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Department of Child Health and School of Biological Sciences, University of Manchester, St. Mary's Hospital, Manchester M13 OJH, and Department of Child Health, St. George's Medical School, London SW17 ORE, United Kingdom
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The microvillous membrane (MVM) potential
(Em) of first
trimester human placental villi was measured and compared with that in
villi from term human placentas. The median
Em in first
trimester villi (
28 mV) was significantly more negative than
that at term (21 mV; P < 0.001). The median
Em measured in
villi from early (weeks 6-11)
first trimester (32 mV) was significantly more negative than
that in late (weeks 12 and
13) first trimester villi (24 mV; P < 0.001). Elevating
extracellular KCl concentration induced a significant depolarization of
Em in both first
trimester and term villi (P < 0.05 and P < 0.001, respectively). The
magnitude of this depolarization was greater in first trimester than at term, indicating that the ion conductance of the MVM changes with gestation. Exposure to ouabain induced a significant depolarization of
Em (3 mV:
P < 0.05) in first trimester villi
but had little effect at term. These results suggest that microvillous
membrane electrophysiology changes with placental development. An
alteration in the relative
K+:Cl
conductance of the MVM is likely to be a major contributor to the
change in the magnitude of
Em.
potassium conductance
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE PLACENTA PLAYS a vital role in the transfer of nutrients and ions between mother and fetus. The major functional components of the human placenta are the villi, which project into the intervillous space where they are bathed in maternal blood. Each villous tree is vascularized by a branch of the umbilical vessels, which divide to form a capillary network/plexus. Maternal blood in the intervillous space and fetal blood in the umbilical capillaries are separated by a multilayer barrier. The outermost maternal-facing layer of this barrier is a continuous uninterrupted sheet of syncytiotrophoblast, which extends over the surface of all villous trees within the placenta, lining the intervillous space (22). This syncytial layer is thought to form the major barrier to transfer of ions between mother and fetus.
As in all epithelia, the electrical potential difference (PD),
individually and in summation, across the microvillous (maternal facing) and basal (fetal facing) plasma membranes of the
syncytiotrophoblast (equivalent to luminal and abluminal plasma
membranes in most other epithelia) are important determinants of the
rate and direction of the transport of ions and of processes involving
the net transfer of charge (e.g., sodium-linked cotransport). With the
use of microelectrode techniques, a small (3 mV) fetal-side
negative PD was shown to exist across the syncytiotrophoblast of
isolated villi from human placenta at term (20). The PD across the
microvillous membrane (MVM) of the syncytiotrophoblast
(Em; inside
negative) has also been measured directly in microelectrode studies.
Carstensen et al. (9) reported 91% of
Em values
measured in fragments of villous tisue from term placenta to fall
between 0 and 20 mV and 78% of
Em values
measured in villous tissue from three immature (12-24 wk)
placentas to fall between 20 and 65 mV. Bara et al. (4)
found an Em of
29 mV in fragments of term human placental tissue, and, in
isolated mature intermediate villi from term placentas, we recorded an
Em of 22
mV (20). Em has
not been measured before 12 wk of pregnancy.
Placental development and differentiation occurs throughout gestation, and many consequent changes in structure and function have been documented. For example, dramatic changes in morphology of the human placental villus are observed as gestation progresses (10), and there is also evidence of changes in ion fluxes, from both in vivo (17) and in vitro (24) studies. Changes in Em and the expression of ion transport proteins are associated with cytotrophoblast cell differentiation (11, 12, 21). Furthermore, the data of Carstensen et al. (9) suggest that there might be a change in Em from 12 wk to term.
The primary aim of this in vitro investigation was to measure, for the first time, Em in villi isolated from first trimester human placentas. Data were compared with measurements made in term human placenta. We found that the first trimester Em was significantly hyperpolarized compared with that at term. Additionally, we report data from experiments designed to provide a basis for the future characterization of this difference in Em. Some of the results have been published in abstract form (6, 18, 19).
