Uptake and intracellular fate of L-DOPA in a human
intestinal epithelial cell line: Caco-2
M. A.
Vieira-Coelho and
P.
Soares-Da-Silva
Institute of Pharmacology and Therapeutics, Faculty of Medicine,
4200 Porto, Portugal
 |
ABSTRACT |
The aim of the present
study was to examine the kinetic characteristics of the
L-3,4-dihydroxyphenylalanine (L-DOPA)
transporter and the fate of newly formed dopamine in Caco-2 cells. In
the presence of 50 µM benserazide (an inhibitor of aromatic
L-amino acid decarboxylase), L-DOPA was rapidly
accumulated in Caco-2 cells. At equilibrium (30 min of incubation) the
intracellular L-DOPA concentration was 10.2 ± 0.1 µM at
a medium concentration of 0.5 µM. In saturation experiments the
accumulation of L-DOPA was saturable with a
Michaelis-Menten constant (Km) of 60 ± 10 µM
and a maximal reaction velocity (Vmax) of 6.6 ± 0.3 nmol · mg protein
1 · 6 min
1; at 4°C the amount of L-DOPA
accumulated in the cells was nonsaturable. When cells were incubated
with increasing concentrations of L-DOPA (10-100 µM)
in the absence of benserazide, a substantial amount of the
L-DOPA that was taken up was decarboxylated to dopamine, with an apparent Km of 27.2 µM. In experiments
performed in cells cultured in polycarbonate filters, the
accumulation of L-DOPA in the presence of benserazide was
greater when the substrate was applied from the basolateral cell border
than when it was applied from the apical cell border. In the absence of
benserazide, L-DOPA applied from the basolateral cell
border resulted in a nonlinear formation of dopamine
(Km = 43 ± 7 µM,
Vmax = 23.7 ± 1.2 nmol · mg
protein
1 · 6 min
1). The
amount of dopamine leaving the cell through the apical cell border was
lower than the amount that escaped through the basolateral cell border,
and the process was saturable (Km = 623 ± 238 µM, Vmax = 0.19 ± 0.02 nmol · mg
protein
1 · 6 min
1). In
conclusion, the data presented here show that Caco-2 cells are endowed
with an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, some
of which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way to
look at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.
L-3,4-dihydrophenylalanine; dopamine; polycarbonate
filter; basolateral cell border; apical cell border; L-amino acid decarboxylase
 |
INTRODUCTION |
THE INTESTINAL TRACT has been shown to be of
crucial importance in the regulation of body fluid and electrolyte
homeostasis, with catecholamines assuming the role of
important regulators of jejunal cell function (2). More recently,
endogenous dopamine in the digestive tract has been suggested to play a
role in regulating sodium absorption (1, 6). The current view of the
intestinal dopaminergic system is that of a local nonneuronal system
that consists of epithelial cells of intestinal mucosa rich in aromatic L-amino acid decarboxylase (AADC) activity and uses
circulating or luminal L-3,4-dihydroxyphenylalanine
(L-DOPA) as a source for dopamine (25). In the intestine,
dopamine is particularly abundant in the mucosal cell layer (4, 5).
Studies on the formation of dopamine from exogenous L-DOPA
along the rat digestive tract showed that the highest AADC activity is
located in the jejunum (28). Because the dopamine produced in this area
is in close proximity to epithelial cells that contain receptors for
the amine, it has been hypothesized that the amine may act as a
paracrine or an autocrine substance (25). A high-salt diet has been
found to constitute an important stimulus for the production of
dopamine in rat jejunal epithelial cells, and this is accompanied, in
20-day-old animals, by a decrease in sodium intestinal absorption (6). This effect is accomplished, at the cellular level, by inhibition of
Na+-K+-ATPase activity (26). The relative
importance of this system in controlling sodium absorption assumes
particular relevance in view of the findings that 40-day-old rats fed a
high-salt diet have a fault in intestinal dopamine production during
salt loading, in contrast to 20-day-old animals (6, 26). The lack of
changes in the jejunal function in response to a high-salt diet
coincides with the period in which the renal function has reached
maturation (15, 16), suggesting the occurrence of complementary
functions between the intestine and the kidney during development. On
the other hand, this intestinal epithelial dopaminergic system presents some similarities to that described for the proximal renal tubules (25), where locally produced dopamine has long been demonstrated to
play a role in the handling of sodium (9, 10, 19).
