H+-linked transport of salicylic acid, an NSAID,
in the human trophoblast cell line BeWo
Akiko
Emoto1,
Fumihiko
Ushigome1,
Noriko
Koyabu1,
Hiroshi
Kajiya2,
Koji
Okabe2,
Shoji
Satoh3,
Kiyomi
Tsukimori3,
Hitoo
Nakano3,
Hisakazu
Ohtani1, and
Yasufumi
Sawada1
1 Department of Medico-Pharmaceutical Sciences, Graduate
School of Pharmaceutical Sciences,. and 3 Department of
Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu
University, Higashi-ku, Fukuoka 812-8582; and 2 Department of
Physiological Science and Molecular Biology, Fukuoka Dental College,
Fukuoka 814-0193, Japan
 |
ABSTRACT |
We investigated the
transport of salicylic acid and L-lactic acid across the
placenta using the human trophoblast cell line BeWo. We performed
uptake experiments and measured the change in intracellular pH
(pHi). The uptakes of [14C]salicylic acid and
L-[14C]lactic acid were temperature- and
extracellular pH-dependent and saturable at higher concentrations. Both
uptakes were also reduced by FCCP, nigericin, and NaN3.
Various nonsteroidal anti-inflammatory drugs (NSAIDs) strongly
inhibited the uptake of L-[14C]lactic acid.
Salicylic acid and ibuprofen noncompetitively inhibited the uptake of
L-[14C]lactic acid.
-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporter
inhibitor, suppressed the uptake of
L-[14C]lactic acid but not that of
[14C]salicylic acid. CHC also suppressed the decrease of
pHi induced by L-lactic acid but had little
effect on that induced by salicylic acid or diclofenac. These results
suggest that NSAIDs are potent inhibitors of lactate transporters,
although they are transported mainly by a transport system distinct
from that for L-lactic acid.
L-lactic acid; blood-placental barrier; monocarboxylate
transporter;
-cyano-4-hydroxycinnamate; intracellular pH
 |
INTRODUCTION |
THE
BLOOD-PLACENTAL BARRIER controls the transfer of nutrients from
the maternal to the fetal circulation (11). The maternal and fetal circulations are separated by chorionic villi consisting of
trophoblast cells. The microvillous membrane of trophoblast cells
directly contacts the maternal circulation and expresses a variety of
transporters that actively transport nutrients such as glucose and
amino acids to the fetus (2, 10, 25). Furthermore, various
exogenous compounds are recognized by these transporters and by efflux
pumps (e.g., P-glycoprotein) that operate to prevent their accumulation
in the fetus (27).
It has been reported that nonsteroidal anti-inflammatory drugs
(NSAIDs), such as salicylic acid, ibuprofen, ketoprofen, and diclofenac, may cause adverse effects such as malformation or constriction of the ductus arteriosus when used during pregnancy (17). On the other hand, acetylsalicylic acid is
administered even to gravidae for the treatment of pregnancy-induced
hypertension and preeclampsia (29). To minimize the risks
of NSAIDs to the gravida and fetus, it is important to understand the
mechanism of their permeation across the placenta. Although the
mechanism has not been investigated in detail, it is expected that
NSAIDs would interact with monocarboxylate transporters (MCTs) and/or other organic anion transporters (OATs), since they possess a carboxyl moiety.
MCT-mediated transport is linked to a proton gradient and is inhibited
by various short-chain monocarboxylic acids and
-cyano-4-hydroxycinnamate (CHC), a classical inhibitor of MCT
(7). Full-length cDNAs of MCT1 ~8 have already been
cloned (7, 19). MCT1, -2, and -4 transport endogenous
monocarboxylic acids such as lactic acid (pKa 3.86) and pyruvic acid,
which are mostly ionized at physiological pH (7). On the
other hand, several exogenous monocarboxylic acids, such as salicylic
acid, benzoic acid, and valproic acid, have been reported to be
transported in the cells expressing rat MCT1 (26).
Similarly, salicylic acid is expected to be transported by MCTs in
humans, although it has not been directly demonstrated whether
NSAIDs, including salicylic acid, are substrates of human MCTs or not.
The aim of this study was to clarify the transport properties of NSAIDs
across the blood-placental barrier and to investigate the possible
contribution of MCTs to the transport of NSAIDs by using BeWo cells, a
human choriocarcinoma-derived model cell line of the blood-placental
barrier (13). We carried out uptake experiments using
radiolabeled drugs and measured intracellular pH (pHi)
change using a pH indicator,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Salicylic acid was employed as a model drug of NSAIDs, and its transport properties were compared with those of
L-lactic acid, a typical physiological substrate of MCTs.
 |
MATERIALS AND METHODS |
Reagents.
[7-14C]salicylic acid (44 mCi/mmol) was purchased from
American Radiolabeled Chemicals (St. Louis, MO).
L-[U-14C]lactic acid sodium salts (116 mCi/mmol) were purchased from ICN Pharmaceuticals (Irvine, CA). DIDS,
FCCP, p-chloromercuribenzosulfonic acid (p-CMBS),
furosemide, probenecid, nigericin, CHC, hexanoic acid, and octanoic
acid were purchased from Sigma Chemical (St. Louis, MO). BCECF-AM was
purchased from Dojindo (Kumamoto, Japan). All other chemicals were of
reagent grade.
Cell culture.
