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Am J Physiol Cell Physiol 283: C822-C830, 2002. First published April 24, 2002; doi:10.1152/ajpcell.00545.2001
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Vol. 283, Issue 3, C822-C830, September 2002

Characteristics of L-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast

Masako Inuyama1, Fumihiko Ushigome1, Akiko Emoto1, Noriko Koyabu1, Shoji Satoh2, Kiyomi Tsukimori2, Hitoo Nakano2, Hisakazu Ohtani1, and Yasufumi Sawada1

1 Department of Medico-Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, and 2 Department of Reproduction and Gynecology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The characteristics of L-lactic acid transport across the trophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake of L-[14C]lactic acid into basal membrane vesicles was Na+ independent, and an uphill transport was observed in the presence of a pH gradient ([H+]out > [H+]in). L-[14C]lactic acid uptake exhibited saturation kinetics with a Km value of 5.89 ± 0.68 mM in the presence of a pH gradient. p-Chloromercuribenzenesulfonate and alpha -cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not. Mono- and dicarboxylic acids suppressed the initial uptake. In conclusion, L-lactic acid transport in the basal membrane is H+ dependent and Na+ independent, as is also the case for the brush-border membrane transport, and its characteristics resemble those of monocarboxylic acid transporters. However, there were several differences in the effects of inhibitors between basal and brush-border membrane vesicles, suggesting that the transporter(s) involved in L-lactic acid transport in the basal membrane of placental trophoblast may differ from those in the brush-border membrane.

human placenta; trophoblast; monocarboxylic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-LACTIC ACID IS UTILIZED as a metabolic energy source in a variety of fetal tissues, such as porcine fetal heart (30), rat fetal brain (3), and ovine fetal liver (10b). Moreover, L-lactic acid is considered to be one of the major carbon sources for fatty acid synthesis, as demonstrated in fetal calves. In humans, the blood level of L-lactic acid in the fetal circulation is higher than in the maternal circulation (19), suggesting that L-lactic acid is also essential for growth of the human fetus.

L-Lactic acid is a monocarboxylic acid (pKa 3.86) that is mostly ionized under physiological conditions. Because the ionized form is not expected to permeate well, a carrier-mediated process is considered to be involved in the permeation of L-lactic acid across the plasma membranes. The transport of L-lactic acid has been well investigated, especially in human erythrocytes, and is ascribed mainly to an H+-dependent system and partly to an anion exchange system (8). In other tissues such as hepatocytes, intestinal brush-border membranes, and skeletal muscles, L-lactic acid was also reported to be transported in an H+-dependent manner (20, 22, 27). On the other hand, L-lactic acid-Na+ cotransport was shown to occur at the renal brush-border membrane (4), and an anion exchange system is involved in L-lactic acid transport in the jejunal basal membrane. Intratissue heterogeneity of the functional properties for L-lactic acid transport was also demonstrated in rat medullary thick limbs of Henle, where the characteristics of L-lactic acid transport in the basal membrane are different from those in the brush-border membrane, i.e., H+ cotransport in the basolateral membrane and organic anion exchange in the brush-border membrane (10). Thus, to fully understand the L-lactic acid transport mechanism across polarized cells, the transport mechanism at each membrane should be separately characterized.

Maternal and fetal circulations are separated by villous tissue in the placenta. The trophoblast, the functional unit of villous tissue, is considered to be the functional entity of the placental barrier. The trophoblast cell consists of brush-border membrane on the maternal side and basal membrane on the fetal side, and it regulates the exchange of various materials, such as nutrients, between mother and fetus. In the brush-border membrane of the trophoblast, H+ gradient-dependent L-lactic acid transport was found to occur (1, 5, 18). In the basal membrane, however, a limited study indicated that an overshoot phenomenon occurs in the presence of an inwardly directed H+ gradient (2). The purpose of the present study was to characterize the transport system of L-lactic acid at the basal membrane of the trophoblast compared with that at the brush-border membrane, which we investigated previously (18), by using membrane vesicles prepared from human placentas.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. L-[U-14C]lactic acid (116 mCi/mmol) was purchased from INC Biomedicals (Costa Mesa, CA), L-[2,3-3H]alanine (52 Ci/mmol) was from Amersham International (Little Chalfont, UK), and [3H]dihydroalprenolol (DHA; 31 mCi/mmol) was from NEN Life Science Products (Boston, MA). Acetic acid, L- and D-lactic acid, propionic acid, pyruvic acid, succinic acid, and glutaric acid were purchased from Nacalai Tesque (Kyoto, Japan). alpha -Cyano-4-hydrocinnamate (4-CHC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and phloretin were purchased from Sigma Chemical (St. Louis, MO). p-Chloromercuribenzenesulfonate (pCMBS) was from both Sigma Chemical and Nacalai Tesque. All other chemicals were of reagent grade.