Several intestinal cell lines are often used as physiological model
systems of intestinal absorptive and secretive function, because in
most cases their utilization enables the evaluation of a given process
in a single population of cells. Caco-2 cells are an established
epithelial cell line derived from a human colon adenocarcinoma that
undergoes enterocyte differentiation in culture (13). This cell line
has been also suggested to possess attributes that make it a suitable
in vitro model system for the investigation of transport across the
small intestinal epithelium (8). To explore further the usefulness of
Caco-2 cells for the study of intestinal dopaminergic physiology, we
have undertaken the study of the kinetic characteristics of the
L-DOPA transporter and the fate of newly formed dopamine in
this cell line. We report here that Caco-2 cells take up
L-DOPA through a saturable, stereoselective, and
temperature-dependent process; in cells cultured in polycarbonate filters, the inward and outward transfers of L-DOPA are
quantitatively more important at the basolateral than at the apical
cell border. The formation of dopamine was found to be a time- and
concentration-dependent process and rapidly saturated, and the newly
formed amine was found to leave the intracellular compartment through
the apical cell border by a saturable process.
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METHODS |
Cell culture.
The Caco-2 cell line was obtained from the American Type Culture
Collection (ATCC, Rockville, MD) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. Caco-2 cells
(ATCC 37-HTB; passages 23-30) were grown in minimal
essential medium (Sigma Chemical, St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml
streptomycin (Sigma Chemical), 20% fetal bovine serum (Sigma
Chemical), and 25 mM HEPES (Sigma Chemical). For subculturing, the
cells were dissociated with 0.05% trypsin-EDTA, split 1:3, and
subcultured in flasks with 75- or 162-cm2 growth areas
(Costar, Badhoevedorp, The Netherlands). For uptake studies the cells
were seeded in collagen-treated 24-well plastic culture clusters (16 mm
ID, Costar) at a density of 40,000 cells/well (2.0 × 104 cells/cm2) or, depending on
the experiment, onto collagen-treated 0.2-µm polycarbonate filter
supports (12 mm ID, Transwell, Costar). The cell medium was changed
every 2 days, and the cells reached confluence after 5-7 days of
initial seeding. For 24 h before each experiment, the cell medium was
free of fetal bovine serum. Experiments were generally performed
2-3 days after cells reached confluence and 7-10 days after
the initial seeding, and each square centimeter contained ~100 µg
of cell protein.
Transport studies.
On the day of the experiment the growth medium was aspirated and the
cells were washed with Hanks' medium at 4°C; then the cell
monolayers were preincubated for 15 min in Hanks' medium at 37°C.
Hanks' medium had the following composition (mM): 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1.0 MgCl2, 0.15 Tris · HCl, and 1.0 sodium
butyrate, pH 7.4. The incubation medium also contained pargyline (100 µM) and tolcapone (1 µM) to inhibit the enzymes monoamine oxidase
and catechol-O-methyltransferase, respectively; in some
experiments, benserazide (50 µM) was also added to the incubation
medium to inhibit AADC. During preincubation and incubation the cells
were continuously shaken and maintained at 37°C.
In the first series of experiments, uptake studies were performed in
cells cultured in collagen-treated plastic supports, the substrates
being applied from the apical cell border only. Uptake was initiated by
the addition of 2 ml of Hanks' medium with a given concentration of
the substrate under study. Initial rate of uptake was determined in
experiments in which cells were incubated with nonsaturating and
saturating concentrations of L-DOPA (0.5, 50, and 500 µM)
for 1, 3, 6, 12, 30, 60, and 120 min. Saturation experiments were
performed in cells incubated for 6 min with increasing concentrations
of the substrate; some experiments were conducted at 4°C. Uptake was
terminated by the rapid removal of uptake solution by means of a vacuum
pump connected to a pasteur pipette followed by two rapid washes with
cold Hanks' medium and the addition of 250 µl of 0.2 mM perchloric
acid; the acidified samples were stored at 4°C before injection into
the high-pressure liquid chromatograph for the assay of
L-DOPA, D-DOPA, and dopamine.
In a second series of experiments, cells were cultured in polycarbonate
supports, the substrates being applied from the apical or the basal
side of the monolayer. The incubation medium used in this series of
experiments was similar to that described above; in some experiments
the medium contained benserazide (50 µM) to inhibit AADC. The upper
and lower chambers contained 100 and 600 µl, respectively. For apical
uptake the uptake solution was added to the upper chamber; for
basolateral uptake the uptake solution was added to the lower chamber.
Cells were preincubated for 30 min and then incubated in the presence
of L-DOPA. [14C]sorbitol (0.4 µM) was used
to estimate paracellular fluxes and extracellular trapping of
L-DOPA during L-DOPA uptake studies. At the end
of incubation, cells were placed on ice, and the medium bathing the
apical and basal cell borders was collected, acidified with 2 M
perchloric acid, and stored at 4°C until assayed for L-DOPA and dopamine. The cells were washed with ice-cold
Hanks' medium and added with 0.2 mM perchloric acid (100 and 500 µl
in the upper and lower chambers, respectively); the acidified samples were stored at 4°C before injection into the high-pressure liquid chromatograph for the assay of L-DOPA and dopamine.