BeWo cells were obtained from American Type Culture Collection
(Rockville, MD). BeWo cells were cultivated in the nutrient mixture
F-12 Ham Kaighn's modification (Sigma Chemical) supplemented with 10%
FCS, 50 µg/ml streptomycin, and 50 U/ml benzylpenicillin K at 37°C
under 95% air-5% CO2. Cells were grown for 4-6 days, and, after reaching confluency, cells were washed with PBS and harvested by exposure to a trypsin-EDTA solution and passed onto new flasks.
Uptake experiment.
In the uptake experiment, BeWo cells were seeded at 2.0 × 105 cells/ml, 200 µl/well, on a 96-well plate
(Becton-Dickinson, Franklin Lakes, NJ) and were cultured for 4 days.
Before the uptake experiments, the culture medium was removed and cells
were washed with 400 µl of prewarmed uptake buffer [20 mM HEPES (for
pH 7.4, 8.0) or MES (for pH 6.0, 5.5), 140 mM NaCl, 5 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, and 5 mM
D-glucose]. Uptake experiments were performed in 100 µl
of the test solution containing 6 µM [14C]salicylic
acid or 3 µM L-[14C]lactic acid at 37 or
4°C. In the concentration dependency and the inhibition studies, each
unlabeled compound was added to the test solution containing
radiolabeled drug, and the pH was adjusted to the desired value with
Tris. To terminate the uptake, test solution was removed by suction,
and cells were washed with 400 µl of ice-cold uptake buffer. In the
inhibition study using a metabolic inhibitor, protonophore, or
ionophore, cells were preincubated with the uptake buffer containing 10 mM NaN3, 50 µM FCCP, or 2.5 µM nigericin for 10 min.
For the quantitation of radiolabeled drugs taken up by cells, cells
were dissolved in 3 M NaOH over 4 h and neutralized with 6 M HCl.
The radioactivity was determined in 4 ml of Clear-sol I (Nakalai
Tesque, Kyoto, Japan) using a liquid scintillation counter (model
LS6500, Beckman Instruments, Fullerton, CA). The amounts of cellular
protein were measured by the method of Lowry et al. (14)
with BSA as a standard, and the mean value was 0.0693 ± 0.0016 mg
protein/well. The initial uptake was expressed as the ratio of cells to
medium (µl · mg protein
1 · time
1) obtained by
dividing the radioactivity taken up per amount of cellular protein by
the concentration of substrate.
Data analysis.
To estimate the kinetic parameters, the initial uptake for 30 s
(J; nmol/mg protein) was fitted to Eq. 1,
consisting of both saturable and nonsaturable components, by using the
nonlinear least-squares regression analysis program MULTI
(30)
|
(1)
|
where C is the concentration of substrate,
Jmax is the maximum uptake rate for the
saturable component, Km is the
half-saturation concentration (mM), and kd is
the first-order rate constant (µl · mg
protein
1 · 30 s
1). The kinetic
parameters in the presence of inhibitor were estimated from the
equation that gave the best fit (either Eq. 2, 3,
or 4).
Competitive inhibition was calculated as
|
(2)
|
Uncompetitive inhibition was calculated as
|
(3)
|
Noncompetitive inhibition was calculated as
|
(4)
|
where Ki is the inhibitory constant and I
is the concentration of inhibitor.
Measurements of pHi.
pHi of BeWo cells was measured using the fluorescent pH
indicator BCECF by a dual-excitation ratiometric method. BeWo cells were cultured on glass coverslips for 2 days and were loaded with 2 µM BCECF-AM in control buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM
D-glucose, and 20 mM HEPES adjusted to pH 7.0 with Tris)
for >1 h at 25°C. The coverslips were then washed with control
buffer for 5 min and used for experiments. Experiments were normally
performed in CO2- and/or HCO
-free conditions to avoid the reduction of pHi resulting from
activation of the CO2/HCO
exchange
mechanism. The coverslips with adherent cells were placed in a
superfusion chamber (volume 1 ml) mounted on the stage of an inverted
fluorescence microscope (TMD-300; Nikon, Tokyo, Japan) and continuously
superfused with control buffer or drug solution (flow rate 1 ml/min).
All experiments were performed at 33-34°C. Excitation
wavelengths were 440 and 495 nm, and the emitted light at 505 nm was
monitored by a photomultiplier (SPEX Industries, Edison, NJ). The ratio of intracellular BCECF fluorescence (405 nm/495 nm) was converted to
pHi by using a calibration curve obtained with nigericin (5 µM) and high-K+ buffer (20 mM HEPES, 140 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, and 5 mM
D-glucose), the pH of which was adjusted to 7.8, 7.4, 6.8, or 6.0 with Tris (21).
Statistical analysis.
Statistical analyses were performed by using Student's
t-test or ANOVA. A difference between means was considered
to be significant when the P value was <0.05.
 |
RESULTS |
Proton-linked uptake of salicylic acid.
To investigate whether salicylic acid is taken up in BeWo cells, we
performed uptake experiments. The intracellular accumulation of
[14C]salicylic acid at 37°C was greater than that at
4°C and increased with a decrease in the extracellular pH at both
temperatures (Fig. 1, A and
C). The initial uptake was reduced by preincubation with 10 mM NaN3, a metabolic inhibitor (Fig. 1E).
Similar results were obtained for the uptake of
L-[14C]lactic acid (Fig. 1, B,
D, and F). At 37°C and extracellular pH 6.0, the uptakes of [14C]salicylic acid and
L-[14C]lactic acid were linear for at least 1 min. The initial bindings were not saturable with regard to the
concentration (data not shown) and were ~5 and 2 µl/mg protein for
salicylic acid and L-lactic acid, respectively.
