Molecular modulation of inward and outward apical transporters
of L-dopa in LLC-PK1 cells
P.
Soares-Da-Silva and
M. P.
Serrão
Faculty of Medicine, Institute of Pharmacology and Therapeutics,
4200 Porto, Portugal
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ABSTRACT |
The present
study examined the nature of the apical inward and outward
L-3,4-dihydroxyphenylalanine (L-dopa)
transporters in LLC-PK1 cells and whether protein kinases
differentially modulate the activities of these transporters. The
apical inward transfer of L-dopa was promoted through an
energy-dependent and sodium-insensitive transporter (Michaelis
constant = 38 µM; maximum velocity = 2608 pmol · mg
protein
1 · 6 min
1). This
transporter was insensitive to N-(methylamino)-isobutyric acid but competitively inhibited by
2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC;
IC50 = 251 µM). Modulators of protein kinase A
(cAMP, forskolin, IBMX, and cholera toxin), protein kinase G (cGMP,
zaprinast, LY-83583 and sodium nitroprusside), and protein kinase C
(phorbol 12,13-dibutirate and chelerythrine) failed to affect the
accumulation of L-dopa. The Ca2+/calmodulin
inhibitors calmidazolium and trifluoperazine inhibited L-dopa uptake (IC50 of 72 and 55 µM,
respectively). The inhibitory effect of calmidazolium on the
accumulation of L-dopa was of the noncompetitive type. The
organic anion inhibitor DIDS, but not p-aminohippurate, and
the protein tyrosine kinase (PTK) inhibitor genistein significantly
increased L-dopa accumulation, which was mainly due to
inhibition of apical outward transfer of L-dopa. It
is concluded that LLC-PK1 cells take up L-dopa
over the apical cell border through the L-type amino acid transporter,
which appears to be under the control of
Ca2+-calmodulin-mediated pathways. The apical outward
transfer of L-dopa may be promoted through a DIDS-sensitive
transport mechanism and appears to be under the tonic control of PTK.
L-3,4-dihydroxyphenylalanine; L-type amino acid
transporter; 4,4'-diisothiocynatostilbene-2,2'-disulfonic
acid-sensitive transporter; Ca2+/calmodulin; protein
tyrosine kinase
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INTRODUCTION |
THE RENAL DOPAMINERGIC
SYSTEM is a local nonneuronal system constituted by epithelial
cells of proximal convoluted renal tubules rich in aromatic
L-amino acid decarboxylase (AADC) activity and using
circulating or filtered L-3,4-dihydroxyphenylalanine
(L-dopa) as a source for dopamine (19, 22,
31). Because the dopamine produced in this area is in close
proximity to renal cells that contain receptors for the amine, it has
been hypothesized that the amine may act as a paracrine or autocrine
substance (30). To overcome technical problems
related to the handling of freshly isolated renal tubular epithelial
cells, LLC-PK1 cells, which express proximal tubule
cell-like properties in vitro (17), have been used to
study dopamine receptors and the renal actions of the amine. These
cells have been also shown to contain AADC and convert
L-dopa to dopamine in a nonsaturable fashion up to 1 mM
L-dopa (5, 11, 12). Newly formed dopamine also
stimulated cAMP accumulation in LLC-PK1, and this effect
was attenuated by an equimolar concentration of carbidopa and blocked
by the D1-antagonist Sch-23390 (12). It
appears, therefore, that in LLC-PK1 cells, as in epithelial
cells of proximal tubules, locally formed dopamine can act as an
autocrine-paracrine substance.
Although the kidney is endowed with one of the highest levels of AADC
in the body and plasma levels of L-dopa are in the
nanomoles per milliliter range (13, 36), the rate-limiting
step for the synthesis of dopamine in renal tissues is still a matter
of debate. However, because Michaelis constant
(Km) values for L-dopa uptake are 10 times lower than Km values for decarboxylation
of L-dopa, it could be possible that L-dopa
uptake rather than decarboxylation may limit the rate of formation of
dopamine. In a previous report (38), we have concluded
that LLC-PK1 cells take up L-dopa through a
saturable, stereoselective, and temperature-dependent process when
applied from the apical and basolateral cell border, this being similar
to that occurring in rat renal proximal tubules (26, 34).
In several epithelia and at the level of brain capillary endothelium,
L-dopa and other large neutral amino acids are transported by the L-type amino acid transporter. This is a
sodium-independent and
2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid
(BHC)-sensitive transporter. Differentiation between A- and L-type
transporters is based on sodium dependence and sensitivity to
N-(methylamino)-isobutyric acid (MeAIB) and insensitivity to
BHC; the Bo,+-type is BHC-sensitive and sodium-dependent
transporter (1, 25, 28, 41).