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Tissue Collection
Experiments were performed on villi isolated from human placentas in the first trimester of pregnancy (weeks 6-13; as determined from the date of the last menstrual period and the appearance of the aborted material) and at term (weeks 38-41; as determined from the date of the last menstrual period, usually confirmed by ultrasound scan). First trimester placental tissue was obtained from St. Mary's Hospital and another local clinic following surgical therapeutic abortions performed for psychosocial reasons under Clause B of the United Kingdom Abortion Act (1967). Term placental tissue was collected from uncomplicated pregnancies delivered vaginally or by cesarean section at St. Mary's Hospital. Samples (~1 cm3) of villous tissue were collected in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Earle's medium [(in mM): 116 NaCl, 5.4 KCl, 1.8 CaCl2, 0.4 MgSO4, 1.0 NaH2PO4, 5.5 glucose, and 5.0 HEPES, pH 7.4 with NaOH] at ambient temperature and washed thoroughly. The tissue was then transferred to an
-Earle's medium [(in
mM): 116 NaCl, 5.4 KCl, 1.8 CaCl2,
0.4 MgSO4, 1.0 NaH2PO4, 5.5 glucose, and 26 NaHCO3]
gassed with 95% O2-5%
CO2 (to pH 7.4) and maintained at
room temperature.
Measurement of Oxygen Consumption
For microelectrode measurements of Em, individual villi were dissected from the placenta and mounted in vitro. Before microelectrode studies were performed, the viability of villi following the isolation procedure was assessed by measuring oxygen consumption by fragments of villous tissue. The method used was based on that described by Arkle et al. (3) for the assessment of isolated pancreatic duct viability, modified to accommodate the larger villous tissue samples.O2 consumption by term placental
villous fragments was compared when the tissue was bathed in either
Earle's medium (composition
as above) or an initially sterile culture medium Dulbecco's modified
Eagle's medium (DMEM) containing
, vitamins, amino acids, and salts
(Life Technologies, Paisley, UK).
O2 consumption by term tissue was
found to be similar in DMEM and
-Earle's medium (see
RESULTS); thus
O2 consumption by first trimester
placental villi was determined in
-Earle's medium only.
Fragments of villous tissue (0.8-14.6 mg wet wt) were dissected
from first trimester or term placentas and maintained in 35-mm culture
dishes (Life Technologies) containing 2 ml of the appropriate incubation medium, pregassed with 5%
CO2 in air. One milliliter of
incubation medium was placed into a 2-ml glass incubation vial (with
Tuf-Bond gas impermeant disk seals; Pierce and Warriner) whose maximum
volume was predetermined by weight. Two fragments of villous tissue,
each suspended in a small volume of medium (10 µl), were transferred
to an incubation vial, and the vial was then filled to the brim with
incubation medium and sealed. Care was taken to seal the tube without
introducing air bubbles. The tissue was incubated in a water bath at
37°C and shaken gently (to mix the medium) for 1.5-4 h. The
suspended fragments "fanned" out in the medium, thereby exposing
a large surface area of the syncytiotrophoblast for
O2 diffusion. Blanks, prepared by
addition of 2 × 10 µl of the medium that bathed the villi in
the 35-mm culture dish, were incubated for the same period of time as
the tissue samples. After a minimum of 1.5 h, an incubation vial was removed from the water bath, the cap loosened slightly to permit withdrawal of fluid, and 0.75 ml of incubation medium was rapidly aspirated using a 1-ml syringe with a large gauge needle to puncture the Tuf-bond disk. The needle was quickly removed, a few drops of
medium expelled to waste, and the sample was injected into the port of
a blood gas analyzer (Corning 158 pH/blood gas analyzer, Corning
Medical Scientific, Medfield, MA) to determine the
PO2. The
villous fragments were kept in the remaining incubation medium in the
vial before determination of their wet weight. Wet (blotted) weight was
determined by transferring the tissue, inevitably with a small volume
of medium, to a filter paper (1 cm diameter). The wet filter paper plus
tissue was placed into a 35-mm culture dish, and the lid was replaced
to minimize evaporative losses. The culture dish, wet filter paper, and
tissue were weighed (A), the tissue was then removed with fine forceps, and the dish and wet filter were
reweighed (B). The blotted weight of
the tissue was given by A B.
The PO2 of
the pregassed (5% CO2 in air)
incubation medium, the blanks, and the samples were determined, and the
fall in
PO2 of the medium
(
PO2)
due to the consumption of O2 by
the tissue was calculated as described by Arkle et al. (3)
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1 · kg
wet wt1) is given by
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1 · mmHg1
and the specific volume of O2 is 1 µmol equivalent to 0.0224 ml.