Therefore, the inhibitory effects of inhibitors and the
concentration dependency of the uptake were analyzed at 30 s.

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Fig. 1.
Time courses of the uptake of [14C]salicylic acid and
L-[14C]lactic acid. Uptakes of
[14C]salicylic acid (6 µM) were measured at 37°C
(A) or 4°C (C) with various pH values as
follows: and , pH 5.5;
and , pH 6.0; and
, pH 7.4. Uptakes of
L-[14C]lactic acid (3 µM) were measured at
37°C (B) or 4°C (D) with same pH values as in
A and C. In the experiment on metabolic
inhibition (E and F), BeWo cells were
preincubated with ( ) or without ( ) 10 mM NaN3 at 37°C and pH 6.0 for 10 min. After
preincubation, the uptakes of [14C]salicylic acid (6 µM; E) and L-[14C]lactic acid (3 µM; F) were measured at 37°C and pH 6.0. Each point
represents the mean ± SE of 3-4 experiments.
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Next, to examine proton influx during the uptake of salicylic acid, we
measured the pHi of BeWo cells using BCECF. When BeWo cells
were superfused with control buffer (pH 7.0), the pHi
ranged from 7.5 to 7.6 [7.36 ± 0.037 (SE), n = 16] at steady state (Fig. 2). Under this
condition, addition of salicylic acid decreased the pHi
(Fig. 2), indicating that the uptake of salicylic acid is accompanied
by proton influx. A similar result was obtained for
L-lactic acid (Fig. 2B).

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Fig. 2.
Decrease in intracellular pH (pHi) induced by salicylic
acid (A) and L-lactic acid (B) under
acidic conditions (pH 7.0). BeWo cells were presuperfused with control
buffer until the pHi became stable (open bars). Next, 10 mM
salicylic acid and 10 mM L-lactic acid were additionally
superfused (filled bars) and washed with control buffer (open bars).
Data are representative of 3-6 experiments. SA, salicylic acid;
LA, L-lactic acid.
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Next, we investigated whether pH-dependent uptakes of salicylic acid
and L-lactic acid are inhibited by a protonophore, FCCP, using radiolabeled drugs. As shown in Fig.
3, the initial uptakes of
[14C]salicylic acid and L-lactic acid were
significantly reduced in the presence of 50 µM FCCP. Both initial
uptakes were attenuated by only 20~30% under a highly acidic
intracellular condition that was attained by preincubation with acidic
buffer (pH 6.0) for 10 min compared with those in the case of
preincubation at pH 7.4 (Fig. 3, insets). Furthermore, we
investigated the effect of the ionophore (K+/H+
antiporter) nigericin. Under the condition of intracellular
K+ concentration ([K+]in) = extracellular K+ concentration
([K+]out), the initial uptakes of
[14C]salicylic acid and L-lactic acid were
also significantly reduced by removing a proton gradient using 2.5 µM
nigericin (Fig. 3, C and D).

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Fig. 3.
pH dependency of initial uptake
of [14C]salicylic acid and
L-[14C]lactic acid. A and
B: BeWo cells were incubated at 37°C for 10 min in uptake
buffer containing 0.5% ethanol with or without 50 µM FCCP. After
preincubation, uptakes of [14C]salicylic acid (6 µM;
A) and L-[14C]lactic acid (3 µM;
B) were measured at 37°C by incubating BeWo cells in
uptake buffer for 30 s in the absence ( ) and
presence ( ) of FCCP. Extracellular pH was 5.5, 6.0, 7.4, or 8.0. Each point represents the mean ± SE of 3 or 4 experiments. The significance of the difference from the value at the
same extracellular pH without FCCP was determined by using Student's
t-test (*P < 0.05). Insets:
effects of preincubation on the initial uptake rates. BeWo cells were
preincubated at pH 6.0 ( ) or 7.4 ( ) for
10 min. After preincubation, the uptakes were measured at 37°C and pH
6.0 for 30 s. Each point represents the mean ± SE of
3-4 experiments. C and D: BeWo cells were
preincubated at 37°C and pH 6.0 for 10 min under each condition as
follows: , control buffer
([K+]in > [K+]out,
[H+]in < [H+]out); , uptake buffer
containing 2.5 µM nigericin ([K+]in > [K+]out,
[H+]in = [H+]out); , uptake buffer
containing 140 mM KCl ([K+]in = [K+]out,
[H+]in < [H+]out); , uptake buffer
containing 140 mM KCl and 2.5 µM nigericin
([K+]in = [K+]out,
[H+]in = [H+]out). After preincubation, the uptakes of
[14C]salicylic acid (10 µM; C) and
L-[14C]lactic acid (10 µM; D)
were measured at 37°C for 30 s. Each point represents the
mean ± SE of 3 or 4 experiments. The significance of
difference from the value in the absence of nigericin was determined by
using Student's t-test (*P < 0.05).
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These results imply that the uptake of salicylic acid, as well as that
of L-lactic acid, is proton gradient dependent.
Concentration dependency of the initial uptake rates.
The concentration dependency of the initial uptake of salicylic acid
was examined at pH 5.5, 6.0, and 7.4. The Eadie-Hofstee plot and
kinetic parameters are shown in Fig. 4
and Table 1, respectively. At each pH,
the uptake of [14C]salicylic acid (Fig. 4A)
was saturable at high concentration, as was that of
L-[14C]lactic acid (Fig. 4B). The
uptake consisted of saturable and nonsaturable components and was
adequately described by Eq. 1. The insets in Fig.