The present study examined the effect of maneuvers that affect cellular
sodium and proton gradients and the sensitivity to inhibitors of amino
acid transport and inhibitors of organic cation and anion transporters
to define the nature of the transporters involved in the apical inward
and outward transfer of L-dopa. The result of maneuvers
that affect molecular mechanisms, namely those concerning protein
kinase A (PKA)-, protein kinase C (PKC)-, protein kinase G (PKG)-,
Ca2+/calmodulin-, and protein tyrosine kinase
(PTK)-mediated pathways on L-dopa uptake, was also
evaluated. It is reported that LLC-PK1 cells take up
L-dopa over the apical cell border through the L-type amino
acid transporter, which appears to be under the control of
Ca2+/calmodulin-mediated pathways. The apical outward
transfer of L-dopa is promoted through a DIDS-sensitive
transport mechanism and appears to be under the tonic control of PTK.
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METHODS |
Cell culture.
LLC-PK1 cells, a porcine-derived proximal renal tubule
epithelial cell line that retains several properties of proximal
tubular epithelial cells in culture (17), were obtained
from the American Type Culture Collection (Rockville, MD).
LLC-PK1 cells (ATCC CRL 1392; passages
198-206) were maintained in a humidified atmosphere of 5%
CO2-95% air at 37°C and grown in Medium 199 (Sigma,
St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml treptomycin (Sigma), 3% fetal bovine serum
(Sigma), and 25 mM HEPES (Sigma). For subculturing, the cells were
dissociated with 0.05% trypsin-EDTA, split 1:4, and subcultured in
Costar 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 (internal
diameter 16 mm, Costar) at a density of 40,000 cells/well or onto
collagen-treated 0.2-µm polycarbonate filter supports (internal
diameter 12 mm Transwell, Costar) at a density 13,000 cells per well
(2.0 × 104 cells cm2). The cell medium
was changed every 2 days, and the cells reached confluence after
3-5 days of incubation. 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 6-8
days after the initial seeding and each squared centimeter contained
~80 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; thereafter, the cell monolayers
were preincubated for 30 min in Hanks' medium at 37°C. The 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 benserazide (50 µM) and tolcapone (1 µM) to inhibit the enzymes AADC and
catechol-O-methyltransferase, respectively. Time-course
studies were performed in experiments in which cells were incubated
with 0.5 µM substrate for 1, 3, 6, and 12 min. Saturation experiments
were performed in cells incubated for 6 min with increasing
concentrations of L-dopa (10-250 µM). In experiments
performed in the presence of different concentrations of sodium, NaCl
was replaced by an equimolar concentration of choline chloride. Test
substances were applied from the apical side only and were present
during the preincubation and incubation periods. During preincubation
and incubation, the cells were continuously shaken and maintained at
37°C. Apical uptake was initiated by the addition of 2 ml Hanks'
medium with a given concentration of the substrate. Uptake was
terminated by the rapid removal of uptake solution by means of a vacuum
pump connected to a Pasteur pipette followed by a rapid wash with cold
Hanks' medium and the addition of 250 µl of 0.2 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.
Previous studies have shown that some of the L-dopa
accumulated in LLC-PK1 cells can leave the cell through
apical outward transporter(s) (38), the inhibition of
which leads to an increase in the cellular accumulation of
L-dopa (39). Thus in experiments designed to
study the effect of drugs that increased the intracellular accumulation
of L-dopa, cells were incubated with 25 µM
L-dopa applied from the basal cell border and uptake
(accumulation in the cell monolayer) and flux (transfer to opposite
chamber) were measured over a 6-min period. Test drugs were applied
from the apical side only and were present during the preincubation and incubation periods. [14C]sorbitol (0.4 µM) was used to
estimate paracellular fluxes and extracellular trapping of
L-dopa during L-dopa uptake studies. Paracellular fluxes were estimated dividing concentration of
[14C]sorbitol in the apical chamber by the concentration
of [14C]sorbitol in the basal chamber. Extracellular
trapping was calculated dividing the amount of
[14C]sorbitol in the cell monolayer by the amount of
[14C]sorbitol in the basal chamber. At the end of
incubation, cells were placed on ice and the medium bathing the apical
cell border was collected, acidified with perchloric acid, and stored
at 4°C till assayed for L-dopa. 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.
Assay of L-dopa.
L-dopa was quantified by means of high-pressure liquid
chromatography with electrochemical detection, as previously reported (38). The high-pressure liquid chromatograph system
consisted of a pump (Gilson model 302; Gilson Medical Electronics,
Villiers le Bel, France) connected to a manometric module (Gilson model 802 C) and a stainless-steel 5-µm ODS column (Biophase; Bioanalytical Systems, West Lafayette, IN) of 25 cm in length; samples were injected
by means of an automatic sample injector (Gilson model 231) connected
to a Gilson dilutor (model 401). The mobile phase was a degassed
solution of citric acid (0.1 mM), sodium octylsulphate (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 (Gilson model 141); the detector cell was
operated at 0.75 V. The current produced was monitored using the Gilson
712 HPLC software. The lower limits for detection of L-dopa
ranged from 350 to 500 fmol.
Transport of p-aminohippurate.