Preparation of basal membrane vesicles and brush-border membrane vesicles of human placental trophoblast. Basal membrane vesicles (BLMVs) of human term placental trophoblast were prepared by the method previously described by Eaton and Oakey (9) with minor modifications. All operations were carried out at 4°C. Normal human term placentas from uncomplicated pregnancies were obtained within 15 min after vaginal or cesarean delivery and placed in 0.9% NaCl at 4°C. After the cord, amniochorion, and decidua were removed, the placental tissue was cut into pieces and villous tissue was collected. The tissue was washed, stirred in phosphate-buffered saline (PBS) for 30 min, and collected on a 250-µm-pore size nylon mesh. The tissue was washed three times with cold buffer A (50 mM Tris · HCl, pH 7.4), collected on a nylon mesh, and divided into 15-g portions. Each portion was sonicated in 100 ml of buffer A for 10 s at 90% maximum amplitude with the use of a sonicator (Vibra-cell VCX400; Sonics and Materials, Newtown, CT). Sonicated tissue was collected on the mesh, washed three times with buffer B (5 mM Tris · HCl, pH 7.4), and then stirred gently for 60 min in 5× buffer B. The tissue was collected on the nylon mesh and washed three times with buffer B. Tissue portions of 18-20 g were resuspended in 100 ml of buffer C (50 mM Tris · HCl, pH 7.4, containing 10 mM EDTA and 250 mM sucrose) and incubated for 30 min with occasional stirring. The portions were then sonicated four times for 10 s at 100% maximum amplitude. Suspensions were strained through the nylon mesh, and the supernatant was centrifuged at 3,430 g for 10 min. The supernatant from this spin was recentrifuged at 80,000 g for 40 min to yield the pellet, which was resuspended in ~12 ml of buffer D (25 mM HEPES-Tris, pH 7.4, containing 1 mM EDTA and 275 mM sucrose) using a homogenizer (Mini D.C. Stirrer; Eyela, Tokyo, Japan). This fraction was designated as crude basal membrane fraction and further purified by centrifugation on a discontinuous Ficoll gradient. Ficoll in the resuspension buffer (10%, wt/vol) was overlaid with 4% Ficoll, and resuspended materials at the top were centrifuged at 90,000 g for 6-8 h. The material at the 4-10% interface was collected and diluted approximately fivefold with buffer D. The pellet (BLMVs) was resuspended in buffer E (25 mM HEPES-Tris, pH 7.4, containing 275 mM sucrose) and passaged through a 25-gauge syringe needle six times. BLMVs were quickly frozen and stored at -80°C for up to 1 mo until use.

Tissue homogenate was prepared by the method previously described by Kelley et al. (15). Approximately 3 g of whole villous tissue were homogenized in 10 ml of buffer E by using a Glass Teflon homogenizer (Physcotron; Micro Teq Nichion, Chiba, Japan) for 2.25 min and further with a homogenizer for eight strokes. The material was filtered through six layers of gauze.

Brush-border membrane vesicles (BBMVs) of human placental trophoblast were prepared by the method previously described by Nakamura et al. (18).

Purity and orientation of BLMVs and BBMVs. Binding activity of DHA as a marker of the basal membrane and alkaline phosphatase (ALP) activity as a marker of the brush-border membrane were assayed as reported by Kelley et al. (15) and Bessey et al. (5a), respectively. P-glycoprotein (P-gp) was also quantitated as a marker of brush-border membrane to assess the purity of BBMVs and BLMVs, because P-gp has been reported to exist only on the brush-border membrane of trophoblast (25). P-gp was quantitated by using Western Blotting with the quantitation software Quantity One (Bio-Rad Laboratories, Hercules, CA). Protein was quantified by the method of Lowry et al. (17) using bovine serum albumin as a standard.

An orientation assay for BLMVs was carried out with the use of concanavalin A-fluorescein as described by Illsley et al. (13). BBMV orientation was determined by measuring nucleotide pyrophosphatase activity (6).

Uptake measurements. The uptake of L-[3H]alanine and L-[14C]lactic acid into BLMVs was measured at 37°C by using a rapid filtration technique (24). Uptake was initiated by the addition of 40 µl of incubation buffer to 10 µl of BLMV suspension containing 40-70 µg of protein. The incubation buffer consisted of, in general, EM buffer (25 mM HEPES-Tris, pH 7.4, or MES-Tris, pH 5.5-6.5, and an appropriate concentration of sucrose to be isotonic) and 100 µM L-[3H]alanine (0.5 µCi/point) or 1 mM L-[14C]lactic acid (0.2 µCi/point). To investigate the concentration-dependent uptake or the effect of inhibitors, unlabeled L-lactic acid or inhibitor was added to this incubation buffer. At the designated time, uptake was terminated by adding 1 ml of ice-cold stop solution, followed immediately by filtration (HAWP 0.45 µm; Millipore Intertech, Bedford, MA). The filter was washed twice with 4 ml of ice-cold stop solution. Stop solution contained, in general, 100 µM L-alanine or 1 mM L-lactic acid and 10 mM sucrose in EM buffer. Nonspecific binding was determined by adding 1 ml of ice-cold stop solution and 40 µl of ice-cold incubation buffer to the ice-cold BLMV suspension, followed by the same treatment as in the uptake experiments.

