElmer Life Sciences) at a final concentration of
5-500 µM ascorbic acid (Sigma) in the presence of 0.1 mM dithiothreitol. For dehydroascorbic acid uptake, 1-10
units of ascorbic acid oxidase (50 units/mg protein, Sigma) was added
to the ascorbic acid mixture and incubated for 5 min at 37 °C before
adding it to the cells at a final concentration of 0.5-12
mM. Uptake was finished by adding 5 volumes of cold stopping solution (NaCl 160 mM, KCl 5 mM,
MgSO4 0.8 mM, CaCl2 1.8 mM, HgCl2 0.2 mM) (37). The cells
were washed twice with cold phosphate-buffered saline (pH 7.4) and
lysed in 300 µl of 10 mM Tris-HCl (pH 8.0) containing
0.2% SDS, and the incorporated radioactivity was determined by liquid
scintillation counting (38). Transport in the absence of sodium ions
was accomplished by replacing the NaCl in the incubation media with 135 mM choline chloride (Sigma). When appropriate, competitors
(deoxyglucose, fructose, sucrose, and
-methyl-D-glucopyranoside) and inhibitors (phloretin or
genistein) were added to the uptake assays simultaneously with the
transported substrate, or the cells were preincubated in their presence
prior to the uptake assay (cytochalasin B and cytochalasin E). Hexose
uptake assays were similarly performed using 1 µCi of
2-[1,2-3H]deoxy-D-glucose (specific activity
26.2 Ci/mmol, PerkinElmer Life Sciences) and 0.1-50 mM
2-deoxy-D-glucose (deoxyglucose) or 1 µCi of
3-O-[methyl-3H]D-glucose
(specific activity 10 Ci/mmol, PerkinElmer Life Sciences) and 0.1-50
mM 3-O-methyl-D-glucose
(methylglucose). Time course experiments measuring the transport of
methylglucose, a nonmetabolizable glucose analog, showed that the rate
of transport for 1 mM substrate was linear for the first
60 s, with an equilibrium being reached in about 30 min. This
allowed us to estimate an intracellular water-exchangeable volume of 2 µl/106 cells.
For RT-PCR, CaCo-2 total mRNA was isolated from cells growing in
monolayers (~5 × 106 cells) with the Micro Poly(A)
PureTM kit (Ambion) according to the manufacturer's
instructions. Total human brain RNA was obtained from
Clontech. For both human brain and CaCo-2 cells,
cDNA synthesis was performed by RT-PCR amplification using the
AdvantageTM RT kit (Clontech) following
the manufacturer's instructions. Expression of hSVCT1 and hSVCT2
transporters in CaCo-2 cells and total human brain was verified by PCR.
Oligonucleotide primer pairs for SVCT1 (forward primer, Fsvct1-733,
5'-ACTCTCCTCCGCATCCAGAT-3'; reverse primer, Rsvct1-1017,
5'-CCAGGCGGGCACAGGCGTAG-3') and SVCT2 (Fsvct2-1025,
5'-AGTATGGCTTCTATGCTCGC-3'; Rsvct2-1464, 5'-TTCCGGATCCTGTGCTGGA-3'; or
Fsvct2-733, 5'-TTGACCATTACACCCACGGT-3'; and Rsvct2-1020,
5'-CATAGAAGCCATACTTTGTG-3') were designed based on
GenBankTM sequence accession numbers AJ269477 and AJ269478,
respectively. PCR amplification was done using 4 µl of cDNA
template (diluted 1:100), 0.3 µM of each primer, 1 unit
Taq polymerase (Invitrogen), 1× PCR buffer, 2.5 mM MgCl2 (Invitrogen), 250 µM
dNTP mix (Roche Molecular Biochemicals), and the following set of
reactions: 1) denaturation step, 4 min at 94 °C; 2) 30 cycles of
45 s at 94 °C, 45 s at 55 °C, 1 min 10 s at
72 °C; and 3) 7 min at 72 °C after the last cycle. The PCR
products were separated by agarose gel electrophoresis and visualized
by staining with ethidium bromide. The 304-, 459-, and 307-bp PCR
products were extracted and purified from agarose gels with the QIAX
Kit (Qiagen), cloned in pBluescript II KS (Stratagene), sequenced, and
analyzed by BLAST using the NCBI server at www.ncbi.nlm.nih.gov/.