AADC preparation and decarboxylation studies.
Caco-2 cells were homogenized in 0.5 M phosphate buffer (pH 7.0) with a
Thomas Teflon homogenizer and kept continuously on ice. Aliquots of 500 µl of cell homogenate plus 400 µl of incubation medium were placed
in glass test tubes and preincubated for 15 min. Thereafter,
L-DOPA (0.1-5.0 mM) was added to the medium for a
further 15 min; the final reaction volume was 1 ml. The composition of
the incubation medium was as follows (in mM): 0.35 NaH2PO4, 0.15 Na2HPO4,
0.11 sodium borate and 0.12 pyridoxal phosphate; tolcapone (1 µM) and
pargyline (100 µM) were also added to the medium. The pH of the
reaction medium was kept constant at an optimal pH of 7.0 (18). During
incubation, cell homogenates were continuously shaken and gassed (95%
O2-5% CO2) and maintained at 37°C.
The reaction was stopped by the addition of 500 µl of 2 M perchloric
acid, and the preparations were kept at 4°C for 60 min. The samples
were then centrifuged (200 g, 2 min, 4°C), and 500-µl
aliquots of the supernatant filtered on Spin-X filter tubes (Costar)
were used for the assay of dopamine.
Assay of L-DOPA, D-DOPA, and
dopamine.
L-DOPA, D-DOPA, and
dopamine were quantified by HPLC with electrochemical detection, as
previously reported (20). The high-pressure liquid chromatograph system
consisted of a pump (model 302, Gilson Medical Electronics, Villiers le
Bel, France) connected to a manometric module (model 802 C, Gilson) and
a 25-cm-long stainless-steel 5-µm ODS column (Biophase, Bioanalytical
Systems, West Lafayette, IN); samples were injected by means of an
automatic sample injector (model 231, Gilson) connected to a dilutor
(model 401, Gilson). The mobile phase was a degassed solution of citric
acid (0.1 mM), sodium octylsulfate (0.5 mM), sodium acetate (0.1 M),
EDTA (0.17 mM), dibutylamine (1 mM), and methanol (8% vol/vol)
adjusted to pH 3.5 with perchloric acid (2 M) and pumped at a rate of
1.0 ml/min. The detection was carried out electrochemically with a glassy carbon electrode, an Ag-AgCl reference electrode, and an amperometric detector (model 141, Gilson); the detector cell was operated at 0.75 V. The current produced was monitored using Gilson 712 HPLC software. The lower limits for detection of L-DOPA,
D-DOPA, and dopamine ranged from 350 to 500 fmol.
Cell water content.
Cell water content was simultaneously measured in parallel experiments
with [14C]inulin as extracellular marker and tritiated
water as total water marker. Intracellular water, obtained by
subtracting extracellular water from total water, was expressed as
microliters of cell water per milligram of protein. Subsequently, the
cells were solubilized by 0.1% (vol/vol) Triton X-100 (dissolved in 5 mM Tris · HCl, pH 7.4), and radioactivity was measured by
liquid scintillation counting.
Protein assay.
The protein content of monolayers of Caco-2 cells was determined by the
method of Bradford (3), with human serum albumin as a standard.
Cell viability.
Caco-2 cells were preincubated for 15 min at 37°C and then incubated
in the absence or the presence of L-DOPA,
D-DOPA, and dopamine for a further 15 or 120 min.
Subsequently, the cells were incubated at 37°C for 2 min with trypan
blue (0.2% wt/vol) 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.
The analysis of the time course of L-DOPA uptake in Caco-2
cells was based on a one-compartment model. The parameters of the following
equation
were
fitted to the experimental data by a nonlinear regression analysis with
use of a computed assisted method (11). Ci and
Co represent the intracellular and extracellular
concentration of the substrate, respectively, kin
and kout rate constants for inward and outward
transport, respectively (in pmol · mg
protein
1 · min
1), and
t the incubation time. Amax is defined as the
factor of accumulation (Ci/Co) at equilibrium
(t
). Michaelis-Menten constants
(Km) and maximal reaction velocities
(Vmax) for the uptake of substrates, as determined
in saturation experiments, and decarboxylation of L-DOPA in
cell homogenates were calculated from nonlinear regression analysis
with use of the GraphPad Prism statistics software package (11). The
rate constant of outward transfer was determined by the slope of the
accumulation of substrates measured by linear regression analysis (12).