To assay the radiolabeled compounds, filters were put into counting vials and mixed with 4 ml of scintillation fluid, Clear-sol I (Nacalai Tesque). Radioactivity was determined using a liquid scintillation counter (LS6500; Beckman Instruments, South Pasadena, CA).

Uptake into BBMVs was determined by the method described by Nakamura et al. (18).

Data analysis. Obtained radioactivity was normalized with respect to the protein amount of vesicles. Data are presented as ratio of vesicle to medium, obtained by dividing the uptake amount by the drug concentration (µl/mg protein), or uptake coefficient (µl · mg protein-1 · 5 s-1). Values were determined by subtracting the nonspecific binding from total uptake, except in the investigation of osmolarity effects.

To determine the kinetic parameters Km, Jmax, and kd for the L-lactic acid uptake, data were fitted to the following Michaelis-Menten equation by using nonlinear least-squares regression analysis (MULTI; Ref. 32)
J=J<SUB>max</SUB><IT>×</IT>S/(<IT>K</IT><SUB>m</SUB><IT>+</IT>S)<IT>+k</IT><SUB>d</SUB><IT>×</IT>S
where J and S represent the transport rate and concentration of substrate, respectively. Jmax (nmol · mg protein-1 · 5 s-1), Km (mM), and kd (µl · mg protein-1 · 5 s-1) represent the maximum uptake rate for a carrier-mediated process, the Michaelis constant, and the rate constant for the nonsaturable component, respectively.

Statistical analysis. Statistical analysis was performed by using Student's t-test or ANOVA followed by Duncan's test. Differences between means were considered to be significant when the P value was <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Purity and orientation of BLMVs and BBMVs of human placental trophoblast. Table 1 shows the protein yield, DHA binding, and enzymatic activities of homogenate and BLMVs. DHA binding, a marker for basal membrane in BLMVs, was 1,490 ± 29 fmol/mg protein, representing 32-fold enrichment over the homogenate (47.2 ± 4.6 fmol/mg protein). ALP activity, a marker for brush-border membrane in BLMVs, was 1.68 ± 0.043 µmol · min-1 · mg protein-1, amounting to threefold enrichment over the homogenate (0.565 ± 0.066 µmol · min-1 · mg protein-1). The amounts of protein in BLMVs and homogenate obtained from 100 g of placental tissue were 2.62 ± 0.20 and 2890 ± 91 mg, respectively, showing that 0.091% of the homogenate was recovered as BLMVs. The level of P-gp in BBMVs was 22-fold higher than in BLMVs (Fig. 1). The ratio of right-side-out BLMVs was 89.6%, whereas 41.9% of BBMVs were right-side-out (Table 1).

                              
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  		Table 1.   Characterization of BLMVs and BBMVs of human placental trophoblast



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  		Fig. 1.   Immunodetection of P-glycoprotein (P-gp). Protein samples, human placental brush-border membrane vesicles (BBMVs; 50 µg), and human placental basolateral membrane vesicles (BLMVs; 50 µg) were resolved by SDS-PAGE on 7.5% polyacrylamide gel and transferred onto Clear Blot Membrane-P (Atto, Tokyo, Japan). A: immunoblots were performed with anti-P-gp mouse monoclonal antibody C219 (TFB, Tokyo, Japan) and developed with the enhanced chemiluminescence detection reagent (ECL; Amersham, Oakville, ON, Canada), as described in a recent report (29). B: P-gp was quantitated by using Western blotting with the quantitation software Quantity One.

Functional validation of BLMVs. Functional activity of BLMVs was evaluated in terms of the Na+-dependent uptake of L-alanine. Time courses of the uptake of 100 µM L-[3H]alanine into BLMVs in the presence of 45 mM Na+ and K+ are shown in Fig. 2. The uptake of L-alanine was significantly stimulated in the presence of an inwardly directed Na+ gradient compared with the K+ gradient.


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  		Fig. 2.   Effect of an inwardly directed Na+ gradient on the uptake of 100 µM L-[3H]alanine into BLMVs of human placental trophoblast. BLMVs were preloaded with 275 mM sucrose and 25 mM HEPES-Tris (pH 7.4). The uptake of L-alanine into BLMVs was performed at 37°C in the presence of 45 mM NaCl () or 45 mM KCl (open circle ) in the buffer containing 25 mM HEPES-Tris (pH 7.4) and sucrose. Inset: effect of osmolarity on the uptake of 100 µM L-[3H]alanine in the presence of an Na+ gradient. The uptake of L-alanine was performed in the presence of various concentrations of sucrose. Each data point represents the mean ± SE of 3 experiments. Significant differences from the control were identified by using Student's t-test (*P < 0.05).

Effect of Na+ gradient on the uptake of L-lactic acid. Figure 3 shows the uptakes of 1 mM L-[14C]lactic acid into BLMVs in the presence of inwardly directed Na+ and K+ gradients. The initial uptake rates were almost identical, and no Na+ dependency of L-lactic acid transport was observed in basal membrane.