4, A and B, show Eadie-Hofstee plots of the saturable components. Both uptakes showed a single saturable component.

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Fig. 4.
Concentration dependency of
initial uptake of salicylic acid and L-lactic acid by BeWo
cells. Uptake of salicylic acid was determined at various pH values and
37°C for 30 s as follows: , pH 5.5;
, pH 6.0; pH 7.4. Total uptake
(J) and the saturable component (Js;
inset) of the initial uptake are shown as Eadie-Hofstee
plots. Js was calculated by subtracting the
nonsaturable component from total uptake. Lines were calculated from
the kinetic parameters obtained as described in MATERIALS AND
METHODS. Each point represents the mean ± SE of 3 or 4 experiments. A: concentrations of salicylic acid (C) were 5, 6, 13, and 59 µM and 0.1, 0.5, 1, 2.5, 5, 10, and 20 mM.
B: concentrations of L-lactic acid were 3, 7, 11, 24, and 54 µM and 0.1, 0.5, 1, 2.5, 10, and 20 mM. Units for
J and Js are
nmol · mg protein 1 · 30 s 1
and for J/C and Js/C
µl · mg protein 1 · 30 s 1.
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Inhibitory effects of various monocarboxylic acids and organic
anions on the initial uptake rates.
To compare the characteristics of the pH-dependent uptake of salicylic
acid with those of L-lactic acid, we investigated the inhibitory effects of various monocarboxylic acids and organic anions
(Table 2). Salicylic acid,
L-lactic acid, pyruvic acid, other short-chain fatty acids
(acetic acid, propionic acid, n-butyric acid),
medium-chain fatty acids (hexanoic acid, octanoic acid), valproic
acid, and aromatic monocarboxylic acids significantly inhibited the
initial uptakes of both [14C]salicylic acid and
L-[14C]lactic acid in a
concentration-dependent manner. These compounds tended to inhibit the
uptake of L-[14C]lactic acid more potently
than that of [14C]salicylic acid. Succinic acid, a
dicarboxylic acid, did not inhibit initial uptake in either case.
Although p-aminohippuric acid, which is a substrate of OATs,
inhibited the uptake of L-[14C]lactic acid,
its potency was weak. Probenecid, which inhibits various OATs,
inhibited both initial uptakes, whereas furosemide did not inhibit the
uptake of [14C]salicylic acid. These results demonstrate
that the uptake of salicylic acid is similar to that of
L-lactic acid in terms of the inhibitory properties of
monocarboxylic acids.
Inhibitory effects of NSAIDs on the initial uptake rates.
We investigated the inhibitory effects of other NSAIDs on the
pH-dependent uptake of [14C]salicylic acid.
Acetylsalicylic acid (1 mM) only slightly inhibited the uptake of
[14C]salicylic acid, whereas ketoprofen, ibuprofen,
diclofenac, and alclofenac markedly inhibited uptake (Table
3). Diclofenac was the most potent
inhibitor among the tested NSAIDs. Although the pattern of inhibitory
properties of these NSAIDs for the uptake of
L-[14C]lactic acid was similar to that for
salicylic acid uptake, the potencies were significantly greater in the
former case.
We also investigated the inhibitory effects of NSAIDs from the
standpoint of pHi. pHi was decreased by the
superfusion of 1 mM diclofenac (Fig. 5,
A and B), the most potent inhibitor of L-[14C]lactic acid uptake. Under this
condition, additional perfusion of 10 mM salicylic acid resulted in
further acidification, which was reversed upon removal of salicylic
acid from the perfusate (Fig. 5A). In contrast, additional
perfusion of 10 mM L-lactic acid after diclofenac and
salicylic acid did not cause further acidification (Fig. 5,
B and C). Conversely, additional perfusion of
salicylic acid (Fig. 5D) and diclofenac (Fig. 5E)
resulted in further acidification after the acidification induced by
L-lactic acid. These results indicate that NSAIDs are
potent inhibitors of the uptake of L-lactic acid.

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Fig. 5.
Mutual inhibitions on decrease of pHi. BeWo cells were
presuperfused with 1 mM diclofenac (A and B), 10 mM salicylic acid (C), or 10 mM L-lactic
acid (D and E) in control buffer (pH 7.0)
(hatched bar). Next, salicylic acid (10 mM; pH 7.0; A),
L-lactic acid (10 mM; pH 7.0; B and
C), or diclofenac (1 mM; pH 7.0; E) was
additionally superfused (filled bar). Data are representative of 3 experiments.
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Kinetic analysis of the mechanism of mutual inhibition between
NSAIDs and L-lactic acid.
Because salicylic acid and L-lactic acid each inhibited
their uptake of the other (Table 2), we investigated the inhibitory kinetics. Eadie-Hofstee plots are shown in Fig.
6. The uptake of salicylic acid was
concentration dependent, with Jmax and
Km values of 114 ± 17.6 nmol · mg protein
1 · 30 s
1
and 4.32 ± 0.722 mM, respectively. L-Lactic acid
competitively inhibited the uptake of [14C]salicylic acid
with a Ki value of 45 mM (Fig. 6A).
The uptake of L-lactic acid was concentration dependent,
with Jmax and Km values of 13.6 ± 1.93 nmol · mg
protein
1 · 30 s
1 and 3.32 ± 0.493 mM, respectively. Salicylic acid noncompetitively inhibited the
uptake of L-[14C]lactic acid with a
Ki value of 0.96 mM (Fig. 6B).