Effect of cyanide and ouabain on O2 consumption. The effects of cyanide (an inhibitor of aerobic metabolism) and ouabain [an inhibitor of Na+-K+-adenosinetriphosphatase (ATPase)] on O2 consumption by first trimester and term villous fragments were investigated to confirm the presence of aerobically respiring tissue and to compare the contribution of Na+-K+-ATPase activity to O2 consumption, respectively.
For each placenta, six control incubations (two villous fragments in each incubation vial), six experimental incubations (two villous fragments in each vial containing incubation medium with either 3 mM cyanide or 0.1 mM ouabain), and six time-matched blank incubations were performed. The mean O2 consumption for control, experimental, and blank incubations for each placenta was calculated, and the data were expressed with n as the number of placentas.
Measurement of Em
Em was measured in villi isolated from first trimester and term placentas using a previously described method (20). Individual placental villi were isolated, placed in a thermostatically regulated heated tissue perfusion chamber (Intracel, Royston, UK), and immobilized using glass suction pipettes. The villi were continuously superfused (1.5 ml/min) with-Earle's medium and were maintained at 37°C and left to equilibrate with the medium for a
minimum of 10 min. In all experiments involving the measurement of
Em, the
incubation media were gassed with 95%
O2-5%
CO2. The perfusion chamber has a
microenvironmental control that allows a stream of gas (95%
O2-5%
CO2) to pass across the surface of medium in the perfusion chamber, ensuring that medium pH is maintained at 7.4. The bath was placed on the stage of an inverting microscope (Nikon Diaphot), and the electrical measurements were made using an
AxoClamp 2B amplifier (Axon Instruments).
Em was measured
between a recording microelectrode (A) positioned in the outermost
layer of the tissue (syncytiotrophoblast) and a similar electrode (B)
placed in the bathing fluid. Both electrodes were pulled from 1.2-mm
(outer diameter) boroscilicate glass with a filament (Clark
Electromedical Instruments, Reading, UK) using a horizontal puller
(Brown and Flaming model P-87, Sutter Instruments, Novato, CA). These
electrodes had resistances of 80-100 M
when filled with 1.5 M
KCl. A and B were in circuit with an Ag:AgCl reference pellet (R)
positioned directly in the bath fluid.
Em was given as
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-Earle's medium, the baseline PD
between bath and recording electrodes was set at 0 mV. An impalement
was taken to be successful if the following criteria were met:
1) a rapid voltage deflection from
baseline occurred on impalement, 2)
maintenance of a stable
Em at a value
±2 mV of the initial deflection for >1 min, and
3) return to 0 ± 2 mV on
withdrawal of the electrode from the tissue.
Villus maturation and development is a dynamic process that is ongoing throughout gestation. Until ~9 wk of gestation, the placenta is composed mainly of mesenchymal villi; from 9 wk onward, mesenchymal villi are gradually transformed into immature intermediate villi (the prevailing villus type until the end of second trimester) and finally stem villi. In the third trimester, mesenchymal villi are transformed into mature intermediate villi, which give rise to terminal villi (10). In first trimester, villi were randomly selected for impalement (no specific villus type was isolated); thus the most prevalent type is likely to have been selected. At term, mature intermediate villi, which comprise the largest fraction of the villus volume at this gestation with the exception of terminal villi (10), were selected for impalement (20). After measurement of Em, the villus was photographed at ×40 and ×100 magnification (Nikon Diaphot microscope fitted with Nikon F-601 camera).
Maximal human chorionic gonadotropin (hCG) secretion occurs at approximately weeks 8-11 of gestation, with concentrations declining rapidly after week 11.5 (8). Because hCG has been shown to affect placental membrane transport (13, 14), Em was compared before and after the peak secretion of this hormone. First trimester Em data were therefore subdivided into measurements made in early (weeks 6-11) and late (weeks 12 and 13) first trimester placental villi.
Effect of ouabain on
Em. The effect
of ouabain on Em
in villi isolated from first trimester and term placentas was studied to compare the relative contribution of the
Na+-K+-ATPase
to Em at these
two stages of gestation. These experiments were performed with a
continuous flow of fluid through the perfusion chamber. A stable
impalement was first achieved (as described above); then, with the
electrode still positioned in the tissue, the inflow to the bath was
exchanged for -Earle's medium
containing either 0.1 or 1 mM ouabain.