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  		Fig. 3.   Effect of an inwardly directed Na+ gradient on the uptake of 1 mM L-[14C]lactic acid into BLMVs of human placental trophoblast. BLMVs were preloaded with 275 mM sucrose and 25 mM HEPES-Tris (pH 7.4). The uptake of L-lactic acid into BLMVs was performed at 37°C in the presence of 100 mM NaCl () or 100 mM KCl (open circle ) in the buffer containing 25 mM HEPES-Tris (pH 7.4) and sucrose. Each data point represents the mean ± SE of 3 experiments.

Effect of pH gradient on the uptake of L-lactic acid. Figure 4 shows the time courses of the uptake of 1 mM L-[14C]lactic acid into BLMVs at extravesicular pH 5.5 and 7.4. When the intravesicular pH was fixed at pH 7.4, the uptake of L-lactic acid into BLMVs was enhanced, and transient uphill transport was observed at extravesicular pH 5.5. The initial uptake rate at pH 5.5 was ninefold higher than that at pH 7.4 when assessed at 30 s. The uptake of L-[14C]lactic acid was markedly stimulated by extravesicular acidification and was significantly suppressed in the presence of 100 mM unlabeled L-lactic acid (Fig. 5).


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  		Fig. 4.   Effect of a pH gradient on the uptake of 1 mM L-[14C]lactic acid into BLMVs of human placental trophoblast. BLMVs were preloaded with 275 mM sucrose and 25 mM HEPES-Tris (pH 7.4). The uptake of L-lactic acid into BLMVs was performed at 37°C in the 25 mM MES-Tris buffer (pH 5.5; ) or 25 mM HEPES-Tris buffer (pH 7.4; open circle ). Inset: effect of osmolarity on the uptake of 1 mM L-[14C]lactic acid in the presence of a pH gradient. The uptake of L-lactic acid was performed in the presence of various concentrations of sucrose. Each data point represents the mean ± SE of 3 experiments. Significant differences from the control were identified by using Student's t-test (*P < 0.05).



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  		Fig. 5.   Effect of extravesicular pH on the uptake of L-[14C]lactic acid into BLMVs of human placental trophoblast. BLMVs were preloaded with 275 mM sucrose and 25 mM HEPES-Tris (pH 7.4). The uptake of L-lactic acid (, 1 mM; open circle , 100 mM) into BLMVs was performed at 37°C in the MES-Tris buffer (pH 5.5, pH 6.5) or HEPES-Tris buffer (pH 7.4) for 5 s. Each data point represents the mean ± SE of 3 experiments. Significant differences from the control were identified by using Student's t-test (*P < 0.05).

Kinetics of the initial uptake of L-lactic acid. Concentration-dependent uptakes of L-[14C]lactic acid into BLMVs at extravesicular pH 5.5 and 7.4 are shown in Fig. 6. The initial uptake clearance was saturable with regard to substrate concentration under an acidic extravesicular condition (pH 5.5). Saturable uptake was only recognized in the presence of a pH gradient and not in its absence. The kinetic parameters Km, Jmax, and kd, calculated from the Michaelis-Menten equation, were 5.89 ± 0.68 mM, 28.77 ± 3.35 nmol · mg protein-1 · 5 s-1, and 0.40 ± 0.07 µl · mg protein-1 · 5 s-1, respectively.


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  		Fig. 6.   Concentration-dependent uptake of L-[14C]lactic acid into BLMVs of human placental trophoblast. BLMVs were preloaded with 275 mM sucrose and 25 mM HEPES-Tris (pH 7.4). The uptake of L-lactic acid (30 µM-100 mM) into BLMVs was performed at 37°C in the 25 mM MES-Tris buffer (pH 5.5; ) or 25 mM HEPES-Tris buffer (pH 7.4; open circle ) for 5 s. Each data point represents the mean ± SD of 3 or 4 experiments.

Effect of inhibitors on the uptake of L-lactic acid. We examined the effects of pCMBS (thiol residue-modifying reagent), 4-CHC (aromatic monocarboxylic acid), phloretin (bioflavonoid), and DIDS (stilbene derivative) on the uptake of 1 mM L-[14C]lactic acid into BLMVs in the presence of a pH gradient. Table 2 shows the results in BLMVs, along with our previous results for BBMVs (18). The initial uptake of L-lactic acid into BLMVs was inhibited by pCMBS and 4-CHC in a concentration-dependent manner but was not affected by phloretin or DIDS. In contrast, the uptake into BBMVs was inhibited by all of the inhibitors investigated.