Transport of p-aminohippurate (PAH) was initiated by adding
Hanks' medium containing [3H]PAH (3 µM) and
[14C]sorbitol (3 µM) to the basal or to the apical side
of the monolayers. [14C]sorbitol was used to estimate
paracellular fluxes and extracellular trapping of
[3H]PAH. For the measurement of transepithelial
transport, the medium in the other side was collected after incubation
for the specified period of time, and the radioactivity was counted. In
time course studies, an aliquot of the medium (100 µl) was collected
every 15 min over a period of 60 min, and the aliquot was replaced with an equal volume of Hanks' medium. The data at 30, 45, and 60 min represent cumulative values. The monolayers were agitated every 5 min
during transport measurement. In some experiments, cell monolayers were
incubated in the presence of unlabeled PAH (1 mM) added from the basal
side. At the end of the transport experiment, the medium was
immediately aspirated and the filter was washed three times with
ice-cold Hanks' medium. 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.
P-glycoprotein activity.
P-glycoprotein activity was measured according to the procedure
described by Holló et al. (15), with minor
modifications. In brief, LLC-PK1 cells cultured in
collagen-treated cover slips were incubated in culture medium for 30 min in the absence or the presence of verapamil (25 µM) and genistein
(100 µM). Thereafter, the cells were transferred to a Perkin-Elmer
cuvette holder (model LS 50) and incubated with Hanks' medium
containing 0.5 µM calcein acetoxymethyl ester (calcein-AM) for a
further 5 min at 37°C with continuous stirring. The 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. Calcein-AM is highly lipid soluble and rapidly
penetrates the plasma membrane and is practically nonfluorescent. By
cleavage of the ester bonds, intracellular esterases transform the dye to a hydrophilic and intensely fluorescent free acid form. Fluorescence was measured in a FluoroMax-2 (Jobin Yvon-SPEX, Edison, NJ)
spectrofluorometer using excitation and emission wavelenghts of 450 and
507 nm, respectively. Time-resolved experiments were started 5 min
after the addition of calcein-AM to the medium bathing the cells and
lasted for 20 min. In experiments using cells pretreated with verapamil
and genistein, the incubation medium also contained verapamil (25 µM)
or genistein (100 µM). Calibration of dye concentration was based on
the measurements of free calcein fluorescence in the same instrument
under identical conditions (in the absence and in the presence of
verapamil or genistein). All experiments were repeated five to seven
times with different batches of cell monolayers.
Protein assay.
The protein content of monolayers of LLC-PK1 cells was
determined by the method of Bradford (2), with human serum
albumin as a standard.
Cell viability.
Cells cultured in collagen-treated plastic supports were preincubated
for 15 min at 37°C and then incubated in the absence or the presence
of L-dopa and test compounds for further 6 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.
Km and maximum velocity
(Vmax) values for the uptake of
L-dopa, as determined in saturation experiments were
calculated from nonlinear regression analysis using the GraphPad Prism
statistics software package (24). P-glycoprotein activity
was determined by the slope of the accumulation of calcein (in pmol/mg
protein) measured by linear regression analysis (24).
Arithmetic means are given with SE. Statistical analysis was performed
by one-way ANOVA followed by Newman-Keuls test for multiple
comparisons. A P value less than 0.05 was assumed to denote
a significant difference.
Drugs.
Actinomycin D, amiloride hydrochloride, BHC, amphotericin B,
calmidazolium, chelerythrine chloride, cholera toxin, cycloheximide, N-6,2'-O-dibutyryl cAMP, cGMP,
1,1'-diethyl-2,4'-cyanine (decynium 24), DIDS, 2,4-dinitrophenol,
forskolin, genistein, genistin, IBMX, L-dopa, MeAIB,
ouabain, phorbol 12,13-dibutyrate (PDBu), 4
-phorbol
12,13-didecanoate (PDDC), sodium nitroprusside, trifluoperazine dihydrochloride, trypan blue, tyrphostin 1 and tyrphostin 25 were purchased from Sigma, St. Louis, MO. LY-83583
[6-(phenylamino)-5,8-quinolinedione] and zaprinast were obtained from
Research Biochemicals International (Natick, MA) and May & Baker
(Dorset, England), respectively. Tolcapone was kindly donated
by the late Professor Mosé Da Prada (Hoffman La Roche, Basle,
Switzerland). [14C]sorbitol (specific activity 990 GBq/mmol) and [3H]PAH (specific activity 1.8-2.2
GBq/mmol) were purchased from New England Nuclear (Boston, MA).
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RESULTS |
The accumulation of a nonsaturating concentration (0.5 µM) of
L-dopa, in time-course experiments, increased linearly with time for several minutes (Fig.
1A). At 6 min when uptake was
linear and considering intracellular water as 7.0 ± 0.7 µl/mg
protein (38), the intracellular L-dopa
concentration was 4.0 ± 0.4 µM at medium concentration of 0.5 µM. This represented a cell concentration of L-dopa that
was 8.0 ± 0.8 times higher than the corresponding medium
concentration. In a subsequent set of experiments, designed to
determine the kinetic parameters of the L-dopa apical
transporter, the cells were incubated for 6 min with increasing
concentrations (1 to 250 µM) of the substrate (Fig. 1B).
