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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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. alpha -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; alpha -cyano-4-hydroxycinnamate; intracellular pH


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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
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MATERIALS AND METHODS
RESULTS
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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)
J<SUB>total</SUB><IT>=J</IT><SUB>max</SUB><IT>×</IT>C<IT>/</IT>(<IT>K</IT><SUB>m</SUB><IT>+</IT>C)<IT>+k</IT><SUB>d</SUB><IT>×</IT>C<IT>=J</IT><SUB>saturable</SUB><IT>+J</IT><SUB>nonsaturable</SUB> (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
J=J<SUB>max</SUB><IT>×</IT>C<IT>/</IT>[<IT>K</IT><SUB>m</SUB><IT>×</IT>(1<IT>+</IT>I<IT>/K</IT><SUB>i</SUB>)<IT>+</IT>C]<IT>+k</IT><SUB>d</SUB><IT>×</IT>C (2)
Uncompetitive inhibition was calculated as
J=J<SUB>max</SUB><IT>×</IT>C<IT>/</IT>[<IT>K</IT><SUB>m</SUB><IT>+</IT>C<IT>×</IT>(1<IT>+</IT>I<IT>/K</IT><SUB>i</SUB>)]<IT>+k</IT><SUB>d</SUB><IT>×</IT>C (3)
Noncompetitive inhibition was calculated as
J=J<SUB>max</SUB><IT>×</IT>C<IT>/</IT>[<IT>K</IT><SUB>m</SUB><IT>×</IT>(1<IT>+</IT>I<IT>/K</IT><SUB>i</SUB>)<IT>+</IT>C<IT>×</IT>(1<IT>+</IT>I<IT>/K</IT><SUB>i</SUB>)]<IT>+k</IT><SUB>d</SUB><IT>×</IT>C (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<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions to avoid the reduction of pHi resulting from activation of the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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
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INTRODUCTION
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RESULTS
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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; open circle  and , pH 6.0; triangle  and black-triangle, 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 (open circle ) 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.

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.

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 (open circle ) 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); open circle , 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).

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; black-triangle 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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Table 1.   Kinetic parameters of initial uptake of salicylic acid and L-lactic acid

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.

                              
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Table 2.   Inhibitory effects of acids on initial uptake of salicylic acid and L-lactic acid

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.

                              
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Table 3.   Inhibitory effects of several NSAIDs on initial uptake of salicylic acid and L-lactic acid

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.

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 (open circle ) 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 (black-triangle) and presence (triangle ) 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.

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).

                              
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Table 4.   Inhibitory effects of several inhibitors on initial uptake of salicylic acid and L-lactic acid

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 alpha -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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient (16). However, we performed experiments under HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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