For quantitative RT-PCR, total RNA was isolated from CaCo-2 cells
cultured in monolayers for 1-20 days (seeded at 3-4 × 105 cells/well in wells coated with collagen from rat
tails) by using the SV Total RNA Isolation SystemTM
(Promega) according to the manufacturer's instructions. cDNA synthesis was done using the 1st strand cDNA synthesis kit for RT-PCR (avian myeloblastosis virus, Roche Molecular Biochemicals) and 1 µg of total RNA using the oligonucleotide primer pairs described above. Each PCR amplification product corresponding to 304 and 307 bp
(for SVCT1 and SVCT2, respectively) was used as the standard for
quantitative RT-PCR. Amplification of
-actin was used as the
internal control. PCR amplification was done with the Light Cycler
(Roche Molecular Biochemicals) using 2 µl of cDNA template, 0.5 µM each primer, 2-3 mM MgCl2
(Invitrogen), and 2 µl of Fast-Start mix (Roche Molecular
Biochemicals) in a final volume of 20 µl under the following reaction
conditions: 1) 10 min at 95 °C; 2) 40 cycles of 5 s at
95 °C, 5-10 s at 65-55 °C (temperature gradient), and 15-20 s
at 72 °C; 3) a final cycle of 5 min at 72 °C. PCR products were
analyzed by 1% agarose gel electrophoresis and stained with SYBR Green (Sigma).
Data are presented as the average ± S.D. and correspond to a
minimum of three assays performed independently in triplicate. Kinetic
parameters were determined using the Michaelis-Menten equation and by
using the linear transformation of Eadie-Hofstee. In experiments that
revealed the presence of more than one kinetic component,
Km and Vmax data were
corrected by using successive iterations (39).
 |
RESULTS |
Time course analysis of vitamin C uptake revealed that CaCo-2
cells take up both reduced and oxidized vitamin C but showed clear
quantitative differences in their capacity to incorporate both forms of
the vitamin (Fig. 1A). The
rate of dehydroascorbic acid incorporation (820 pmol/min per
106 cells) was 10-fold greater than the rate of ascorbic
acid incorporation (80 pmol/min per 106 cells). Uptake
of ascorbic acid was sodium-dependent, as shown by a
greater than 90% decrease in the rate of uptake (to 1.5 pmol/min per 106 cells) when choline chloride replaced NaCl in the
incubation buffer (Fig. 1B). In contrast, the rate of
dehydroascorbic acid was similar in the presence (810 pmol/min per
106 cells) or absence (805 pmol/min per
106 cells) of NaCl (Fig. 1C).

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Fig. 1.
Uptake of vitamin C in CaCo-2 cells.
A, time course of AA ( ) or DHA ( ) uptake.
B, ascorbic acid uptake in the presence ( ) or in the
absence ( ) of NaCl (replaced with choline chloride). C,
dehydroascorbic acid uptake in the presence ( ) or in the absence
( ) of NaCl. CaCo-2 cells were plated in 12-well plates, and uptake
was measured at 24 h. Uptake experiments were performed at
37 °C using 50 µM AA or DHA. Data represent the
mean ± S.D. of experiments performed in triplicate.
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|
A time course analysis of dehydroascorbic acid uptake using substrate
concentrations from 50 µM to 30 mM revealed
that the uptake rate was constant for at least 3 min at each tested
concentration, indicating that these values represent real transport
rates (Fig. 2A). A study of
dehydroascorbic acid transport dependence on the substrate
concentration showed that the rate of transport approached saturation
at 20 mM dehydroascorbic acid (Fig. 2B).