Furthermore, ibuprofen, which inhibited the uptake of
L-[14C]lactic acid (Table 3),
noncompetitively inhibited the uptake of
L-[14C]lactic acid, with a
Ki value of 0.17 mM, as did salicylic acid (Fig.
6C). These findings indicate that NSAIDs and
L-lactic acid do not share a common transporter.

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Fig. 6.
Eadie-Hofstee plots for initial uptake of nonsteroidal
anti-inflammatory drugs (NSAIDs) and L-lactic acid by BeWo
cells. Uptake was determined at pH 6.0 and 37°C for 30 s. Total
uptake (J) and the saturable component
(Js; insets) of the initial uptake
are shown as Eadie-Hofstee plots. Js was
calculated by subtracting the nonsaturable component from total uptake.
Lines were calculated from the kinetic parameters obtained as described
in MATERIALS AND METHODS. A: salicylic acid
uptake measured in the absence ( ) and presence
( ) of 30 mM L-lactic acid. The
concentrations of salicylic acid were 0.5, 1, 2.5, 5, and 20 mM.
B: L-Lactic acid uptake was measured in the
absence ( ) and presence ( ) of 2 mM
salicylic acid. The concentrations of L-lactic acid were
0.1, 0.5, 1, 5, 10, and 20 mM. C: L-Lactic acid
uptake was measured in the absence ( ) and presence
( ) of 0.2 mM ibuprofen. The concentrations of
L-lactic acid (C) were 0.1, 0.5, 1, 5, 10, and 20 mM. Each
point represents the mean ± SE of 4 experiments. Units for
J and Js are nmol · mg
protein 1 · 30 s 1 and for
J/C and Js/C µl · mg
protein 1 · 30 s 1.
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Effects of MCT inhibitors on the initial uptake rates.
Finally, to assess whether proton-linked MCTs are involved in the
pH-dependent transport of NSAIDs, we performed further studies using
MCT inhibitors, i.e., p-CMBS, CHC, and phloretin, which are
classical inhibitors of L-lactic acid transport.
These compounds markedly inhibited the initial uptake of
L-[14C]lactic acid in a
concentration-dependent manner, whereas that of
[14C]salicylic acid was hardly inhibited (Table
4).
We also examined whether CHC attenuates the intracellular acidification
induced by L-lactic acid, salicylic acid, and diclofenac. Superfusion of 10 mM CHC (pH 7.0) decreased the pHi and
resulted in a steady-state pH of 6.73 ± 0.047 (n = 12, mean ± SE; Fig. 7). Under
this condition, further addition of 10 mM salicylic acid significantly
decreased the pHi in the presence of CHC (Fig. 7A). In contrast, L-lactic acid (10 mM) only
slightly decreased the pHi in the presence of CHC (Fig.
7B). Diclofenac (1 mM) clearly decreased the pHi
in the presence of CHC (Fig. 7C).

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Fig. 7.
Effects of -cyano-4-hydroxycinnamate (CHC) on pHi
decrease induced by salicylic acid (10 mM; A),
L-lactic acid (10 mM; B), and diclofenac (1 mM;
C). BeWo cells were presuperfused with 10 mM CHC in control
buffer (pH 7.0; hatched bars), and a drug was superfused additionally
(filled bars). Data are representative of 3 experiments.
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 |
DISCUSSION |
In this study, to investigate whether NSAIDs are transferred
across the placenta by monocarboxylic acid transporters, we compared the transport properties of salicylic acid and L-lactic
acid by using BeWo cells. This is the first report to characterize the transport characteristics of salicylic acid at the human
blood-placental barrier. From the results of experiments with
radiolabeled drugs, the uptakes of both salicylic acid and
L-lactic acid were 1) temperature dependent and
NaN3 sensitive, 2) FCCP and nigericin sensitive, and 3) concentration dependent. Moreover, the measurement of
pHi revealed that both drugs decreased the pHi
of BeWo cells. These findings suggest that salicylic acid and
L-lactic acid are taken up in BeWo cells via proton-linked
active transport systems.
Transport pathway of L-lactic acid.
Various monocarboxylic acids, and well-known inhibitors of lactate
transport (p-CMBS, CHC, phloretin; see Ref.
18), inhibited the uptake of L-lactic acid by
BeWo cell. The Km value for the uptake of
L-lactic acid was estimated to be 2~5 mM, which coincided well with the values reported in Xenopus laevis oocytes
expressing human MCT1 and MCT2 (6.0 and 6.5 mM, respectively; see Ref.
12). We also revealed that mRNA of MCT1, -3, -5, -6, and
-7 are expressed in BeWo cells by using RT-PCR and DNA sequencing (data
not shown). Thus we conclude that the uptake of L-lactic
acid into BeWo cells is mediated by MCT, although the problem of which
MCT(s) transports L-lactic acid should be solved in the
future by using the expression system.
Interestingly, the initial uptake of
L-[14C]lactic acid was activated by
preincubation with uptake buffer (Fig. 1, B and
F), but this phenomenon was not the case for salicylic acid
(Fig. 1, A and E). A speculative explanation for
this phenomenon is that lactate efflux occurred during preincubation
and its intracellular concentration became low, so lactate was easily
taken up in the cells.
Proton influx was directly demonstrated by measurement of
pHi under an inwardly directed proton gradient. The
pHi was further decreased by superfusion of
L-lactic acid (Fig. 2B), indicating that influx
of proton into BeWo cells accompanied that of L-lactic acid. This proton influx was largely suppressed by CHC (Fig.