Arithmetic means are given with SE. Statistical analysis was performed
by one-way ANOVA followed by Newman-Keuls test for multiple
comparisons. P < 0.05 was assumed to denote a significant
difference.
 |
RESULTS |
To determine kin and kout,
Caco-2 cells were incubated with L-DOPA (0.5, 50, or 500 µM) for 1, 3, 6, 12, 30, 60, and 120 min in the presence of
benserazide. As shown in Fig.
1, uptake of L-DOPA in Caco-2 cells was linear with time for up to 12 min of incubations in the presence of 0.5, 50, or 500 µM
L-DOPA and occurred at a kin of 4.2 ± 0.2, 217.4 ± 9.8, and 333.6 ± 23.9 pmol · mg protein
1 · min
1,
respectively. The kout was 0.61 ± 0.03, 31.1 ± 1.4, and 47.7 ± 3.4 pmol · mg
protein
1 · min
1 for 0.5, 50, and 500 µM L-DOPA, respectively. The equilibrium factor
(Amax) declined from 20.4 ± 0.3 at 0.5 µM
L-DOPA to 13.5 ± 0.8 and 4.4 ± 0.1 at 50 and 500 µM
L-DOPA, respectively. The intracellular water content of
cell monolayers was 7.0 ± 0.6 µl/mg protein (n = 5). At
equilibrium (60 min of incubation), the intracellular L-DOPA concentration was 10.2 ± 0.1, 674.2 ± 37.3, and
2,184.7 ± 59.6 µM at medium concentrations of 0.5, 50, and 500 µM. This represented a cell concentration of L-DOPA that
was 19.1 ± 0.8, 12.7 ± 0.9, and 2.8 ± 0.1 times higher than the
corresponding medium concentration.

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Fig. 1.
Time course of L-3,4-dihydroxyphenylalanine
(L-DOPA) accumulation in Caco-2 cells. Cells were incubated
at 37°C with 0.5 (A), 50 (B), or 500 (C) µM L-DOPA. Exponential saturation curve was
fitted to experimental data. At equilibrium (60 min of incubation),
intracellular L-DOPA concentration was 10.2 ± 0.1, 674.2 ± 37.3, and 2184.7 ± 59.6 µM at medium concentrations
of 0.5, 50, and 500 µM. This represented a cell concentration of
L-DOPA that was 19.1 ± 0.8, 12.7 ± 0.9, and
2.8 ± 0.1 times higher than corresponding medium concentration.
Symbols represent means of 4 experiments/group; vertical lines show
SE.
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On the basis of these results, a 6-min incubation was used in all
subsequent experiments designed to determine the kinetic parameters for
the uptake of L-DOPA. The accumulation of
L-DOPA from the apical cell border was found to be
dependent on the concentration used and to be saturable at 250 µM
(Fig. 2). Nonlinear analysis of the
saturation curves revealed a Km of 60 ± 10 µM
and a Vmax of 6.6 ± 0.3 nmol · mg
protein
1 · 6 min
1. In
experiments carried out at 4°C the amount of L-DOPA
accumulated in the cells was markedly lower than that observed at
37°C and was found to be nonsaturable (Fig. 2). Caco-2 cells
incubated at 37°C with increasing concentrations of
D-DOPA, instead of L-DOPA, were found to
accumulate trace amounts of the D-isomer; the cellular accumulation of D-DOPA at the highest concentration used
was ~5% of the corresponding L-isomer (data not shown).
The experiments shown in Figs. 1 and 2 were performed in the presence
of benserazide (50 µM) to avoid the intracellular decarboxylation of
incorporated L-DOPA by AADC. The effectiveness of
benserazide in inhibiting L-DOPA decarboxylation was very
high, since no traces of dopamine were found in these samples.

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Fig. 2.
Concentration-dependent accumulation of L-DOPA in Caco-2
cells. Cells were incubated for 6 min at 37°C ( ) or 4°C ( ),
and increasing concentrations (1-500 µM) of substrate were
applied from apical border. Symbols represent means of 4 experiments/group; vertical lines show SE. Linear coefficient
(r2) values were as follows for
L-DOPA at 4°C: r2 = 0.995, n = 24.
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Incubation of homogenates of Caco-2 cells with
L-DOPA (0.1-5.0 mM) resulted in a
concentration-dependent formation of dopamine (Fig.
3). The decarboxylation process was
nonsaturable up to 1 mM L-DOPA and showed a trend for
saturation at 2 mM L-DOPA. Nonlinear analysis of the
saturation curves revealed a Km of 1.0 ± 0.4 mM and a Vmax of 22.5 ± 0.6 nmol · mg
protein
1 · h
1.

