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
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ABSTRACT |
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
-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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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).
-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)
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 |
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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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.
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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
( ) 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).
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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
( ) in the buffer containing 25 mM HEPES-Tris (pH 7.4)
and sucrose. Each data point represents the mean ± SE of 3 experiments.
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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;
). 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; , 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).
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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; ) for 5 s. Each data point represents the
mean ± SD of 3 or 4 experiments.
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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
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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
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DISCUSSION |
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
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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
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.
 |
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