Transport data analysis using the Eadie-Hofstee method revealed the
presence of at least two different functional components, involved in
dehydroascorbic acid transport by CaCo-2 cells, each having different
affinities for the substrate (Fig. 2C). The higher affinity
component had an apparent Km of 0.7 mM
and a Vmax of 15 nmol/min per 106
cells for the transport of dehydroascorbic acid. In comparison, the
lower affinity component had an apparent transport
Km of 4.5 mM and a
Vmax of 30 nmol/min per 106 cells
(Fig. 2C). Competition and inhibition experiments revealed that 50 mM deoxyglucose decreased dehydroascorbic acid
transport by more than 90%, whereas 50 mM sucrose, 50 mM fructose, or 10 mM
-methyl-D-glucoside had no effect. Furthermore, 20 µM cytochalasin B, but not cytochalasin E, inhibited
transport by more than 85% (Fig. 2D). In parallel
experiments, we studied the transport of deoxyglucose. Time course
analysis using deoxyglucose concentrations from 1 to 40 mM
defined a temporal window of 1 min for the transport assays (Fig.
2E). Dose-response analysis (Fig. 2, F-G)
revealed the presence of at least two different kinetic components
involved in deoxyglucose transport by CaCo-2 cells. The higher affinity component had apparent transport Km and
Vmax values of 0.9 mM and 15 nmol/min per 106 cells, respectively. The lower affinity
component had Km and Vmax
values of 23 mM and 90 nmol/min per 106 cells,
respectively. The transport and kinetic data are compatible with
expression of the transporters GLUT3 and GLUT2 in CaCo-2 cells.
Expression of GLUT3 and GLUT2 was confirmed by RT-PCR analysis using
primers specific for the different transporter isoforms and by
immunolocalization experiments using antibodies specific for the
glucose transporter isoforms GLUT1 to GLUT5 (data not shown). These
experiments also revealed that CaCo-2 cells express GLUT1, a
transporter of intermediate affinity, and a closer analysis of the
deoxyglucose transport data reveals the possible presence of a third
functional component (Fig. 2G) that would be consistent with
the presence of GLUT1 in these cells. No such third component is
evident from the dehydroascorbic transport data (Fig. 2C), which is consistent with the similar transport Km of GLUT1 and GLUT3.

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Fig. 2.
Dehydroascorbic acid and deoxyglucose
transport in CaCo-2 cells. A, time course of 1 ( ), 10 ( ), or 30 mM ( ) DHA uptake. B,
dose-response analysis of DHA transport. C, Eadie-Hofstee
plot of substrate dependence for DHA transport. D, effect of
glucose transporter substrates (2-deoxy-D-glucose, sucrose,
and fructose) and inhibitors (cytochalasins B and E) on DHA transport.
Transport in the presence of the test compounds is expressed as percent
of control (transport in the absence of substrates or inhibitors).
E, time course of 1 ( ), 20 ( ), or 40 mM
( ) 2-deoxy-D-glucose (DOG) uptake.
F, dose-response analysis of 2-deoxy-D-glucose
transport. G, Eadie-Hofstee plot of substrate dependence for
2-deoxy-D-glucose transport. CaCo-2 cells were plated in
12-well plates, and uptake of DHA or 2-deoxy-D-glucose was
measured at 24 h. Uptake experiments were performed at room
temperature. Data represent the mean ± S.D. of experiments
performed in triplicate.
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|
Next we examined the characteristics of ascorbic acid transport in
CaCo-2 cells based on the initial evidence of its sodium dependence.
Uptake proceeded in a linear fashion for at least 6 min at a substrate
concentration of 10-500 µM (Fig.
3A). A detailed dose-response
study showed that the transport of ascorbic acid approached saturation
at about 500 µM substrate (Fig. 3B). Analysis of the transport data by the Eadie-Hofstee method revealed the presence
of at least two different functional components, involved in ascorbic
acid transport by CaCo-2 cells, each with different affinities for the
substrate (Fig. 3C). The higher affinity component had an
apparent transport Km of 8 µM and a
Vmax of 40 pmol/min per 106 cells,
whereas the lower affinity component had apparent Km and Vmax values of 125 µM and 670 pmol/min per 106 cells, respectively. Analysis of the
sodium effect on ascorbic acid transport at 10 µM,
conditions at which greater than 70% of the total transport is
expected to be mediated by the higher affinity transporter, revealed
that this transporter was strongly activated by sodium (Fig.