                              
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  		Table 2.   Effects of various potential inhibitors on the uptake of L-[14C] lactic acid into BLMVs and BBMVs of human placental trophoblast

Effects of mono- and dicarboxylic acids on the uptake of L-lactic acid. Table 3 shows the effects of mono- and dicarboxylic acids on the uptakes of 1 mM L-[14C]lactic acid into BLMVs and BBMVs in the presence of a pH gradient, including data from our previous report on BBMVs (18). Monocarboxylic acids (acetic acid, L-lactic acid, D-lactic acid, propionic acid, and pyruvic acid) and dicarboxylic acids (succinic acid and glutaric acid) all suppressed the uptake of L-lactic acid in a concentration-dependent manner. With regard to BLMVs, all the carboxylic acids at high concentration (50 mM) suppressed the uptake, whereas only L- and D-lactic acid inhibited the uptake at low concentration (5 mM). There was no statistically significant difference in the inhibition of the uptake of L-lactic acid between L- and D-lactic acid, suggesting that the inhibitory effect of lactic acid is not stereoselective.

                              
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  		Table 3.   Effects of several mono- and decarboxylic acids on the uptake of L-[14C] lactic acid into BLMVs and BBMVs of human placental trophoblast


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of BLMVs. In this study, we initially assessed the binding of DHA, a marker for basal membrane, and the enzymatic activity of ALP, a marker for brush-border membrane, to confirm the purity of BLMVs isolated from human placental syncytiotrophoblast. The enrichment of DHA binding was 32-fold, considerably higher than that of ALP activity, which was only threefold (Table 1). It has been reported that the enrichment of DHA binding and ALP activity were 18.5- to 45.0-fold and 0.60- to 5.01-fold, respectively (9, 10a, 12, 15, 17a, 17b, 24a). Our results fall within the above ranges and indicate that the basal membrane was selectively recovered. Moreover, we demonstrated that P-gp was expressed only in the BBMVs, not in the BLMVs (Fig. 1). The absence of P-gp in the BLMVs also indicates that the contamination of brush-border membrane in the BLMVs is negligible. The orientation of BLMVs, assessed by the use of concanavalin A, was 90% right-side-out. Na+-dependent uptake of L-alanine was observed (Fig. 2), as expected (12). The uptake of L-alanine declined with increase in the osmolarity of external buffer (Fig. 2), suggesting that L-alanine was not only adsorbed onto vesicles but actually transported into the intravesicular space. Therefore, BLMVs prepared in this study should be a suitable model to elucidate the characteristics of transport across the basal membrane of the human placental syncytiotrophoblast.

Mechanism of L-lactic acid uptake into BLMVs. In the presence of a pH gradient, the uptake of L-lactic acid into BLMVs showed an overshoot phenomenon (Fig. 4), i.e., L-lactic acid was transiently accumulated inside the vesicles against the concentration gradient. The uptake of L-lactic acid at the steady state was reduced with increase in the osmolarity of the external buffer (Fig. 4), suggesting that L-lactic acid was not only adsorbed onto the surface of vesicles but also transported into the vesicles. The initial uptake rate of L-lactic acid increased with decrease in the extravesicular pH (Fig. 5). Although the simple diffusion of L-lactic acid, a monocarboxylic acid with a pKa of 3.86, increased at lower pH, the uptake of L-[14C]lactic acid in the presence of 100 mM L-lactic acid did not show pH dependency. Therefore, a pH-dependent transport system was demonstrated to be involved in the uptake of L-lactic acid, in addition to simple diffusion. pCMBS and 4-CHC, which are known to interact with H+-linked L-lactic acid carriers, reduced the uptake of L-lactic acid into BLMVs in the presence of a pH gradient (Table 2). These results indicate that a H+ gradient-dependent carrier for L-lactic acid exists on the basal membrane of human placental trophoblast.

Comparison of the uptake properties of L-lactic acid between BLMVs and BBMVs of human placental trophoblast. In BBMVs of human placental trophoblast, L-lactic acid was reported to be transported in an Na+-independent and H+-dependent manner (5). This is consistent with our present findings (Figs. 3-5). The uptake properties of L-lactic acid into BLMVs were similar to those into BBMVs in many respects: the uptake was saturable, H+ gradient dependent, and inhibited by mono- and dicarboxylic acids including D-lactic acid (Fig. 6 and Table 3). The Km value for L-lactic acid in BLMVs was determined to be 5.89 mM (Table 4), which is close to the Km value in BBMVs [1.71 mM (18) and 4.1 mM (5)]. Therefore, brush-border and basal membranes in the human placental trophoblast may share common functional properties for the transport of L-lactic acid. However, the inhibitory effects of pCMBS, 4-CHC, and acetic acid in BLMVs were weaker than those in BBMVs, and phloretin and DIDS were ineffective in BLMVs (Tables 2 and 3). Therefore, the H+-dependent transport system for L-lactic acid in basal membrane seems to differ in some respects from that in brush-border membrane.

                              
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  		Table 4.   A comparison of the characteristics of L-lactic acid transport in the placenta and other tissues using vesicles

Physiological implications. The physiological concentration of L-lactic acid in the fetal circulation was reported to be ~2 mM (19), whereas the Km value of L-lactic acid transport in the basal membrane was 5.89 mM, which implies that this transport system may be functional under physiological conditions.