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Fig. 3.
Saturation curve of aromatic L-amino acid decarboxylase
(AADC) activity in homogenates of Caco-2 cells. AADC activity is shown
as rate of formation of dopamine vs. concentration of
L-DOPA. Symbols represent means of 5 experiments/group;
vertical lines show SE.
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As shown in Fig. 4, decarboxylation of
L-DOPA (0.5 µM) in monolayers of Caco-2 cells was a
time-dependent process, and most of the incorporated L-DOPA
was decarboxylated to dopamine; only small amounts of
L-DOPA remained in the intracellular compartment without
undergoing decarboxylation to dopamine. Most of the newly formed amine
escaped into the medium bathing the apical cell border. The amount of
newly formed dopamine that remained in the cell attained equilibrium 30 min after the addition of the substrate. In saturation experiments
(Fig. 5) a 6-min incubation period was chosen. In this set of experiments the formation of dopamine from increasing concentrations of L-DOPA (10-100 µM)
followed nonlinear kinetics. Again, most of the incorporated
L-DOPA was decarboxylated to dopamine; only small amounts
of L-DOPA escaped decarboxylation (Fig. 5A). The
process of L-DOPA decarboxylation in Caco-2 cells was, however, rapidly saturated at low concentrations of
L-DOPA with an apparent Km of 27.2 ± 3.8 µM and a Vmax of 6.4 ± 0.3 nmol · mg protein
1 · 6 min
1. Some of newly formed dopamine escaped into the
incubation medium, and this process was nonsaturable, the rate constant
of outward transfer being 4.4 ± 0.6 mmol
1 (Fig.
5B).

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Fig. 4.
Time-dependent formation of dopamine in monolayers of Caco-2 cells
incubated with 0.5 µM L-DOPA. Values are intracellular
( ) and extracellular ( ) levels of dopamine formed from added
L-DOPA and intracellular levels of L-DOPA
( ). Inset, nonlinear accumulation of newly formed dopamine
( ) in intracellular compartment and intracellular levels of
L-DOPA ( ). Each point represents mean of 4 experiments/group; vertical lines show SE.
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Fig. 5.
A: intracellular levels of L-DOPA taken up ( )
and newly formed dopamine ( ) and extracellular levels of dopamine
( ) in Caco-2 cells. B: extracellular levels of newly formed
dopamine plotted against intracellular dopamine. Cells were
preincubated in absence of benserazide for 30 min and incubated for 6 min with increasing concentrations of L-DOPA; substrate was
applied from apical cell border. Symbols represent means of 4 experiments/group; vertical lines show SE.
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The next series of experiments was performed in cells cultured in
polycarbonate filters. L-DOPA was applied from the apical or the basal border; intracellular L-DOPA and
L-DOPA that had escaped into the basal or the apical
bathing fluid were also measured. Paracellular leakage measured by the
fluxes of [14C]sorbitol from either side was minimal and
represented 0.1% of the amount applied at the cell surface. The first
series of experiments was carried out in the presence of benserazide
(50 µM), and L-DOPA (0.5 µM) was applied from the
apical or the basal cell border for increasing periods of time
(1-60 min). As shown in Fig. 6, L-DOPA applied from the apical or the basal cell border was
rapidly accumulated in Caco-2 cells, equilibrium being attained at 12 min of incubation, and no flux of L-DOPA across the cell
monolayer was detected. The kin,
kout, and Amax for the apical
application of L-DOPA (38.7 ± 18.0
pmol · mg
protein
1 · min
1,
11.1 ± 5.2 pmol · mg
protein
1 · min
1, and
42.7 ± 5.6, respectively) were lower (P < 0.05) than
those observed for the basal application (183.5 ± 50.8
pmol · mg
protein
1 · min
1,
52.4 ± 16.5 pmol · mg
protein
1 · min
1, and
64.7 ± 5.4, respectively) of the substrate. When the substrate was
applied from the apical side, at equilibrium (30 min of incubation), the intracellular concentration of L-DOPA was also lower
than that observed for the basal application of the substrate
(15.9 ± 3.0 vs. 31.7 ± 1.5 µM) at medium concentration of 0.5 µM. In benserazide-treated cells the accumulation of
L-DOPA applied from the basal side was also greater than
that from the apical side; Vmax values were substantially greater for the basal application than for the apical application (78.1 ± 5.4 and 14.6 ± 2.1 nmol · mg
protein
1 · 6 min
1), as
revealed by nonlinear analysis (Fig.