3D). The sodium effect was of a cooperative nature as
indicated by the sigmoidal shape of the dose-dependent curve, a conclusion that was corroborated when the transport data were
utilized to construct a Hill plot that resulted in a straight line
having a Hill coefficient (the slope of the Hill graph) of 1.8 (Fig.
3F). Similar results were obtained when using 500 µM ascorbic acid, a concentration at which it is expected
that greater than 70% of the total transport would be mediated by the
lower affinity transporter. The dose-dependent curve was
sigmoidal (Fig. 3E) with a Hill coefficient of 1.9 (Fig.
3F). Competition and inhibition experiments revealed that
deoxyglucose, sucrose, fructose, and cytochalasin B failed to affect
ascorbic acid transport, indicating that the ascorbic acid transporters
expressed by CaCo-2 cells are functionally unrelated to the
dehydroascorbic acid transporters.

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Fig. 3.
Ascorbic acid transport in CaCo-2 cells.
A, time course of 10 ( ), 50 ( ), or 500 µM ( ) AA uptake. B, dose-response analysis
of AA transport. C, Eadie-Hofstee plot of the substrate
dependence for AA transport. D, effect of sodium on
transport of 10 µM AA. E, effect of sodium on
transport of 500 µM AA. F, Hill plots for the
sodium effect on transport of 10 ( ) or 500 µM ( )
AA. CaCo-2 cells were plated in 12-well plates, and uptake was measured
at 24 h. Uptake experiments were performed at 37 °C. Data
represent the mean ± S.D. of experiments performed in
triplicate.
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We performed RT-PCR with primers specific for SVCT1 and SVCT2, the two
human isoforms currently cloned, in order to confirm the existence of
two sodium-dependent ascorbic acid transporters in CaCo-2
cells, as well as to identify them. RT-PCR experiments yielded
amplification products having the expected sizes for SVCT1 (304 bp) and
SVCT2 (459 or 307 bp) (Fig. 4 and data
not shown). The specificity of the amplification reaction was verified
by performing the RT-PCR assays using human brain for SVCT2 (Fig. 4)
and human intestinal cells for SVCT1 (data not shown) as the RNA
sources. PCR amplification products were isolated and submitted to
automated sequencing, which confirmed their identities as corresponding to products amplified from the coding sequences of SVCT1 and SVCT2.

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Fig. 4.
RT-PCR analysis of SVCT1 and SVCT2 expression
in CaCo-2 cells. PCR analysis was performed using CaCo-2 cDNA
(lanes 2-4) or total human brain cDNA (lanes
5-7) as templates with specific primers for SVCT1 (lanes
2 and 5) or SVCT2 (lanes 3 and
6). Amplification of glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) (lanes 4 and 7)
was used as an internal control. The products were separated on 1.5%
agarose gel and stained with ethidium bromide. DNA bands in lane
1 correspond to size markers.
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When cultured in vitro for several days and allowed to reach
confluence, CaCo-2 cells underwent further differentiation along the
enterocyte pathway which is accompanied by changes in the expression of
proteins that are considered markers of terminal differentiation.
CaCo-2 cells cultured for 15 days reached a post-confluence state that
was characterized by a notable increase in their capacity to transport
ascorbic acid. Transport studies using 10 and 500 µM
ascorbic acid revealed that the increase in transport was fundamentally associated with the increased expression of the lower affinity component. At 10 µM ascorbic acid, the transport rate
first increased from 11.5 pmol/min per 106 cells at day 2 post-seeding (pre-confluent cells) to 15.5 pmol/min per 106
cells at day 6, and then decreased to 9 pmol/min per 106
cells at day 10 to reach a lower value of 5 pmol/min per
106 cells at day 15 (Fig. 5,
A and B). In contrast, the transport rate at 500 µM ascorbic acid increased from 70 pmol/min per
106 cells at day 2 to 160 pmol/min per 106
cells at day 6 and reached a value of 250 pmol/min per 106
cells at day 15 (Fig. 5, C and D). Dose-response
analysis using increasing concentrations of ascorbic acid revealed that
the transport rate at saturation was about 2-fold higher in
post-confluent (15-day culture) as compared with pre-confluent (2-day
culture) cells (Fig. 5E). Analysis of the transport data by
the Eadie-Hofstee method confirmed the previous observation by
revealing a 1.7-fold increase in the Vmax of
transport in 15-day-old cells compared with those at 2 days (Fig.