Nonlinear analysis of the saturation curve for L-dopa
revealed a Km value (in µM) of 47 ± 8 and a Vmax value (in pmol · mg
protein
1 · 6 min
1) of 3,069 ± 224.

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Fig. 1.
A: time course of
L-3,4-dihydroxyphenylalanine (L-dopa) in renal
tubular epithelial cells (LLC-PK1). Cells were incubated at
37°C with 0.5 µM of L-dopa and results are levels (in
pmol/mg protein) of substrate accumulated. Each point are the mean of 8 experiments/group; vertical lines show SE. B:
Concentration-dependent accumulation of L-dopa in
LLC-PK1 cells. Cells were incubated for 6 min at 37°C and
increasing concentrations (1 to 250 µM) of the substrate were applied
from the apical border. Results reflect levels (in pmol · mg
protein 1 · 6 min 1) of accumulated
L-dopa. Each point is mean of 4 experiments/groups;
vertical lines show SE.
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To evaluate the metabolic requirements for L-dopa uptake,
cells were pretreated with the uncoupling agent 2,4-dinitrophenol or
incubated at 4°C. Pretreatment with 2,4-dinitrophenol (1 mM) resulted
in a marked reduction in L-dopa (2.5 µM) uptake (Table 1). Similarly, the effect of reducing
temperature from 37° C to 4°C during preincubation and incubation
was a marked reduction in L-dopa (2.5 µM) accumulation
(Table 1).
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Table 1.
Effect of manoeuvres that affect energy-dependent process,
transepithelial flux of sodium ions and protons and of modulators
of PKA, PKG, PKC, and PTK on the uptake of L-dopa (2.5 µM) in LLC-PK1 cells
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Reducing extracellular sodium (from 140 mM to 70, 35 and 0 mM) did not
affect the accumulation of L-dopa (Fig.
2A). Moreover, in the absence
of extracellular sodium (replaced by an equimolar concentration of
choline), Km and Vmax
values for L-dopa were similar to those observed in the
presence of sodium (Table 2). Maneuvers
that affect transepithelial flux of sodium, such as acidification of
the extracellular milieu (from pH 7.4 to pH 6.9 or pH 6.4) and the
addition of amphotericin B (2.5 µg/ml), amiloride (100 µM), or
ouabain (500 µM), failed to affect the accumulation of
L-dopa (Table 1). MeAIB (1 mM) failed to affect the uptake of L-dopa, whereas BHC produced a concentration-dependent
inhibition of L-dopa uptake (IC50 = 251 ± 26 µM) (Fig. 2B). The inhibitory effect of 1 mM BHC on the accumulation of L-dopa was of the competitive type, as evidenced by the increase in Km but not
Vmax values for L-dopa uptake (Table
2). Taken together, these results suggest that the apical inward
transfer of L-dopa may be promoted through the
BHC-sensitive and sodium-independent L-type amino acid transporter.

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Fig. 2.
Effect of increasing medium concentrations of sodium
(A; 0, 20, 40, 60, 120 and 140 mM) and of
N-(methylamino)-isobutyric acid (MeAIB; B) and
2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC) on accumulation
of L-DOPA (2.5 µM) in LLC-PK1 cells. Symbols
are mean of 4-8 experiments/group; vertical lines show SE.
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Table 2.
Km and Vmax values for L-dopa
uptake in LLC-PK1 cells under control conditions, in
absence of sodium; presence of BHC, DIDS, genistein, calmidazolium;
or exposed for 24 h in presence of L-dopa
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Separate experiments were designed to evaluate whether the presence of
a saturating concentration of L-dopa (250 µM) in the growth medium for 24 h affect the L-dopa transporter.
Cells were cultured as described in METHODS, and the growth
medium was substituted 24 h before experiments by fetal bovine
serum (FBS)-free growth medium with added L-dopa (250 µM)
or the vehicle. On the day of the experiments, cells were washed with
Hanks' medium and were then preincubated for 30 min followed by
incubation with increasing concentrations of L-dopa (10 to
250 µM) for 6 min. Nonlinear analysis of the saturation curve for
L-dopa revealed similar Km and
Vmax values for vehicle- and
L-dopa-treated cells (Table 2).
Because neither the affinity nor the number of transport units was
affected by prolonged exposure to a saturating concentration of
L-dopa, we were then interested to know to which extent
inhibition of synthesis of new transporter units would affect the
uptake of L-dopa. For this purpose, confluent
monolayers were exposed for 3 h to the DNA transcription
inhibitor actinomycin D (0.5 µg/ml) and the protein synthesis
inhibitor cycloheximide (70 µM) in FBS-free growth medium. Then the
cells were allowed to recover for 12 h or 24 h in FBS-free
growth medium before L-dopa uptake experiments took place.