Em was measured for at least 1 min before addition of ouabain and during a 10-min exposure to ouabain. The electrode was then removed from the tissue, and the impalement was considered acceptable if the potential difference returned to 0 ± 2 mV. In this way, each villus acted as
its own control, and data were paired.
The effect of KCl on
Em. The change
in Em in response
to elevating extracellular KCl concentrations was used to assess the relative
K+:Cl
conductance of the MVM in villi from first trimester and term placentas. In these experiments, the isolated villus was equilibrated by superfusion with -Earle's
medium at 37°C (as described in Measurement of
Em). The flow of fluid through the perfusion chamber
was then stopped, and a stable impalement (meeting
criteria
1 and
2 above) was achieved. With the
electrode still positioned in the tissue, 1 ml of
-Earle's medium (at 37°C)
containing 50, 100, 200, or 400 mM KCl was rapidly injected into the
static bath, and the maximum change in
Em was noted
(this was usually achieved within 1-2 min). The injectate was
diluted ~1:1 with the existing bath fluid, and a sample of this bath
fluid was collected to allow the final extracellular K+ concentration to be measured by
flame photometry (Corning Flame Photometer, Corning Medical Scientific,
Medfield, MA). In some experiments, the electrode was then removed from
the tissue (n = 6); in other
experiments, a recovery procedure was performed (n = 18). For these latter
experiments, the flow of control
-Earle's medium through the
perfusion chamber was resumed following collection of bath fluid for
analysis. This allowed the extracellular ion concentration and
(assuming the electrode was still in position within the tissue)
Em to recover
back toward control values. Such a repolarization of
Em was achieved
in every case. In these experiments, junction potentials that might
arise due to bath solution changes were estimated as the PD [(A R) (B R)] with the electrodes in
-Earle's medium minus the PD when
the bath fluid was exchanged for the experimental medium. Using this two-electrode arrangement, the junction potentials were less than 1 mV
with all concentrations of KCl used, and the reported values are
therefore not corrected.
Chemicals
All reagents were analytic grade from standard suppliers. Ouabain was purchased from Sigma (Poole, UK), and sodium cyanide was from British Drug Houses (Poole, UK).Statistics
A Kolmogorov-Smirnov goodness of fit test was performed to determine whether data were normally distributed. Em data were found to deviate from normality and are therefore expressed as median values and statistical comparisons between the distribution of Em values for first trimester versus term and early versus late first trimester placentas were made using a Mann-Whitney U-test.Paired Student's t-tests were used to analyze the effect of ouabain and cyanide on O2 consumption and the effect of ouabain on Em. Control data from first trimester and term placentas were compared using an unpaired Student's t-test.
Regression analyses were used to fit linear models to
O2 uptake data obtained when
incubating with -Earle's medium
and DMEM and to the membrane depolarization induced by increasing
extracellular KCl concentration in first trimester and term
villi. The 95% confidence interval for the difference between the slopes of the appropriate regression lines was calculated to determine whether the slopes of the regression lines differed significantly (2).
An analysis of variance was used to determine whether increasing extracellular KCl concentration significantly altered Em in first trimester and term villi. An analysis of variance (using log change in KCl concentration as a covariate) was also performed to compare the change of Em in response to altering extracellular KCl concentration in first trimester versus term villi.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Oxygen Consumption
O2 uptake. O2 uptake and consumption by placental villi were measured to confirm their viability following the isolation procedure. The initial PO2 of the incubation medium was 193 ± 1 mmHg for DMEM and 183 ± 3 mmHg for-Earle's. The fall in
PO2
in the time-matched blank incubations was 0.0797 ± 0.01 mmHg/min
for DMEM, equivalent to an average
O2 utilization of 0.22 nmol/min
and 0.02 ± 0.005 mmHg/min for
-Earle's medium (equivalent to
0.05 nmol/min). This indicates that leakage of gas from the incubation
vial and/or consumption of
O2 by aerobic bacteria was
insignificant. Figure 1 shows
O2 uptake by term placental villous fragments incubated with either DMEM or
-Earle's medium. These
comparisons were made using term placenta because this tissue was more
readily available than first trimester placenta. A linear relationship
between O2 uptake and duration of
incubation was observed with both incubation media. The 95% confidence
interval calculated for the difference between the slopes of the two
regression lines (0.022-0.112) indicates that
O2 uptake by placental villous fragments was similar in DMEM or
Earle's medium. This suggests
that tissue metabolism and thus viability were equally well maintained
in both incubation media.