7A). In both cases the uptake was
a saturable process with Km of 601 ± 67 and 482 ± 118 µM for the basal and apical application,
respectively. A considerable amount of intracellular
L-DOPA, applied from the basal or the apical side, left the
cell. In both experimental conditions (apical and basal application of
L-DOPA) the outward transfer of intracellular
L-DOPA was nonsaturable (Fig. 7B). However, the
rate constant of outward transfer of intracellular L-DOPA was greater at the basolateral cell border than at the apical cell
border (11.9 ± 1.9 and 3.7 ± 0.2 mmol
1). The
rate constant of outward transfer was determined by the slope of the
levels of L-DOPA in the incubation medium measured by
linear regression analysis (12).

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Fig. 6.
Time course of L-DOPA accumulation in Caco-2 cells cultured
in polycarbonate filters. Cells were incubated at 37°C with 0.5 µM
L-DOPA. Exponential saturation curve was fitted to
experimental data. L-DOPA applied from apical ( ) or
basal cell border ( ) was rapidly accumulated in Caco-2 cells,
equilibrium being attained at 12 min of incubation; no flux of
L-DOPA across cell monolayer could be detected.
kin, kout, and Amax
values for apical application of L-DOPA were lower than
those observed for basal application of substrate. Symbols represent
means of 4 experiments/group; vertical lines show SE.
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Fig. 7.
A: saturation curves of L-DOPA accumulation in
Caco-2 cells. Cells were incubated for 6 min, and increasing
concentrations of L-DOPA (0.5-500 µM) were applied
from apical ( ) or basal ( ) cell border. Symbols represent means
of 4 experiments/group; vertical lines show SE. B:
extracellular levels of extruded L-DOPA plotted against
intracellular levels of intracellular L-DOPA; substrate was
applied from apical ( ) and basal ( ) cell border, respectively.
Cells were preincubated in presence of benserazide for 30 min and
incubated for 6 min with increasing concentrations of
L-DOPA. Symbols represent means of 4 experiments/group;
vertical lines show SE. Linear coefficient values are as follows:
L-DOPA apical, r2 = 0.909, n = 24; L-DOPA basal, r2 = 0.980, n = 32.
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In the absence of benserazide, Caco-2 cells cultured in polycarbonate
filters and incubated with L-DOPA (applied from either cell
side for 6 min) converted the substrate to dopamine, some of which
remained in the intracellular compartment, and a small amount left the
cell. Figure 8 shows intracellular levels
of newly formed dopamine in Caco-2 incubated with increasing
concentrations of L-DOPA (5-250 µM). The
intracellular accumulation of dopamine differed markedly according to
the side of the cell used for L-DOPA application (Fig. 8).
Levels of newly formed dopamine were higher and showed a trend for
saturation (apparent Km = 43 ± 7 µM) when L-DOPA was applied from the basal side. In contrast, the
levels of newly formed dopamine were lower when L-DOPA was
applied from the apical side, and the process of amine formation was
nonsaturable up to 250 µM L-DOPA. Table
1 shows the percentage of intracellular L-DOPA decarboxylated to dopamine when the substrate was
applied from either side. The amount of intracellular
L-DOPA that undergoes decarboxylation to dopamine was
approximately the same over a wide range of substrate concentrations,
although at the highest concentrations of extracellular
L-DOPA the decarboxylation was less pronounced. On the
other hand, the decarboxylation of L-DOPA was greater when
L-DOPA was applied from the basal side
(P < 0.05) than from the apical side (Table 1).

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Fig. 8.
Intracellular levels of newly formed dopamine in Caco-2 cells loaded
with L-DOPA applied from apical ( ) or basal ( ) cell
border. Cells were preincubated in absence of benserazide for 30 min
and incubated for 6 min with increasing concentrations of
L-DOPA. Symbols represent means of 4 experiments/group;
vertical lines show SE.
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As mentioned above, a small amount of newly formed dopamine escaped
into the incubation medium. As shown in Fig.
9, the amount of dopamine that escaped
through the apical cell border was lower than the amount leaving the
cell through the basolateral cell border and was a saturable process;
nonlinear analysis of the saturation curve revealed a
Km of 623 ± 238 µM and a
Vmax of 0.19 ± 0.02 nmol · mg
protein
1 · 6 min
1. The
dopamine leaving the cell through the basolateral cell border was not
directly related to the intracellular levels of the amine. In these two
examples, dopamine levels in the medium were measured in experiments in
which cells were incubated with L-DOPA from the opposite
cell border.

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Fig. 9.