7B). The portion of L-lactic acid-induced
acidification that was resistant to CHC may be accounted for the
concentration of substrate used in this study; we used 10 mM
L-lactic acid to allow clear detection of the change in pH
by BCECF, and, at this concentration, the nonsaturable component is
estimated to amount to 40-50% of total uptake (Table 1). Thus the
aforementioned decrease in the pHi may result from a
pathway independent of the proton gradient, such as the passive
diffusion of protonated lactic acid.
Measurement of pHi also revealed that CHC itself potently
decreased the pHi (Fig. 7). This finding is consistent with
the fact that CHC competitively inhibits the transport of lactate (6). Therefore, at least in BeWo cells, CHC may be
transported by MCT and also may competitively inhibit MCT.
Transport pathway of NSAIDs.
Various monocarboxylic acids specifically inhibited the uptake of
salicylic acid into BeWo cells. In this respect, salicylic acid and
L-lactic acid share similar characteristics. On the other hand, L-lactic acid-induced intracellular acidification was
strongly attenuated by preincubation with salicylic acid (Fig.
5C) or diclofenac (Fig. 5A), but not vice versa
(Fig. 5, D and E). These findings suggest that
NSAIDs and L-lactic acid are transported by distinct pathways. However, this seems to be inconsistent with the result that
the uptake of salicylate was inhibited by lactate competitively (Fig.
6A). This discrepancy might be caused because the affinity of lactate to salicylate transporter was quite low, with a
Ki value of 45 mM. The value is quite high
compared with the physiological concentration of lactate (<10 mM), and
we did not use such a high concentration in this study. Thus, under the
current experimental concentration range, the component of lactate
transport by salicylate transporter may overlap with the nonsaturable
component, and it might not be detected at the concentration used in
this study.
The difference between the pathway of L-lactic acid and
salicylic acid could be classified in terms of the sensitivity to CHC.
In other words, L-lactic acid was transported mainly via a
pathway sensitive to CHC, whereas the NSAIDs were transported via a
proton-linked pathway insensitive to CHC (Fig.
8). Indeed, the uptake of salicylic acid
was not inhibited by CHC or other MCT inhibitors, which potently
inhibited the uptake of L-lactic acid (Table 4).
Correspondingly, in the measurement of pHi, CHC did not
attenuate the decrease in pH induced by 10 mM salicylic acid, a
concentration at which the saturable component was estimated to amount
for 85% of total uptake (Table 1 and Fig. 4). Therefore, the saturable
component of the uptake of salicylic acid is considered not to be
strongly suppressed by CHC. This is remarkably different from the case
of L-lactic acid uptake. With regard to diclofenac, we only
performed a measurement of pHi, since radiolabeled
diclofenac was not available; hence, we could not directly investigate
uptake. The intracellular acidification induced by diclofenac (1 mM;
Fig. 5), which implies that diclofenac was taken up with protons in BeWo cells, was not suppressed by CHC (10 mM; Fig. 7C). Thus
diclofenac may also be transported via the CHC-insensitive pathway, as
well as salicylic acid (Fig. 8).

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|
Fig. 8.
Schematic diagram of suggested proton-linked transport mechanisms
from the apical membrane (maternal side) of BeWo cells, a model of the
blood-placental barrier. Proton-linked transport mechanisms can be
classified into CHC-sensitive and CHC-insensitive pathways.
A: L-lactic acids are mainly transported by the
CHC-sensitive pathway, possibly monocarboxylate transporters (MCTs).
NSAIDs inhibit the CHC-sensitive pathway of L-lactic acid.
B: NSAIDs are mainly transported by the CHC-insensitive
pathway, which may involve H+-linked transport and passive
diffusion. LA , ionized L-lactic acid; LAH,
protonated L-lactic acid; SA , ionized
salicylic acid; SAH, protonated salicylic acid.
|
|
Inhibitory character of NSAIDs on the uptake of
L-lactic acid.
In this study, several NSAIDs potently inhibited the uptake of
L-[14C]lactic acid (Table 3). These results
are consistent with previous reports that diclofenac (0.05 mM)
inhibited the Na+-dependent and pH-independent transport of
lactate by 85% in pigmented rabbit conjunctiva and that ibuprofen (5 mM) inhibited the pH-dependent transport of lactate by 70% in Caco-2
cells (8, 15).
It is not yet known whether these NSAIDs share common transporters with
L-lactic acid. Therefore, there are two possible
explanations for the inhibitory effects of NSAIDs on the uptake of
L-lactic acid. One possibility is that NSAIDs directly
inhibit lactate transporters. The other possibility is that the proton
gradient (the driving force for lactate transporters) is attenuated by the proton-linked influx of NSAIDs, resulting in a decrease in the
uptake of L-lactic acid. However, the initial uptake of
L-[14C]lactic acid was only attenuated by
20-30% under a highly acidic intracellular condition attained by
preincubation with acidic buffer (pH 6.0) for 10 min, compared with the
case of preincubation at pH 7.4 (Fig. 3, A and B;
insets). Under this experimental condition, components
insensitive to nigericin, which removes the transmembrane proton
gradient, remained (Fig. 3, C and D). These
results indicate that intracellular acidification alone is not
sufficient to suppress the uptake of L-lactic acid.
Therefore, it is conceivable that salicylic acid and diclofenac may
directly inhibit the lactate transport system.
Nature of the CHC-insensitive pathway.
The CHC-insensitive transport system was demonstrated to play an
important role in NSAID transfer across the blood-placental barrier.