As shown in Fig. 3, exposure to both
actinomycin D and cycloheximide was found to significantly reduce the
ability of LLC-PK1 cells to accumulate L-dopa
(2.5 µM). The decrease in the ability to take up L-dopa
was proportional to the extent of the interval between exposure to
actinomycin D and cycloheximide and the incubation with
L-dopa. The microscopic appearance of vehicle (0.1%
DMSO)-treated cells was similar to those exposed to actinomycin D or
cycloheximide. Thus the more intense effect of actinomycin D at 24 h than at 12 h may be not related to unspecific toxicity, but
indicates a slow rate of synthesis of transporters. Though less
evident, the effects of cycloheximide point in the same direction.

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Fig. 3.
Effect of pretreatment of LLC-PK1 cells with
(A) actinomycin D or (B) cycloheximide on the
accumulation of L-dopa (2.5 µM). Cells were exposed for
3 h to actinomycin D (0.5 µg/ml) or cycloheximide (70 µM) in
FBS-free growth medium; then, cells were allowed to recover for 12 or
24 h in FBS-free growth medium before incubation with
L-dopa. Columns are mean of 8 experiments/group; vertical
lines show SE.
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Though L-dopa exists as a Zwitteron, it was decided to test
the effects of decynium 24 and DIDS, inhibitors of the organic cation
and organic anion transporters, respectively. Decynium 24 failed to
affect the accumulation of L-dopa, whereas DIDS produced a
concentration-dependent increase in the accumulation of a nonsaturating concentration (2.5 µM) of L-dopa (Fig.
4). As shown in Table 2, pretreatment of
cells with 1 mM DIDS was found to markedly increase (P < 0.05) the accumulation of increasing concentrations of
L-dopa without changes in Km values.
In contrast, the organic anion PAH was found to produce a slight
(P = 0.09) decrease (~20% reduction) in the
accumulation of L-dopa (Fig. 4). Though DIDS is a
well-known inhibitor of the organic anion transporter
(16), it is unlikely that its effects upon
L-dopa uptake might be attributed to the interference with
this transporter. First, this cell line has been shown by several
groups to have lost the organic anion transporter (16, 23,
27). Second, movement of organic anions is usually performed in
the opposite direction (basal-to-apical) to that imposed here for
L-dopa. On the other hand, it is difficult to conceive a
model in which DIDS promotes the entry of L-dopa into the
cell through the apical cell border. By contrast, previous studies have
shown that LLC-PK1 cells are able to extrude
L-dopa through apical outward transporter(s)
(38), the inhibition of which leads to an increase in the
cellular accumulation of L-dopa (39). Thus to
clarify these issues it was decided to evaluate whether DIDS decreases
the basal-to-apical flux of L-dopa, and second if
LLC-PK1 cells are endowed with the organic anion
transporter. Cells cultured in polycarbonate filters were incubated
with L-dopa (25 µM) applied from the basal cell border
and the basal-to-apical flux measured. As shown in Fig.
5, DIDS (1 mM) markedly reduced the
basal-to-apical flux of L-dopa and increased the
accumulation of L-dopa in the cell. By contrast, as shown
in Fig. 6, the transepithelial transport
and the cell accumulation of [3H]PAH was close to that of
[14C]sorbitol, indicating that the apparent transport and
accumulation of [3H]PAH represents nonspecific transfer
and/or trapping. The basal-to-apical transport and cell accumulation of
[3H]PAH and [14C]sorbitol were not affected
by unlabeled PAH. These two sets of experiments indicate that
LLC-PK1 cells were devoid of the organic anion transporter,
and the increase in L-dopa accumulation by DIDS depended on
its ability to reduce the outward transfer of L-dopa
through the apical cell border but was unrelated to its properties as
organic anion inhibitor.

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Fig. 4.
Effect of decynium 24, DIDS, and
p-aminohippurate (PAH) on accumulation of L-dopa
(2.5 µM) in LLC-PK1 cells. Symbols are mean of 4 to 8 experiments/group; vertical lines show SE. * Significantly different
from corresponding control values (P < 0.05).
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Fig. 5.
Effect of DIDS (1 mM) on the apical flux and cell
accumulation of L-dopa (25 µM) applied from basolateral
cell border in LLC-PK1 cells. Columns are mean of 4 experiments/group; vertical lines show SE. * Significantly different
from corresponding control values (P < 0.05).
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Fig. 6.
Effect of unlabeled PAH (1 mM) on basal-to-apical
transports (A) and cell accumulation of 3 µM
[3H]PAH and [14C]sorbitol (B)
across LLC-PK1 monolayers. Each point or column is mean of
4 separate experiments.
