Biochemicdl Society Transactions ( 1 991 ) 19 4 0 9 s
Proton gradient-dependent active transport of Llactate in basal plasma membrane vesicles
isolated from syncytiotrophoblast human placenta
Sara R. ALONSO DE LA TORRE, Maria A. SERRANO, Tomas CAROPATON and JOGM. MEDINA
Departamento de Bioquimica y Biologia Molecular. Facultad de
Farmacia. Universidad de Salamanca. Aptdo. 449. 37080 Salamanca.
Spain.
The occurrence of a L-lactate carrier in human placenta was
reported by Balkovetz et al. (1) who characterized L-lactate transport in
isolated microvillous (maternal side) membrane vesicles from human
placenta. However, the basal (fetal side) plasma membrane of the
syncytiotrophoblast may represent a potential site for physiological
control of L-lactate transport between the mother and thg-fetus (2).The
aim of the present work was to characterize the properties of L-lactate
transport across the basal plasma membrane of human placenta
syncytiotrophoblast in an attempt to understandthe implications of this
L-lactate transport system in the homeostasis of L-lactate.
Basal membrane vesicles from syncytiotrophoblast human term
placenta (kindly supplied by the Ginaecology and Obstetrics Unit of the
'Virgen de la Vega' Hospital, Salamanca, Spain) were prepared
according to the method of Kelley et al. (3). High partial purification of
BLMV was confirmed by the presence of basal and apical
syncytiotrophoblast membrane markers, i.e., dihydroalprenolol binding
and alkaline phosphatase activity .L-lactate uptake by basal membrane
veslcles was determined at 37" C using a rapid filtration technique as
described by Hopfer et al. (4). Absolute velocity data as a function of the
substrate concentration were fitted by iteration to a complex equation
involving the sum of one non-saturable and one saturable component
(5.6).
The transport of L-lactate in BLMV from human placenta was
examined in the presence and absence of an inwardly-directed pH
gradient. The results (Fig.1) show that in the presence of an inwardlydirected proton gradient (pHirr7.4, p W . 4 ) 1 mM L-lactate uptake was
rapid; furthermore a transient accumulation 'overshoot' was observed
(5 to 6-fold higher at 30 s than the equilibrium ). Variations in the
magnitude of the pH gradient across the BLMV (pHir7.4. p h = 6 . 6 )
slgnificantly decreased the initial rate and peak uptake. In addition, in
0
2
90
Incubation time min
Flgure 1 . Effect of Increasing Inwardly-directed p r o t o n
gradlent o n L-lactate transport I n BLMV f r o m human
placenta.
BLMV were preloaded with a buffer containing 500 mM sorbitol, 20mM
Tris/Mes (pH 7.4) . Incubation was performed at 37°C in buffers
containing 1 mM L-lactate. 499 mM sorbitol, 20mM TridMes adjusted at
pH 5.4 (upper curve) or pH 6.4 (lower curve). Each data point
represents the mean SE of four experiments.
*
Abbrevlat1ons.- BLMV. basal membrane vesicles; DIDS, 4,4'diisothiocyanostilbene-2,2'-disulfonic acid; MES, 2-(N-morpholino)
ethanesutfonic acid; p-CMBS, p-chloromercuriphenylsulfonic acid SH,
sulfhydryl; TRIS, 2-amino-2-hydroxymethylpropane-l,3-diol.
the absence of a pH gradient (pHin-7.4, pHd-7.4) the initial uptake rate
was relatively slower and no 'overshoot' was observed (data not
shown).These results together with the cation-independency of Llactate transport (data not shown) suggest the presence of a coupled
H+/L-lactate- symport activity in BLMV from human placenta. The
ocurrence of an electroneutral L-lactate/H+ cotransport in BLMV was
reinforced by the fact that in the presence or absence of a pH gradient
and and outwardly directed K+gradientvalynomicin does not effect Llactate uptake significantly (data not shown). L-lactate uptake was
insensitive to DIDS, suggesting that L-lactate transport in BLMV is not
achived by an L-lactate/OH- antiporter system. Instead, our results are
consistent with the idea that L-lactate transport in BLMV occurs through
a cotransport system coupled to a proton gradient, as has also been
found in the microvillous membrane of human placenta (11.
The temperature dependence of L-lactate transport across
BLMV from human placenta is strong as can be inferred by the 12-fold
increase in L-lactate uptake observed when temperature rises from 4" to
37" C (resuts not shown). In addition, L-lactate (1 mM) uptake measured
in the presence of an inwardly directed proton gradient (pHin-7.4,
pHout-5.4) in BLMV preincubated for 45 min at 25" with 0.55 mM pCMBS decreased to about 60% of the controls (0.77+_0.02 vs
1.96f0.03 nmol/mg prot/lOs. n-3) suggesting that p-CMBS has a
direct effect on the carrier, presumably by modifying its SH-groups.
These evidences points to the existence of a carrier-mediated pathway
for L-lactate uptake in BLMV from human placenta.
L-lactate uptake was a saturable function of its own concentration
suggesting that L-lactate uptake across BLMV from human placenta
must be the result of a carrier-mediated process. The best fit of these
results yields a kd value of 0 nl. si. m g l . a Vmax of 1.3 nmol. mg-1
protein. 51and an apparent Km value of 13 mM. Linearization of the
data according to the Eadie-Hofstee transformation indicates that there
is only one transport system with a very low affinity for L-lactate. Since
diffusion component is very low, it can be concluded that L-lactate
crosses the basal membrane of human placenta trophoblast through a
single carrier-mediated system which elicits a small affinity towards Llactate. However, the low affinity of basal membrane L-lactate carrier
would guarantee a roughly first order process and hence a strong
dependence of the L-lactate transport rate on the actual lactate
concentrations.
L-lactate preloaded inside the vesicles significantly stimulates
(52%) the L-lactate uptake in the presence of a pH gradient (2.60k0.14
vs 1.71+_0.04nmol/mg proteidl0s in control, n=3) and in its absence
(data not shown). The trans-stimulation of L-lactate transport suggests
the occurrence of a countertransport system that under the appropriate
biochemical conditions can transport L-lactate in both directions usit?g
the same carrier. Although our membrane vesicles preparation is mixed
in orientation (7) our results mainly mimic L-lactate transport from
syncytiotrophoblast cytosol to the fetal blood. This system may be
responsible for the transport of both L-lactate from the very active
placental glycolysis (8) and those of maternal origin. However, when Llactate concentration increases as at the end of gestation, the
reversibility of the basal membrane L-lactate carrier may also accomplish
L-lactate transport from fetus to placenta and hence to the mother's
blood for gluconeogenesis (9).
In conclusion, our results suggest that the transport of L-lactate
across syncytiotrophoblast basal membrane from human placenta is
accomplished by a carrier-mediated low-affinity system driven by a
proton gradient. The reversibility of the carrier suggests that the
vectorial transport of L-lactate from placenta to fetus or viceversa
depends on the concentration of L-lactate in the compartments of the
conceptus.
AcknowledgementsThis work was partially supported by a grant
from CICYT, Spain. S.R.A.T. is recipient of a fellowship from FISSS.
Spain.
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