The candidates for transporters of salicylic acid are MCTs, anion
exchanger, and OATs. Several studies have been conducted to investigate
whether MCT is involved in the transport of salicylic acid. Tamai et
al. (26) reported that, in rat MCT1-expressing cells,
salicylic acid is transported by rat MCT1, along with
L-lactic acid and benzoic acid. Utoguchi et al.
(28) showed that, in BeWo cells, the uptake of
L-lactic acid was competitively inhibited by benzoic acid,
an aromatic monocarboxylic acid similar to salicylic acid. However, in
this study, inhibition of the uptake of
L-[14C]lactic acid by salicylic acid was
noncompetitive (Fig. 6). In addition, differences in the sensitivity to
MCT inhibitors were demonstrated (Table 4). On the basis of these
results, salicylic acid may not be a substrate of human MCT, or it may
have a quite low affinity for MCT as a substrate, even though it is a
potent inhibitor. It was also reported that L-lactic acid
has different affinities for each MCT isoform and that each MCT isoform
has a different pattern of sensitivity to MCT inhibitors (CHC,
phloretin, and p-CMBS; see Refs. 5, 7, and
20). Because MCT1 and -4 are sensitive to CHC, salicylic
acid may be a substrate of some other MCT isoform(s) expressed in BeWo cells.
With regard to the involvement of an anion exchanger, it has been
reported that both lactic acid and salicylic acid are transported by
anion exchangers,
which are sensitive to DIDS and activated by a
HCO
gradient (16). However, we
performed experiments under HCO
-free conditions in
this study, so the contribution of this anion exchanger may be
neglected under our conditions.
Last, a few reports have also been demonstrated that NSAIDs are
transported or interacted with OAT. Apiwattanakul et al.
(1) and Sekine et al. (22) demonstrated by
using rat OAT1- and rat OAT2-expressing X. laevis oocytes
that salicylic acid was a substrate of both transporters. However,
human OAT1, -2, -3, and -5 mRNA, except for OAT4, were little expressed
in placental tissue by Northern hybridization (9, 24).
Although human OAT4 is expressed, salicylate has been reported not to
inhibit the uptake of [3H]estrone sulfate by
OAT4-expressed oocytes (3). Moreover, there has been no
report indicating that OATs are dependent on a proton gradient.
Therefore, the involvement of OATs in the pH-dependent transport of
salicylic acid is unlikely.
Permeation through placenta and exposure to the fetus.
Kinetic analysis revealed that a portion of the saturable
component is ~50% at the therapeutic plasma concentration of
salicylic acid (~100 µM; see Table 1), showing that the
nonsaturable component is also substantial. It may be possible that the
blood-placental barrier may be not so tight for the permeation of
salicylate. On the other hand, assuming that extracellular pH is
7.0-7.3, the saturable component of initial uptake of salicylic
acid seems to be much larger (Fig. 3 and Table 1). Indeed, the
microacidic environment is formed by an Na+/H+
antiporter that is localized on the brush-border membrane of BeWo cells
(23).
Therefore, it should not be definitely concluded that the uptake of
salicylate by active transporters is not significant in the exposure
and/or the adverse effects to the fetus, although the contribution of
the nonsaturable component was first determined quantitatively in this
study. To fully understand the transplacental transfer of salicylic
acid, the transfer from trophoblast cells to the fetus and subsequent
steps should be taken into consideration in addition to the transfer
across trophoblast cells that we investigated here.
In conclusion, NSAIDs potently inhibited the transport of
L-lactic acid across BeWo cells. However, the transport
system of NSAIDs is essentially CHC insensitive and apparently is
different from that of L-lactic acid.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Sawada, Dept. of Medico-Pharmaceutical Sciences, Graduate School of
Pharmaceutical Sciences, Kyushu Univ., 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan (E-mail:
sawada{at}phar.kyushu-u.ac.jp).
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.
First published December 19, 2001;10.1152/ajpcell.00179.2001
Received 17 April 2001; accepted in final form 17 December 2001.
 |
REFERENCES |
1.
Apiwattanakul, N,
Sekine T,
Chairoungdua A,
Kanai Y,
Nakajima N,
Sophasan S,
and
Endou H.
Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis oocytes.
Mol Pharmacol
55:
847-854,
1999[Abstract/Free Full Text].
2.
Barros, LF,
Yudilevich DL,
Jarvis SM,
Beaumont N,
and
Baldwin SA.
Quantitation and immunolocalization of glucose transporters in the human placenta.
Placenta
16:
623-633,
1995[ISI][Medline].
3.
Cha, SH,
Sekine T,
Kusuhara H,
Yu E,
Kim JY,
Kim DK,
Sugiyama Y,
Kanai Y,
and
Endou H.
Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta.
J Biol Chem
275:
4507-4512,
2000[Abstract/Free Full Text].
4.
Doughty, LM,
Glazier JD,
Powell TL,
Jansson T,
and
Sibley CP.
Chloride transport across syncytiotrophoblast microvillous membrane of first trimester human placenta.
Pediatr Res
44:
226-232,
1998[Abstract].
5.
Grollman, EF,
Philip NJ,
McPhie P,
Ward RD,
and
Sauer B.
Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast.
Biochemistry
39:
9351-9357,
2000[ISI][Medline].
6.
Halestrap, AP.
Transport of pyruvate and lactate into human erythrocytes.
Biochem J
156:
193-207,
1976[ISI][Medline].
7.
Halestrap, AP,
and
Price NT.
The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.
Biochem J
343:
281-299,
1999[ISI][Medline].
8.
Horibe, Y,
Hosoya K,
Kim KJ,
and
Lee VHL
Carrier-mediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva.