Fetal blood is slightly more acidic (pH 7.3) than maternal blood (pH 7.4) (19). In addition, an Na+/H+ exchanger (NHE-2) exists on the basal membrane of human placental syncytiotrophoblast and generates a local H+ gradient across the membrane. This H+ gradient would provide a driving force for L-lactic acid transport across the trophoblast basal membrane. Taking into consideration the direction of the H+ gradient under physiological conditions and the orientation of the prepared BLMVs, the direction of uptake observed in this study corresponds to the transfer of L-lactic acid from fetus to mother. Accumulation of L-lactic acid in the fetus can cause damage to the heart and cerebral tissue during hypoxia and induces acidosis in the fetus. Therefore, the L-lactic acid transport system in the basal membrane may help to prevent excessive increase of L-lactic acid in the fetus. The lower affinity for L-lactic acid on the fetal side compared with the maternal side of the trophoblast is consistent with this hypothesis. Indeed, at the end of gestation, L-lactic acid produced in human fetus is transferred to the placenta (19). On the other hand, the fetus utilizes L-lactic acid for metabolic energy and for fatty acids synthesis. L-Lactic acid in the maternal circulation or produced in the placenta by glycolysis might be transferred to the fetus under certain conditions. Indeed, L-lactic acid can be transported in both directions by the same carrier under certain circumstances in rat BBMVs (1), supporting the idea that the transport system observed in this study in BLMVs may possibly transfer L-lactic acid bidirectionally across the placenta. At any rate, transporters in the brush-border and basal membranes of placental trophoblast may cooperatively regulate the fetal concentration of L-lactic acid.

Comparison with other tissues. In the brush-border membrane of kidney cortex, Na+-L-lactic acid cotransport is a major mechanism for the transport of L-lactic acid (4). The lack of Na+-dependency in human trophoblast BLMVs (Fig. 2) indicates that the L-lactic acid transport system in basal membrane of trophoblast is distinct from that in brush-border membrane of kidney cortex.

An H+ gradient-dependent transport of L-lactic acid has also been observed in the plasma membrane of other mammalian tissues. As summarized in Table 4, the Km value was similar to that in the small intestinal BBMVs [12.7 mM (27)], large intestinal BBMVs [14.8 mM (22)], and erythrocytes [10 mM (8)] but lower than that in skeletal muscle vesicles [40.1 mM (23); 20.9 mM (14)], and cardiac muscle vesicles [16-40 mM (28)].

pCMBS, an irreversible inhibitor, has been demonstrated to specifically inhibit H+-linked L-lactic acid transport in erythrocytes (8), whereas 4-CHC is a reversible inhibitor for H+-linked transporters (20). The uptake of L-lactic acid into BLMVs of placental trophoblast in the presence of an H+ gradient was reduced by 10 mM pCMBS and 12.5 mM 4-CHC by only 19 and 27%, respectively (Table 2), whereas in porcine large intestinal BBMVs, lower concentrations of pCMBS (0.5 mM) and 4-CHC (5 mM) have been reported to reduce the transport by 83 and 69%, respectively (22). In rat skeletal muscle vesicles, 0.5 mM pCMBS and 10 mM 4-CHC inhibit the uptake by 83 and 69%, respectively (23). Therefore the transport system for L-lactic acid in BLMVs of placental trophoblast appears to be less sensitive to these inhibitors (Table 4). These variations may be due to tissue and/or species differences.

Phloretin, a reversible inhibitor, has been reported to decrease L-lactic acid transport in a variety of mammalian epithelial cells (20). In contrast, the H+-L-lactic acid cotransporter in the basal membrane of rat medullary thick limbs of Henle was reported to be insensitive to phloretin (10) (Table 4). In this study, the transport of L-lactic acid in the basal membrane of placental trophoblast was not inhibited by phloretin and is therefore similar to that in the basal membrane of rat medullary thick limbs of Henle.

The anion exchange system (Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger), which is potently inhibited by DIDS, is an another candidate for L-lactic acid uptake in erythrocytes (8). However, the intravesicular or extravesicular buffer did not contain any driving force for an anion exchanger, bicarbonate or chloride, under our experimental conditions, so the anion exchanger is not expected to have contributed to the uptake of L-lactic acid. Moreover, the expression level of anion exchanger (AE1) in basal membrane of human placental syncytiotrophoblast has been reported to be low. Consequently, the anion exchange mechanism may make little contribution to the transport of L-lactic acid.

The inhibitory effect of D-lactic acid was not significantly different from that of L-lactic acid in the presence of an H+ gradient (Table 3). However, the stereoselective transport of lactic acid has been reported in the skeletal muscle (23) and the liver (20) but not in the cardiac muscle (28) (Table 4). Hence, in this respect, L-lactic acid transport in the basal membrane of placental trophoblast is different from that in skeletal muscle or liver and similar to that in cardiac muscle.

Dicarboxylic acids such as succinic acid and glutaric acid were found to reduce the uptake of L-lactic acid into BLMVs in the presence of an H+ gradient, with comparable inhibitory potency to that of monocarboxylic acids (Table 3). These results are in contrast with the transport properties of L-lactic acid in many other tissues, where the transport is inhibited by monocarboxylic acids but not by dicarboxylic acids (20). However, the inhibition of L-lactic acid transport by dicarboxylic acids was demonstrated in rabbit small intestinal BBMVs (26) (Table 4). The nature of the inhibition by dicarboxylic acids remains to be evaluated.