-Earle's medium was subsequently
used as the incubation medium for assessment of
O2 consumption by first trimester
villous fragments and as the control bathing solution in all the
microelectrode studies.
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O2 consumption and the effect of cyanide and ouabain. As shown in Fig. 2, A and B, O2 consumption by first trimester villous fragments was significantly higher than that of villous fragments from term placentas (P < 0.001; unpaired Student's t-test). A significant reduction in O2 consumption by first trimester (88%) and term (92%) villi was observed in the presence of cyanide (Fig. 2A). Exposure to ouabain (0.1 mM) reduced O2 consumption in first trimester placental villi by 22% (Fig. 2B, P < 0.001; paired Student's t-test). In contrast, O2 consumption by villi from term placentas was unchanged by exposure to ouabain.
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Microelectrode Studies
Villi impaled. Examples of the placental villi impaled during microelectrode studies are shown in Fig. 3. Figure 3, A and B, shows a villus isolated from a first trimester placenta (at 8 wk of gestation), magnified ×40 and ×100, respectively. Figure 3, C and D, shows a mature intermediate villus isolated from a term placenta magnified ×40 and ×100, respectively. There are considerable differences in the size and morphology of villi from the two different stages of gestation. The term mature intermediate villus is smaller in diameter and length, has several terminal villi projecting from its surface, and, as shown clearly in Fig. 3D, is well vascularized by fetal blood vessels. In contrast, first trimester villi are more poorly vascularized, show limited branching, and terminal villi are absent. These characteristic features are common to first trimester villi, in general, irrespective of villus type (10).
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Em in first
trimester and at term. We have previously measured
Em in 200 mature
intermediate villi from term human placenta (20). During the course of
the present study, another 27 such measurements were made. The median
value for our previous 200 measurements was 22 mV, and the value
for the additional 27 measurements was similar at 21.5 mV. We,
therefore, compared the control measurements of
Em in first
trimester villi made in the present study with the total pool
(n = 227) of measurements made at
term.
The distributions of
Em measured in
villi from the first trimester and at term are shown in Fig.
4, A and
B, respectively. These data were not
normally distributed (Kolmogorov-Smirnov goodness of fit test). The
median Em
measured in first trimester placental villi (28 mV; range
17 to 87 mV) was significantly more negative than that
measured at term (21 mV; range 12 to 60:
P < 0.001; Mann-Whitney
U-test). In the first trimester villi,
34% of all Em
values exceeded 35 mV; in contrast, only 12% of measurements made in term villi were more negative than this potential.
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Em measurements
made in villi from placentas at 6-11 and 12-13 wk of
gestation are shown in Fig. 5,
A and
B, respectively. These data do not
follow a normal distribution (Kolmogorov-Smirnov goodness of fit test).
The median Em in
early first trimester (32 mV; range 17 to 87 mV)
was significantly more negative than in late first trimester villi
(24 mV; range 18 to 50 mV: P < 0.001; Mann-Whitney
t-test).
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Factors contributing to the difference in Em between first trimester and term villi. 1) ROLE OF NA+-k+-atpase in maintaining em. The contribution of the Na+-K+-ATPase to Em was assessed directly by measuring Em before and after exposure to ouabain. The effect of ouabain on Em in villi isolated from first trimester and term placentas is shown in Fig. 6. These data were normally distributed (Kolmogorov-Smirnov goodness of fit test). Furthermore, 0.1 and 1 mM ouabain induced similar effects on Em, and the data have been pooled. Exposure to ouabain resulted in a significant depolarization of Em (3 mV: P < 0.05; paired Student's t-test) in villi from first trimester placentas. This effect of ouabain (0.1 and 1 mM) was maximal within 3 min of application, and, thereafter, no further change was observed up to 10 min. In contrast, ouabain had no effect on Em in term villi.