Invest Ophthalmol Vis Sci
39:
1436-1443,
1998[Abstract].
9.
Hosoyamada, M,
Sekine T,
Kanai Y,
and
Endou H.
Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney.
Am J Physiol Renal Physiol
276:
F122-F128,
1999[Abstract/Free Full Text].
10.
Kekuda, R,
Prasad PD,
Fei FYJ,
Torres-Zamorano V,
Sinha S,
Yang-Feng TL,
Leibach FH,
and
Ganapathy V.
Cloning of the sodium-dependent, broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line.
J Biol Chem
271:
18657-18661,
1996[Abstract/Free Full Text].
11.
Knipp, GT,
Audus KL,
and
Soares MJ.
Nutrient transport across the placenta.
Adv Drug Delivery Res
38:
41-58,
1999[ISI][Medline].
12.
Lin, RY,
Vera JC,
Chaganti RSK,
and
Golde DW.
Human monocarboxylate transporter 2 (MCT2) is a high affinity pyruvate transporter.
J Biol Chem
273:
28959-28965,
1998[Abstract/Free Full Text].
13.
Liu, F,
Soares MJ,
and
Audus KL.
Permeability properties of monolayers of the human trophoblast cell line BeWo.
Am J Physiol Cell Physiol
273:
C1596-C1604,
1997[Abstract/Free Full Text].
14.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin-phenol reagent.
J Biol Chem
193:
265-275,
1951[Free Full Text].
15.
Ogihara, T,
Tamai I,
Takanaga H,
Sai Y,
and
Tsuji A.
Stereoselective and carrier-mediated transport of monocarboxylic acids across Caco-2 cells.
Pharmacol Res
13:
1828-1832,
1996.
16.
Ogihara, T,
Tamai I,
and
Tsuji A.
Structural characterization of substrates for the anion exchange transporter in Caco-2 cells.
J Pharm Sci
88:
1217-1221,
1999[ISI][Medline].
17.
Stensen, M,
and
Rosalind-Goldman R.
Treatment of inflammatory rheumatic disorders in pregnancy.
Drug Saf
19:
389-410,
1998[ISI][Medline].
18.
Poole, RC,
and
Halestrap AP.
Transport of lactate and other monocarboxylates across mammalian plasma membranes.
Am J Physiol Cell Physiol
264:
C761-C782,
1993[Abstract/Free Full Text].
19.
Price, NT,
Jackson VN,
and
Halestrap AP.
Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past.
Biochem J
329:
321-328,
1998[ISI][Medline].
20.
Rahman, B,
Schneider HP,
Bröer A,
Deitmer JW,
and
Bröer S.
Helix 8 and Helix 10 are involved in substrate recognition in the rat monocarboxylate transporter MCT1.
Biochemistry
38:
11577-11584,
1999[ISI][Medline].
21.
Roos, A,
and
Boron WF.
Intracellular pH.
Physiol Rev
61:
296-434,
1981[Free Full Text].
22.
Sekine, T,
Cha SH,
Tsuda M,
Apiwattanakul N,
Nakajima N,
Kanai Y,
and
Endou H.
Identification of multispecific organic anion transporter 2 expressed predominantly in the liver.
FEBS Lett
429:
179-182,
1998[ISI][Medline].
23.
Silva, NLCL,
Wang H,
Harris CV,
Singh D,
and
Fliegel L.
Characterization of the Na+/H+ exchanger in human choriocarcinoma (BeWo) cells.
Pflügers Arch
433:
792-802,
1997[ISI][Medline].
24.
Sun, W,
Wu RR,
van Peolje PD,
and
Erion MD.
Isolation of a family of organic anion transporters from human liver and kidney.
Biochem Biophys Res Commun
283:
417-422,
2001[ISI][Medline].
25.
Tadokoro, C,
Yoshimoto Y,
Sakata M,
Fujimiya M,
Kurachi H,
Adachi E,
Maeda T,
and
Miyake A.
Localization of human placental glucose transporter 1 during pregnancy. An immunohistochemical study.
Histol Histopathol
11:
673-681,
1996[ISI][Medline].
26.
Tamai, I,
Sai Y,
Ono A,
Kido Y,
Yabuuchi H,
Takanaga H,
Satoh E,
Ogihara T,
Amano O,
Izeki S,
and
Tsuji A.
Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1.
J Pharm Pharmacol
51:
1113-1121,
1999[ISI][Medline].
27.
Ushigome, F,
Takanaga H,
Matsuo H,
Yanai S,
Tsukimori K,
Nakao H,
Uchiumi T,
Nakamura T,
Kuwano M,
Ohtani H,
and
Sawada Y.
Human placental transport of vinblastine, vincristine, digoxin and progesterone : contribution of P-glycoprotein.
Eur J Pharmacol
408:
1-10,
2000[ISI][Medline].
28.
Utoguchi, N,
Magnusson M,
and
Audus KL.
Carrier-mediated transport of monocarboxylic acids in BeWo cell monolayers as a model of the human trophoblast.
J Pharm Sci
88:
1288-1292,
1999[ISI][Medline].
29.
Walsh, SW,
and
Wang Y.
Maternal perfusion with low-dose aspirin preferentially inhibits placental thromboxane while sparing prostacyclin.
Hypertens Pregnancy
17:
203-215,
1998[ISI].
30.
Yamaoka, K,
Tanigawara Y,
Nakagawa T,
and
Uno T.
A pharmacokinetic analysis program (MULTI) for microcomputer.
J Pharmacobio-Dyn
4:
879-885,
1981[Medline].
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