Relationship to monocarboxylic acid transporters. Several monocarboxylic acid transporters (MCTs), which transport monocarboxylic acids including L-lactic acid, have been cloned. The presence of mRNAs of MCT1, -2, -4, -5, -6, -7, and -8 was confirmed in human placenta (21), and MCT4 protein was also detected by Western blotting (31). Functional analyses have mainly dealt with MCT1, -2, -3, and -4. Broer et al. (7) reported that L-lactic acid transport via rat MCT1 and MCT2 was H+ dependent and inhibited by 4-CHC and monocarboxylic acids, and rat MCT1 was also sensitive to pCMBS. In addition, the Km values of human MCT1 and MCT2 for the transport of L-lactic acid were determined to be 6.0 and 6.5 mM, respectively (16). The present results show some similarities, including H+-dependency, sensitivity to pCMBS and 4-CHC, and Km value, suggesting that MCTs may transport L-lactic acid across the basal membrane of trophoblast. The uptake of L-lactic acid into BLMVs of trophoblast was inhibited by pCMBS and 4-CHC but not by phloretin, an inhibitor for rat MCT1 (7). pCMBS inactivates rat MCT1 without affecting MCT2 activity (7). Chick MCT3 was reported to be resistant to pCMBS, 4-CHC, and phloretin (11). These reports suggest that each isoform of MCTs may possess a distinct sensitivity to inhibitors. None of the known isoforms seems to show L-lactic acid transport properties identical with those observed in this study. Therefore, the transport of L-lactic acid across the basal membrane of placental trophoblast may be mediated by an as yet uncharacterized MCT isoform(s).

In conclusion, the basal membrane of human term placental trophoblast possesses an H+-dependent L-lactic acid transport system whose properties are similar to those of MCTs and lacks an Na+-dependent transport system. In addition, the characteristics of the L-lactic acid transport in the basal membrane are similar to those in the brush-border membrane. However, the difference in the sensitivity of H+-dependent L-lactic acid transport to phloretin and DIDS between the basal and the brush-border membranes implies that distinct transporters of L-lactic acid may exist on each side of the trophoblast.


    ACKNOWLEDGEMENTS

We thank Dr. B. M. Eaton (Chelsea and Westminster Hospital, London, UK) for helpful advice on the preparation of basal membrane vesicles and assay of the orientation. We also thank Dr. N. P. Illsley (New Jersey Medical School, Newark, NJ) for kind advice on assay of the orientation.


    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 address: 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.

April 24, 2002;10.1152/ajpcell.00545.2001

Received 14 November 2001; accepted in final form 18 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alonso de la Torre, SR, Serrano MA, Alvarado F, and Medina JM. Carrier-mediated L-lactate transport in brush-border membrane vesicles from rat placenta during late gestation. Biochem J 278: 535-541, 1991[Medline].

2.   Alonso de la Torre, SR, Serrano MA, Caropaton T, and Medina JM. Proton gradient-dependent active transport of L-lactate in basal plasma membrane vesicles isolated from syncytiotrophoblast human placenta. Biochem Soc Trans 19: 409S, 1991[Medline].

3.   Arizmendi, C, and Medina JM. Lactate as an oxidizable substrate for rat brain in vitro during the perinatal period. Biochem J 214: 633-635, 1983[ISI][Medline].

4.   Barac-Nieto, M, Murer H, and Kinne R. Lactate-sodium cotransport in rat renal brush border membranes. Am J Physiol Renal Fluid Electrolyte Physiol 239: F496-F506, 1980[Abstract/Free Full Text].

5.   Balkovetz, DF, Leibach FH, Mahesh VB, and Ganapathy V. A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles. J Biol Chem 263: 13823-13830, 1988[Abstract/Free Full Text].

5a.   Bessey, OA, Lowry OH, and Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 164: 321-329, 1946[Free Full Text].

6.   Bohme, M, Mullaer M, Leier I, Jedlitschky G, and Keppler D. Cholestasis caused by inhibition of the adenosine triphosphate-dependent bile salt transport in rat liver. Gastroenterology 107: 255-265, 1994[ISI].

7.   Broer, S, Broer A, Schneider HP, Stegen C, Halestrap AP, and Deitmer JM. Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes. Biochem J 341: 529-535, 1999[Medline].

8.   Deuticke, B, Beyer E, and Forst B. Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties. Biochim Biophys Acta 684: 96-110, 1982[Medline].

9.   Eaton, BM, and Oakey MP. Sequential preparation of highly purified microvillous and basal syncytiotrophoblast membranes in substantial yield from a single term human placenta: inhibition of microvillous alkaline phosphatase activity by EDTA. Biochim Biophys Acta 1193: 85-92, 1994[Medline].

10.   Eladari, D, Chambrey R, Irinopoulou T, Leviel F, Pezy F, Bruneval P, Paillard M, and Podevin R. Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle. J Biol Chem 274: 28420-28426, 1999[Abstract/Free Full Text].