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Up to this stage, the combined results revealed that in
LLC-PK1 cells the apical inward transfer of
L-dopa was promoted through the L-type amino acid
transporter and intracellular L-dopa could be extruded by a
DIDS-sensitive apical transporter that lacked the properties of an
organic anion transporter. The next series of experiments explored the
role of intracellular regulatory pathways in the cellular handling of
L-dopa applied from the apical cell border. Involvement of
a PKA-mediated pathway in the regulation of L-dopa uptake
was tested by examining the effect of pretreating LLC-PK1
cells for 30 min with compounds that are known to increase intracellular cAMP levels. Dibutyryl cAMP (0.5 mM), forskolin (50 µM), IBMX (0.1 mM), and cholera toxin (5 µg/ml) failed to affect
the accumulation of a nonsaturating concentration of L-dopa (Table 1). In another series of experiments, we tested the involvement of a PKG-mediated pathway in the regulation of L-dopa
uptake. cGMP (1 mM), zaprinast (30 µM), LY-83583 (30 µM), and
sodium nitroprusside (100 µM) failed to affect the accumulation of
L-dopa (Table 1). The possible role of PKC in the
regulation of L-dopa uptake in LLC-PK1 cells
was tested by examining the effect of pretreating cells with either PKC
activators or inhibitors. The PKC activator (1 µM), the inactive
phorbol ester PDDC (1 µM), and the PKC inhibitor chelerythrine (50 µM) were found to fail to affect the accumulation of
L-dopa (Table 1).
In another study, we tested the involvement of PTK in the regulation of
L-dopa uptake by LLC-PK1 cells. The PTK
inhibitors genistein and tyrphostin 25 were found to increase the
accumulation of L-dopa (2.5 µM), whereas their negative
controls genistin and tyrphostin 1 were devoid of effect (Fig.
7). At the highest concentrations (300 µM), genistein and tyrphostin 25 significantly (P < 0.05) increased L-dopa accumulation by 112 ± 23% and
37 ± 4%, respectively. As shown in Table 2, pretreatment of
cells with 300 µM genistein was found to significantly increase
(P < 0.05) the maximal accumulation (Vmax) of increasing concentrations of
L-dopa without significant changes in
Km values. To test whether the increased
accumulation of L-dopa-induced by genistein was due to a
reduced outward transfer of intracellular (recently accumulated)
L-dopa, cells cultured in polycarbonate filters were
incubated with L-dopa (25 µM) applied from the basal cell
border and the basal-to-apical flux measured. As shown in Fig.
8, genistein (100 µM) markedly reduced
the basal-to-apical flux of L-dopa and increased the
accumulation of L-dopa in the cell, suggesting that PTK may
be tonically active in promoting the phosphorylation of the
L-dopa outward transfer. Because P-glycoprotein is one
of the transporters involved in the apical outward transfer of
L-dopa, it was felt worthwhile to evaluate the effect of
genistein upon P-glycoprotein activity in LLC-PK1 cells.
P-glycoprotein activity was measured according to the procedure
described by Holló et al. (15), using calcein as
substrate for P-glycoprotein. Figure 9
shows the accumulation of calcein in LLC-PK1 cells in control conditions and in the presence of verapamil (25 µM) or genistein (100 µM) during a 20-min incubation period. As can be observed in this figure, the P-glycoprotein inhibitor verapamil, but
not genistein, markedly (P < 0.05) increased the rate
of accumulation of calcein. The lack of effect of genistein on calcein
accumulation suggests that the increased accumulation of
L-dopa by genistein may be not related to inhibition of
P-glycoprotein activity.

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Fig. 7.
Effect of active (genistein and tyrphostin 25) and
inactive (genistin and tyrphostin 1) PTK inhibitors on accumulation of
L-dopa (2.5 µM) in LLC-PK1 cells. Columns
or symbols are mean of 4-8 experiments/group; vertical lines show
SE. * Significantly different from corresponding control values
(P < 0.05).
|
|

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Fig. 8.
Effect of genistein (300 µM) on apical flux
(A) and cell accumulation (B) of
L-dopa (25 µM) applied from basolateral cell border in
LLC-PK1 cells. Columns are mean of 4-8
experiments/group; vertical lines show SE. * Significantly different
from corresponding control values (P < 0.05).
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Fig. 9.
Effect of verapamil (25 µM) and genistein (100 µM) on
accumulation of calcein in LLC-PK1 cells incubated in
presence of calcein (0.5 µM) for 20 min. Values are percent of
calcein levels at t = 0 min. Symbols are means of
5-7 experiments/group; SE values are indicated by the broken
lines.
|
|
In the final series of experiments, the role of
Ca2+/calmodulin-mediated pathways in the regulation of
L-dopa by LLC-PK1 cells was tested by examining
the effect of pretreating the cells with the calmodulin inhibitors
calmidazolium and trifluoperazine. Both compounds produced
concentration-dependent inhibition of L-dopa (2.5 µM)
uptake with IC50s of 71.5 ± 1.2 µM and 54.7 ± 1.0 µM, respectively (Fig. 10). The
inhibitory effect of 30 µM calmidazolium on the accumulation of
L-dopa was of the noncompetitive type, as evidenced by the
decrease in Vmax without changes in
Km values for L-dopa uptake (Table
2).

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Fig. 10.
Effect of calmidazolium and trifluoperazine on the
accumulation of L-dopa (2.5 µM) in LLC-PK1
cells. Columns or symbols are mean of 4-8 experiments/group;
vertical lines show SE.
|
|
 |
DISCUSSION |
The results presented here show that LLC-PK1 cells
transport L-dopa quite efficiently through the apical cell
border, and several findings demonstrate that this uptake process is a
facilitated mechanism. First, steady-state uptake of nonsaturating
concentrations of L-dopa showed a linear dependence on
incubation time. The efficiency of the L-dopa transport in
LLC-PK1 cells can be also evidenced by the ratio of
L-dopa concentration in cellular water to medium concentration. The intracellular L-dopa concentration at 6 min, when uptake was linear, was eight times greater than that which could be expected by passive equilibration of L-dopa.