5F). Moreover, whereas two functional components were
present in 2-day-old cells, only the lower affinity component was
present at 15 days. At 2 days, the higher affinity component had
apparent Km and Vmax values
of 8 µM and 40 pmol/min per 106 cells,
respectively, whereas the lower affinity component had Km and Vmax values of 125 µM and 80 pmol/min per 106 cells,
respectively. In contrast, 15-day-old cells displayed a lower affinity
component with apparent Km and
Vmax values of 110 µM and 180 pmol/min per 106 cells, respectively.

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Fig. 5.
Ascorbic acid transport in pre-and
post-confluent CaCo-2 cells. A, transport of 10 µM AA in CaCo-2 cells cultured for 2 ( ), 6 ( ), 10 ( ), and 15 ( ) days. B, rate of AA transport from data
in A. C, transport of 500 µM AA in CaCo-2
cells cultured for 2 ( ), 6 ( ), 10 ( ), and 15 ( ) days.
D, rate of AA transport derived from data in C. E, dose-response analysis of AA transport in CaCo-2 cells cultured
3 ( ) or 15 ( ) days. F, Eadie-Hofstee plot of the
substrate dependence for AA transport in 3- ( ) or 15-day ( )
cells. Transport was measured at 37 °C. Data represent the mean ± S.D. of experiments performed in triplicate.
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We interpreted our transport data as indicating that the post-confluent
differentiation of CaCo-2 cells is accompanied by a marked increase in
the expression of functional SVCT1 transporters, changes that occurred
independently of any change in SVCT2 expression. We further analyzed
this issue using RT-PCR with primers specific for SVCT1 and SVCT2 and
RNA prepared from pre- and post-confluent cells. These experiments
revealed that post-confluence was associated with an increase in the
steady-state SVCT1 mRNA levels, as indicated by a marked increase
in the intensity of the amplification product without detectable
changes in SVCT2 mRNA (data not shown). To confirm these findings,
we performed quantitative RT-PCR for SVCT1 and SVCT2 using RNA isolated
from CaCo-2 cells maintained in culture from 2 to 20 days. We used
changes in GLUT1, GLUT2, and GLUT5 RNA as markers for post-confluent
CaCo-2 cell differentiation (25, 26) with
-actin as an internal
control. A standard curve was constructed optimizing the PCR for each
transporter and for
-actin, and the respective cross-over points
were used for quantification. The control experiments revealed that
GLUT1 and GLUT5 were initially present in CaCo-2 cells at equivalent
levels of expression for the first 4 days of culture. However,
post-confluent differentiation of the CaCo-2 cells was accompanied by a
marked increase in the amount of GLUT5 mRNA. At day 15, the amount
of GLUT5 mRNA was ~8-fold greater than at day 1 (Fig.
6A). Further control
experiments revealed that
-actin expression was maintained at
constant levels during the duration of the experiment (Fig.
6A). Parallel experiments revealed that SVCT1 and SVCT2 are
independently regulated during the post-confluent differentiation of
the CaCo-2 cells (Fig. 6B). SVCT1 levels increased as
differentiation proceeded, and at day 20 there was 5-fold more SVCT1
mRNA than at day 2. On the other hand, there was no increase in
SVCT2 mRNA levels during differentiation, confirming the results of
the functional studies indicating that SVCT1 was selectively
up-regulated during post-confluent differentiation of the CaCo-2
cells.

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Fig. 6.
Quantitative analysis of hexose and ascorbic
acid transporter expression in post-confluent CaCo-2 cells.