Outward transfer of newly formed dopamine in Caco-2 cells through
apical ( ) and basal ( ) cell border. Cells were preincubated in
absence of benserazide for 30 min and incubated for 6 min with
increasing concentrations of L-DOPA applied from opposite
cell border. A: saturation curves of dopamine apical outflow in
Caco-2 cells plotted against intracellular dopamine. B: levels
of dopamine basal outflow in Caco-2 cells plotted against intracellular
dopamine. Symbols represent means of 4 experiments/group; vertical
lines show SE.
|
|
 |
DISCUSSION |
The data presented here show that Caco-2 cells efficiently take up
L-DOPA, and several findings demonstrate that this uptake process was a carrier-mediated mechanism. First, steady-state uptake of
nonsaturating concentrations of L-DOPA showed a curvilinear dependence on incubation time. Second, at an initial rate of uptake (6 min of incubation) the cellular transport of L-DOPA showed a curvilinear dependence on L-DOPA medium concentration,
suggesting that the uptake was saturable. Third, the cellular transport
of L-DOPA was nearly abolished at 4°C, demonstrating that
the uptake was energy dependent. The efficiency of this
L-DOPA transport in Caco-2 cells can also be evidenced by
the ratio of L-DOPA concentration in cellular water to
medium concentration. The intracellular L-DOPA concentration at equilibrium was higher than that which could be
expected by passive equilibration of L-DOPA. At steady
state of L-DOPA uptake the mean intracellular concentration
of L-DOPA was 50 times higher than the L-DOPA
concentration in the incubation medium. Finally, the finding that
D-DOPA transport was nonsaturable indicates that the uptake
of L-DOPA is stereoselective, in agreement with previous
evidence on the uptake of L-DOPA obtained in other epithelia (14, 20-22, 27).
When nonsaturating concentrations (0.5 µM) of
L-DOPA are used in time course experiments, the
result is a rapid accumulation of the substrate until equilibrium is
reached, at ~30 min of incubation. Under these experimental
conditions and with 1 mg of cell protein, Caco-2 cells cleared through
the L-DOPA transporter ~8.5 ± 0.4 µl · mg
protein
1 · min
1 of incubation
medium containing 0.5 µmol/l L-DOPA (=4.2 ± 0.2 pmol · mg
protein
1 · min
1). The
clearance values were markedly reduced to 4.4 ± 0.2 and 0.7 ± 0.1 µl · mg
protein
1 · min
1 when
half-saturating and nearly saturating concentrations of L-DOPA, 50 and 500 µM, respectively, were used. Under
similar experimental conditions we were able to demonstrate that OK and LLC-PK1 cells take up 36 and 7 µl · mg
protein
1 · min
1,
respectively, of a solution containing 0.5 µmol/l L-DOPA
(23, 27). Other authors (17, 24) used the same approach to study the
uptake of different substrates and found that the
L-arginine and glucose transport systems in OK cells clear
~130 and 5.9 µl/min incubation medium of L-arginine and
-methylglucosidase, respectively. On the other hand,
Amax values for L-DOPA in Caco-2 cells
(20.4 ± 0.3) were similar to those described for
LLC-PK1 cells (15.9 ± 0.9) but lower than those
for OK cells (75.0 ± 5.0) (23, 27). Therefore, it can be
suggested that the L-DOPA transporter in Caco-2 cells is
quite efficient and the clearance values are within the range observed
for other substrates and the same substrate (L-DOPA) in
other cell types. This, however, does indicate that L-DOPA is neither free in cellular water nor distributed to a single compartment. This type of data gives only an indication concerning the
kinetic characteristics of the transporter but is unable to provide
information related to the intracellular distribution of the substrate
or even its compartmentalization. Intracellular concentration of
L-DOPA of 10 µM at a medium substrate concentration of
0.5 µM assumes that L-DOPA is free in intracellular water
and is, therefore, a mean value of substrate concentration inside the
cell. If we hypothesize that intracellular L-DOPA is
unequally distributed, then this value can vary (even higher
or substantially lower) according to the degree of compartmentalization
of the substrate. The possibility that intracellular L-DOPA
may be subjected to some sort of compartmentalization is discussed
below.