Second, at 6 min of incubation the cellular transport of
L-dopa showed a curvilinear dependence on substrate medium
concentration, suggesting that the uptake was saturable. Thirdly, both
low temperature and the uncoupling agent 2,4-dinitrophenol markedly
inhibited the uptake of L-dopa. Finally, inhibition of
synthesis of new transporter units by the DNA transcription inhibitor
actinomycin D and the protein synthesis inhibitor cycloheximide
significantly reduced the ability of LLC-PK1 cells to
accumulate L-dopa.
The sensitivity of L-dopa uptake to BHC, but not to MeAIB,
supports the view that L-dopa inward transfer in
LLC-PK1 cells is promoted neither by the A- nor the
ASC-type amino acid transporter, but most probably by the L-type amino
acid transporter. Other evidence agreeing with this suggestion is that
sodium in the incubation appeared to play no role in L-dopa
uptake, as indicated by the lack of effect of sodium removal on uptake.
Moreover, maneuvers that alter sodium gradients, such as those
resulting from the application of amiloride (an inhibitor of the
Na+/H+ exchanger), ouabain (an inhibitor of the
Na+-K+-ATPase), and amphotericin B (a sodium
ionophore), or changes in extracellular pH, were found not to alter
L-dopa uptake. The L-type, leucine preferring, amino acid
transporter is facilitative, sodium independent, and blocked by BHC but
not by MeAIB (1, 25, 28). Another point suggesting that
L-dopa in LLC-PK1 cells is transported through
the L-type amino acid transporter concerns the similarity
of Km values for L-dopa uptake and
IC50 values for BHC when acting as an inhibitor for
L-dopa uptake. Several findings suggest that the
type of inhibition by BHC on L-dopa uptake is of the
competitive type. Only Km values, but not
Vmax values, for L-dopa uptake were
changed when saturation experiments were performed in the presence of a
concentration of the inhibitor equal to IC50 values. The
finding that L-dopa inward transport in LLC-PK1
cells is promoted through the sodium-insensitive L-type amino acid
transporter contrasts with the role of sodium in the formation of renal
dopamine. In fact, studies from several groups have shown that the
increase in the renal delivery of sodium constitutes the most powerful
stimulus for the production of renal dopamine (14, 29,
32). Studies from our group using human and rat kidney slices
also showed that L-dopa uptake is a sodium-dependent and
ouabain-sensitive process (33, 35), suggesting that the sodium-dependent increase in urinary dopamine depends on enhanced uptake of L-dopa into tubular epithelial cells. More
recently, we have shown that OK cells are endowed with sodium-dependent and sodium-independent L-dopa transporters (9,
10), which is currently being investigated in more detail.
Altogether, this suggests that LLC-PK1 cells may lack or
have loss the sodium-dependent transporter.
The finding that inhibition of the organic cation and the organic anion
transporters by decynium 24 and DIDS, respectively, failed to reduce
L-dopa accumulation indicates that these transporters are
not involved in L-dopa uptake in LLC-PK1 cells.
By contrast, DIDS markedly increased L-dopa accumulation;
this was a concentration-dependent effect, at the highest concentration
(1 mM) resulting in an increase in Vmax values
for L-DOPA uptake without changes in
Km values (Table 1). Because in these
experiments cells were cultured in plastic supports, the most likely
explanation is that enhanced accumulation resulted from a reduced
apical outward transfer of L-dopa. This is in agreement
with findings in experiments carried out in cells cultured in
polycarbonate filters, where DIDS produced a marked reduction in
basal-to-apical flux of L-dopa and an increase in
L-dopa accumulation.
After defining the mechanism of uptake, we then examined the regulation
of L-dopa transport in LLC-PK1 cells. We
concentrated on intracellular regulatory pathways that have been shown
to play an important role in the regulation of uptake of other
substrates by epithelial cells (PKA-, PKG-, PKC-,
Ca2+/calmodulin- and PTK-mediated pathways). Using specific
modulators of these pathways, we found that PKA-, PKG- and PKC-mediated
pathways appear to have no role in regulating L-dopa uptake
in LLC-PK1 cells. In contrast, antagonists of
Ca2+/calmodulin-mediated pathways, such as calmidazolium
and trifluoperazine, caused a significant and concentration-dependent
reduction in L-dopa uptake. The inhibitory effect of
calmidazolium was accompanied by a marked decrease in
Vmax values without changes in
Km values, which is compatible with a
noncompetitive inhibitory profile. This would suggest that
calmidazolium might reduce the number of L-type amino acid
transporters in the apical membrane. Similar findings were observed in
a model of renal epithelial cell model, the HK-2 cells, where
calmidazolium and other inhibitors of
Ca2+/calmodulin-mediated pathways, such as trifluoperazine
and KN-62, were found to markedly reduce riboflavin accumulation
(21). In contrast to that observed for inhibitors of
Ca2+/calmodulin-mediated pathways, PTK inhibitors genistein
and tryphostin 25 were found to markedly increase L-dopa
accumulation in LLC-PK1 cells. This was a
concentration-dependent effect, and the inactive analogs genistin and
tyrphostin 1 failed to affect the accumulation of L-dopa in
LLC-PK1 cells cultured in plastic supports. When tested in
cells cultured in polycarbonate filters, the effect of genistein was a
marked reduction in basal-to-apical flux of L-dopa,
this being accompanied by a large increase in the accumulation of
L-dopa. This indicates that PTK-mediated pathways do not
interfere with the inward movement of L-dopa but exert a
tonic stimulatory effect upon its outward transfer.