A, quantitative RT-PCR analysis of GLUT1 ( ), GLUT5 ( ),
and actin ( ) mRNA expression in CaCo-2 cells grown to
confluence. CaCo-2 cells were cultured in 6-well plates, and RNA was
prepared at days 1, 4, 6, 14, and 20 after seeding and used to
synthesize the corresponding cDNAs. Expression of the test proteins
was assessed by quantitative PCR using specific primers and real time
PCR with the Light CyclerTM. B, quantitative
RT-PCR analysis of SVCT1 ( ) and SVCT2 ( ) mRNA expression in
CaCo-2 cells grown to confluence. Details are the same as in
A.
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When CaCo-2 cells were cultured in permeable filter inserts (transwell
system), they formed an impermeable barrier consisting of highly
polarized cells tightly packed and with the establishment of tight
junctions. Under these conditions, the culture medium present in the
upper compartment of the transwell system was in contact only with the
apical region of the cells and did not contact the basolateral plasma
membrane, whereas the culture medium present in the lower compartment
was in contact with the basolateral region of the cell and did not
contact the apical plasma membrane. We hypothesized whether the
post-confluent differentiation of CaCo-2 cells growing in transwells
was accompanied not only by an increased expression of SVCT1 but also
by the specific sorting of the transporters to the apical or to the
basolateral membrane compartments. The transport experiments revealed
that there was no transport of reduced vitamin C through the
basolateral plasma membrane and that ascorbic acid was exclusively
taken up through the apical plasma membrane (Fig.
7A). Kinetic analysis of
ascorbic acid transport revealed that transport approached saturation
at about 400 µM substrate (Fig. 7B), and
analysis of the data by the Eadie-Hofstee transformation revealed that
the transport of ascorbic acid at the apical membrane compartment was
mediated by a single component with an apparent Km
of 105 µM and an apparent Vmax of 350 pmol/min × 106 cells (Fig. 7, B and
C). Overall, our data can be interpreted as indicating that
SVCT1 is selectively expressed and sorted to the apical plasma membrane
of the CaCo-2 cells during differentiation and polarization.

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|
Fig. 7.
Ascorbic acid transport in polarized CaCo-2
cells. A, transport of AA in polarized CaCo-2 cells cultured
in two-chamber permeable membrane inserts. CaCo-2 cells were seeded on
collagen-coated transwell inserts in 6-well plates and allowed to grow
for 15 days. Uptake of AA was measured from the apical ( ) or
basolateral ( ) transwell compartment. B, dose-response
analysis of apical AA transport in 15-day cells. C,
Eadie-Hofstee plot of the substrate dependence for apical AA transport
in 15-day cells. Transport was measured at 37 °C. Data represent the
mean of two independent experiments.
|
|
 |
DISCUSSION |
The human colon carcinoma cell line CaCo-2 is a valuable cell
system to model the mechanism and regulation of nutrient transport in
human intestinal epithelium (30, 31, 33). Therefore, we selected this
cell system to perform a detailed characterization of vitamin C
transport in enterocyte-like cells. We concluded that CaCo-2 cells
acquire both forms of vitamin C, the oxidized form (dehydroascorbic
acid) and the reduced form (ascorbic acid). CaCo-2 cells acquire
oxidized vitamin C with the participation of two, and perhaps three
different transporter activities. The fact that sodium was not required
for transport discards the participation of a sodium-glucose
co-transporter (SGLT) in this process. Results of competition
experiments, showing that deoxyglucose, but not
-methyl-D-glucoside, blocked the transport of
dehydroascorbic acid, and the strong inhibitory effect of cytochalasin
B indicated that dehydroascorbic acid transporters expressed in CaCo-2
cells correspond to facilitative glucose transporters. This conclusion is consistent with previous evidence indicating that CaCo-2 cells express facilitative glucose transporters (GLUTs) and that oxidized vitamin C is transported exclusively by members of this transporter family (2, 14, 16, 17). RT-PCR corroborated the expression of GLUT1,
GLUT2, GLUT3, and GLUT5 in CaCo-2 cells. GLUT5 is a fructose
transporter that does not transport glucose or dehydroascorbic acid. On
the other hand, apparent Km values for the transport of dehydroascorbic acid and deoxyglucose suggest that the higher affinity transporter present in CaCo-2 cells corresponds to GLUT3. Although GLUT1 is expressed in CaCo-2 cells, its transport
Km for both dehydroascorbic acid and deoxyglucose is
similar to that of GLUT3 and cannot be differentiated from this
transporter using functional assays. A third, low affinity
dehydroascorbic acid transporter detected in CaCo-2 cells was
identified as GLUT2 based on transport and RT-PCR analysis. Although
the ability of GLUT2 to transport dehydroascorbic acid is still under
review, we have obtained evidence from studies using primary rat
hepatocytes indicating that GLUT2 is a low affinity, high capacity
transporter of dehydroascorbic acid.2
We also determined the presence of two Na+-ascorbic acid
co-transporters in CaCo-2 cells with different affinities for ascorbic acid transport. The lower affinity component had an apparent
Km of 125 µM, which is similar to the
Km described for the cloned human SVCT1 expressed in
Xenopus laevis oocytes. Based on
Km data and RT-PCR results, we propose that the low affinity ascorbic acid transporter expressed in CaCo-2 cells is SVCT1.