The accumulation of L-DOPA in experiments performed in
cells cultured in polycarbonate filters differed markedly from that observed in cells cultured in collagen-treated plastic. This was particularly evident for the apical application of the substrate, the
main difference being that Km values for
L-DOPA were approximately eight times higher in cells
cultured in polycarbonate filters than in cells cultured in plastic;
cells grown in collagen-treated plastic also accumulated more
L-DOPA. The most likely explanation for this discrepancy
concerns the possibility that large quantities of L-DOPA
can be easily extruded from the cell at the basal cell side; a fully
functional basal side provides an easy way to extrude intracellular
L-DOPA (much more important than extrusion at the apical
cell border), thereby reducing the saturability of the system. In fact,
the outward transfer of intracellular L-DOPA through the
basolateral cell border was markedly greater than that observed through
the apical side, although at both cell sides the outward transfer was a
nonsaturable process. By contrast, the apical and basal uptakes of
L-DOPA were found to be saturable processes with similar
Km values (482 ± 118 and 601 ± 67 µM), but Vmax values were substantially greater for the
basal than for the apical application (78.1 ± 5.4 and
14.6 ± 2.1 nmol · mg protein
1 · 6 min
1). Taken
together these data suggest that inward and outward transfers of L-DOPA are quantitatively more important at the
basolateral than at the apical cell border. This is in agreement with
the data of Hidalgo and Brochardt (7), who used phenylalanine, a large
neutral amino acid, as the substrate. Obviously, this would favor the
accumulation of L-DOPA at the basal pole of the cell, even with the assumption that there are no intracellular stores
for the substrate. Time course experiments in cells cultured in
polycarbonate filters also show that intracellular L-DOPA
is greater when a nonsaturating concentration of the substrate (0.5 µM) is applied from the basal side than from the apical side.
Another crucial step in the whole process of dopamine formation is the
decarboxylation of intracellular L-DOPA. Experiments conducted in cell homogenates showed that Caco-2 cells are endowed with
a high AADC activity and the efficiency of decarboxylation process, as
indicated by Km values, is quite similar to that
observed for the rat renal and jejunal epithelial cells (25). When cell monolayers were loaded with L-DOPA, in the absence of
benserazide, Caco-2 cells also synthesized dopamine. The formation of
dopamine was a time- and concentration-dependent process and rapidly
saturated, with an apparent Km of 27 µM; most of
the L-DOPA taken up was, in fact, decarboxylated to
dopamine. Again it is interesting to observe (Table 1) that
L-DOPA applied from the basal side is considerably more
decarboxylated than that applied from the apical side. This would fit
the hypothesis that L-DOPA uptake at the basolateral pole
is quantitatively more important than that at the apical cell border,
as discussed above. Because AADC is a cytosolic enzyme, these cells are
believed to synthesize dopamine as a result of the availability of
L-DOPA in the cytosol.
The intracellular fate of newly formed dopamine is another interesting
point to discuss. In the first set of experiments in cells cultured in
collagen-treated plastic, the outward transfer of newly formed dopamine
through the apical cell border was found to be a diffusional process.
On the other hand, in polycarbonate-cultured cells loaded with
L-DOPA from the basolateral cell border, the apical outward
transfer of newly formed dopamine was found to be a saturable process.
The magnitude of the outward transfer of newly formed dopamine differed
markedly depending on the technique used, being considerably greater in
cells cultured on plastic. In polycarbonate-cultured cells the outward
transfer of newly formed dopamine through the basolateral cell border
(evaluated in cells loaded with L-DOPA from the apical
side) was 5-10 times greater than that through the apical cell
border (evaluated in cells loaded with L-DOPA from the
basal side) and did not depend on the intracellular concentration of
dopamine. This, again, shows that the basolateral cell border in Caco-2
cells is quite permeable and may constitute an important route for
intracellular molecules to leave this compartment. It is possible that
the presence of a fully functional basolateral cell border may explain,
as well, the discrepant outward transfer of dopamine through the apical cell border observed in Caco-2 cells cultured on plastic and
polycarbonate filters. Although the present study was intended to
define the nature of the apical outward dopamine transfer, the finding
that this is a process with a Km of 623 ± 238 µM suggests that this does not correspond to the dopamine transporter
found in neuronal and nonneuronal cells.
In conclusion, the data presented here show that Caco-2 cells are
endowed with an efficient L-DOPA uptake system and
considerable AADC activity. Intracellular L-DOPA was found
to be rapidly converted to dopamine, some of which diffuses out of the
cell. The utilization of Caco-2 cells cultured on polycarbonate filters
probably provides a better way to look at processes such as the outward
transfer of intracellular molecules, namely, the outward transfer of
newly formed dopamine. Our observations also support the use of Caco-2 cells as in vitro models for the study of the intestinal dopaminergic physiology, although this may not reflect what happens in the intact
tissue.
 |
ACKNOWLEDGEMENTS |
This study was supported by Fundação Ciência e
Tecnologia Grant PECS/C/SAU/29/95.
 |
FOOTNOTES |
Address reprint requests to P. Soares-da-Silva.
Received 7 October 1997; accepted in final form 25 March 1998.
 |
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