The apical outward transfer of L-dopa in
LLC-PK1 cells has been suggested to represent a powerful
mechanism for the clearance of intracellular L-dopa in
steady-state conditions (removing ~13% of intracellular
L-dopa at a rate of 1.0 pmol · mg
protein
1 · min
1; Ref. 38). However,
this outward transfer of L-dopa is considerably less
dynamic than the inward transfer at initial rate of uptake (3.6 pmol · mg protein
1 · min
1;
Ref. 38). The data presented here suggest that this outward transfer is
a DIDS-sensitive mechanism and is susceptible of modulation by
PTK-mediated pathways. Sensitivity of L-dopa outward
transfer to DIDS would suggest the involvement of an organic anion
transporter; 1) transport from the basal to the apical cell
side corresponds to the secretion in renal tubules and 2)
DIDS is a well-known inhibitor of the organic anion transporter
(16). However, this is quite unlikely to be the case,
because this cell line has been shown by several groups to have lost
the organic anion transporter (16, 23, 27). As has been
previously reported by others (16), the
basal-to-apical transport and accumulation of [3H]PAH
was not higher than that of [14C]sorbitol, indicating
that the apparent transport and accumulation represents
nonspecific uptake and/or trapping. In agreement with these
results is the finding that PAH failed to increase L-dopa accumulation in LLC-PK1 cells (Fig. 4). It is likely,
therefore, that DIDS may have exerted its effects through inhibition of
an apical outward transporter rather than the organic anion
transporter. This would agree with the results recently reported for
the apical secretion of ciprofloxacin by human intestinal Caco-2
epithelia, in which a novel DIDS-sensitive transport mechanism has been
suggested to be involved (3). In addition, there is
evidence in the literature showing that DIDS and probenecid block
transport of several substrates in LLC-PK1 cells (4,
6, 7, 20, 40). The inhibitory effect of PTK inhibitors genistein
and tyrphostin 25 upon the apical secretion of L-dopa may
be to some extent connected to this DIDS-sensitive transport mechanism.
In fact, genistein has recently been found to effectively inhibit the
secretion of anionic substrates of liver canalicular multispecific
organic anion transporter in Wistar rats (18) and diminish
PAH uptake in S2 segments of rabbit proximal tubules
(8). On the other hand, there is evidence suggesting that
the apical outward transfer of L-dopa in
LLC-PK1 cells may be promoted in part through
P-glycoprotein (37, 39). Therefore, an alternative
explanation for the inhibitory effect of genistein upon the apical
secretion of L-dopa could be that resulting from inhibition
of P-glycoprotein phosphorylation by PTK. Verapamil, which inhibits
P-glycoprotein activity and markedly decreases secretion of
L-dopa in LLC-PK1 cells (37, 39),
was found to markedly increase calcein accumulation. However, the finding that genistein failed to affect the accumulation of calcein favors the view that modulation of L-dopa outward transfer
by PTK may not involve phosphorylation of P-glycoprotein.
In conclusion, it is suggested that LLC-PK1 cells take up
L-dopa over the apical cell border through the L-type amino
acid transporter, which appears to be under the control of
Ca2+/calmodulin-mediated pathways. The apical outward
transfer of L-dopa may be promoted through a DIDS-sensitive
transport mechanism and appears to be under the tonic control of PTK,
the inhibition of which leads to increases in the intracellular
accumulation of L-dopa. Taken together the apical membrane
in LLC-PK1 cells is endowed with different transporters for
the handling of L-dopa at its cytoplasmic and extracellular
sides. The fact that all experiments were performed in conditions of
AADC inhibition may not reduce the relevance of these findings, because
L-dopa uptake process does not rate limit the formation of
dopamine; large amounts of taken up L-dopa are not
converted to dopamine (38). On the other hand, substance
that interfere with the outward transfer of L-dopa may even
be beneficial to increase its availability for decarboxylation, leading
to enhanced formation of dopamine.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P
Soares-da-Silva, Institute of Pharmacology & Therapeutics, Faculty of
Medicine, 4200 Porto, Portugal (E-mail:
Patricio.Soares{at}mail.telepac.pt).
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.
Received 29 December 1999; accepted in final form 31 May 2000.
 |
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