This is consistent with the fact that SVCT1 was cloned from a CaCo-2
cell cDNA library (20). The second higher affinity transporter of
ascorbic acid detected in CaCo-2 cells had an apparent transport
Km of 8 µM, which is similar to that
described for the cloned human SVCT2 transporter (21) expressed in
Xenopus oocytes. RT-PCR analysis confirmed the expression of
SVCT2 in CaCo-2 cells, supporting the concept that it corresponds to
the higher affinity transporter of ascorbic acid present in these cells.
A central finding of this study is the evidence, obtained from
functional assays coupled to quantitative RT-PCR analysis, indicating
that SVCT1 and SVCT2 expressions are independently regulated in CaCo-2
cells during cell differentiation. Cells cultured for long periods
showed a severalfold increase in SVCT1 mRNA expression without
appreciable changes in SVCT2 mRNA levels. By using GLUT5 expression
as a differentiation marker, we confirmed that SVCT1 mRNA
expression was up-regulated coincidentally with the maturation of
post-confluent cells. The net result is an increase in the amount of
functional SVCT1 protein present at the plasma membrane as revealed by
a marked increase in ascorbic acid transport. Furthermore, post-confluent CaCo-2 cells growing on permeable filter inserts showed
selective sorting of SVCT1 to the apical membrane compartment. This is
similar to the glucose transporter pattern developed by CaCo-2 cells
grown under conditions that led to the expression of enterocyte-like
properties, with polarized distribution of glucose transporters at the
apical and/or basolateral surface of the cell, whereas the
Na+-dependent transporter SGLT1 displays a
unique subcellular distribution in the apical membrane compartment (24,
30, 40). In the normal absorptive epithelia of the human small
intestine, glucose uptake from the lumen of the gut is mediated by
SGLT1, and glucose efflux from these cells into the blood is mediated
by GLUT2. Thus, both CaCo-2 cells and normal human enterocytes are
characterized by the differential subcellular distribution of SGLT and
GLUTs, which is a key element in the vectorial transfer of sugars from the intestinal lumen to the blood. In this context, our data open up
the possibility that the polarized distribution of vitamin C
transporters at the apical or basolateral membrane compartment of the
intestinal enterocytes may play an important role in the vectorial
transport of vitamin C across the intestinal barrier.
 |
FOOTNOTES |
*
This work was supported in part by Grants 3990007, 3000024, 1990333, and 1020451 from FONDECYT, Chile, and grant 201034006-1.4 from
the Dirección de Investigación, Universidad de
Concepción, Concepción, Chile.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.

To whom correspondence should be addressed. Tel.: 56-41-203817;
Fax: 56-41-216558; E-mail: juvera@udec.cl.
Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M205119200
2
J. G. Cárcamo and J. C. Vera,
unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
AA, ascorbic acid;
DHA, dehydroascorbic acid;
RT, reverse transcriptase;
GLUT, glucose
transporter;
SGLT, sodium-glucose co-transporter.
 |
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