10a.   Glazier, J, Ayuk P, Grey AM, and Sides K. Syncytiotrophoblast basal plasma membrane isolation. Placenta 19: 443-444, 1998[ISI][Medline].

10b.   Gleason, CA, Rudolph CD, Bristow J, Itskovitz J, and Rudolph AM. Lactate uptake by the fetal sheep liver. J Dev Physiol 7: 177-183, 1985[ISI][Medline].

11.   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. Biochem J 39: 9351-9357, 2000.

12.   Hoeltzli, SD, and Smith CH. Alanine transport systems in isolated basal plasma membrane of human placenta. Am J Physiol Cell Physiol 256: C630-C637, 1989[Abstract/Free Full Text].

13.   Illsley, NP, Wang ZQ, Gray A, Sellers MC, and Jacobs MM. Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1029: 218-226, 1990[Medline].

14.   Juel, C. Muscle lactate transport studied in sarcolemmal giant vesicles. Biochim Biophys Acta 1065: 15-20, 1991[Medline].

15.   Kelley, LK, Smith CH, and King BF. Isolation and partial characterization of the basal cell membrane of human placental trophoblast. Biochim Biophys Acta 734: 91-98, 1983[Medline].

16.   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].

17.   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].

17a.   Marin, JJ, Serrano MA, el-Mir MY, Eleno N, and Boyd CA. Bile acid transport by basal membrane vesicles of human term placental trophoblast. Gastroenterology 99: 1431-1418, 1990[ISI][Medline].

17b.   Monte, MJ, Rodriguez-Bravo T, Macias RI, Bravo P, el-Mir MY, Serrano MA, Lopez-Salva A, and Marin JJ. Relationship between bile acid transplacental gradients and transport across the fetal-facing plasma membrane of the human trophoblast. Pediatr Res 38: 156-163, 1995[ISI][Medline].

18.   Nakamura, H, Ushigome F, Koyabu N, Sato S, Tsukimori K, Nakano H, Ohtani H, and Sawada Y. Proton gradient-dependent transport of valproic acid in human placental brush-border membrane vesicles. Pharm Res 19: 154-161, 2002[ISI][Medline].

19.   Piquard, F, Schaefer A, Dellenbach P, and Haberey P. Lactate movements in the term human placental in situ. Biol Neonate 58: 61-68, 1990[ISI][Medline].

20.   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].

21.   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].

22.   Ritzhaupt, A, Wood IS, Ellis A, Hosie KB, and Shirazi-Beechey SP. Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate. J Physiol 513: 719-732, 1998[Abstract/Free Full Text].

23.   Roth, DA, and Brooks GA. Lactate transport is mediated by membrane-bound carrier in rat skeletal muscle sarcolemmal vesicles. Arch Biochem Biophys 279: 377-385, 1990[ISI][Medline].

24.   Russel, FGM, van der Linden PEM, Vermeulen WG, Heijn M, van Os CH, and van Ginneken CAM Na+ and H+ gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex. Biochem Pharmacol 37: 2639-2649, 1988[ISI][Medline].

24a.   Serrano, MA, Bravo P, el-Mir MY, and Marin JJ. Influence of hydroxylation and conjugation in cross-inhibition of bile acid transport across the human trophoblast basal membrane. Biochim Biophys Acta 1151: 28-34, 1993[Medline].

25.   St-Pierre, MV, Serrano MA, Macias RI, Dubs U, Hoechli M, Lauper U, Meier PJ, and Marin JJ. Expression of members of the multidrug resistance protein family in human term placenta. Am J Physiol Regul Integr Comp Physiol 279: R1495-R1503, 2000[Abstract/Free Full Text].

26.   Tamai, I, Ogihara T, Takanaga H, Maeda H, and Tsuji A. Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane. Biochim Biophys Acta 1468: 285-292, 2000[Medline].

27.   Tiruppathi, C, Balkovetz DF, Gnapathy V, Miyamoto Y, and Leibach FH. A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles. Biochem J 256: 219-223, 1988[ISI][Medline].

28.   Trosper, TL, and Philipson KD. Lactate transport by cardiac sarcolemmal vesicles. Am J Physiol Cell Physiol 252: C483-C489, 1987[Abstract/Free Full Text].

29.   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].

30.   Werner, JC, and Sicard RE. Lactate metabolism of isolated, perfused fetal, and newborn pig hearts. Pediatr Res 22: 552-556, 1987[ISI][Medline].

31.   Wilson, MC, Jackson VN, Heddle C, Price NT, Pilegaard H, Juel C, Bonen A, Montgomery I, Hutter OF, and Halestrap AP. Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3. J Biol Chem 273: 15920-15926, 1998[Abstract/Free Full Text].

32.   Yamaoka, K, Tanigawara Y, Nakagawa T, and Uno T. A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharmacobio-Dyn 4: 879-885, 1981[Medline].

Am J Physiol Cell Physiol 283(3):C822-C830
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