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2) EFFECT OF ELEVATING EXTRACELLULAR KCL
CONCENTRATION ON eM. The relationship
between Em and
extracellular KCl concentration was assessed in villi from first
trimester and term placentas to estimate the relative
K+:Cl
conductance of the MVM. In this study, we measured the change in
Em in response to
elevating KCl concentrations in the bathing solution. The results of
these experiments are presented in Fig. 7.
On inspection, three of the first trimester data points (indicated by
asterisks in Fig. 7) appeared to be outliers from the sample population. When the ratio depolarization divided by change in KCl
concentration was calculated for the other 21 first trimester data
points, the ratios of the three apparent outliers results were found to
lie more than 5 SDs away from the sample mean (9.57 ± 3.23, mean ± SD). On this basis, these three data points were excluded from
further statistical analyses.
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Increasing K+ concentration in the bathing fluid (by addition of KCl) depolarized the MVM of both first trimester and term villi (P < 0.05 and P < 0.001, respectively; analysis of variance). However, the degree of depolarization induced by changing extracellular KCl concentration was greater in villi from the first trimester than in those from term (P < 0.001; analysis of variance; because changing extracellular KCl concentrations also influences depolarization as shown above, this variable was used as a covariate in this analysis).
Regression analysis was used to fit linear models of depolarization with change in KCl concentration to data from first trimester and term villi (Fig. 7). The 95% confidence interval calculated for the difference between first trimester and term regression lines was 3.8 to 18.3. Zero does not lie near to the middle of this confidence interval, indicating that the slope of the regression lines are different.
The increase in osmolality associated with addition of KCl is unlikely to have affected Em because preliminary experiments showed that increasing extracellular osmolality to the same extent by addition of raffinose, sucrose, or mannitol had no significant effect on Em (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Oxygen Consumption
The present study is the first to use microelectrodes to measure the electrical properties of first trimester placental tissue. Before performing the electrophysiological studies, we determined tissue viability following collection by measuring the oxygen consumption rate of first trimester and term villi. The rate of O2 consumption measured in term placental fragments in our study (136 µmol · kg1 · min1)
is close to measurements made by Leichtweiss et al. (27) and Edwards et
al. (17) in the in vitro perfused human placenta (156 ± 0.06 and
186 ± 0.02 µmol · kg1 · min1,
respectively). However, higher rates of
O2 consumption by the perfused
human placental cotyledon have also been reported (28). The rate at
which the human placenta consumes
O2 in vivo is unknown. However,
O2 consumption by the perfused
human placenta compares reasonably well with in vivo data from the cow,
sheep, goat, and mare (7).
An interesting observation revealed by the viability studies was that
villous tissue from first trimester placenta consumed O2 at a faster rate than that from
term placenta (301 ± 19 vs. 136 ± 11 µmol · kg1 · min1).
A decrease in the rate of placental
O2 consumption (measured in tissue
slices in vitro) as gestation progresses has been reported previously
(30). Throughout gestation, the placental dry-to-wet weight ratio
almost doubles. Thus the fraction of solid material comprising the
placenta per kilogram wet weight is lower in first trimester than at
term (30), and expression of O2
consumption data per gram wet weight is likely to have underestimated
the actual difference between first trimester and term
O2 consumption rates. However,
differences in the proportions of various cells in the villus, such as
fetal red cells, may have contributed to the measured difference in
O2 consumption between first
trimester and term placentas. A significant reduction in
O2 consumption by both first
trimester and term placental villi was observed in the presence of
cyanide. This suggests that O2
consumption by villous fragments from both gestational ages is
dependent on the presence of aerobically respiring tissue. In summary,
these experiments demonstrate that the placental tissue used in this study had an active metabolism and that tissue viability was similar to
that of the in vitro perfused human placenta. Exactly how
representative this is of in vivo metabolism has yet to be established.
The rate of O2 consumption
measured in the human placenta in vitro is low in comparison with other
isolated epithelial tissues that have high rates of salt and water
secretion or absorption such as the pancreas and kidney. Isolated human
pancreatic ducts have been shown to consume
O2 at a rate of 1,013 µmol · kg wet wt1 · min1
(3). The value measured in human pancreatic duct was in turn similar to
rates measured in isolated rat pancreatic cells and whole rat pancreas
(3). Liver and kidney cells have been shown to consume
O2 at a rate of ~2
µmol · g1 · min1
(7), with the isolated rabbit proximal tubule consuming 20-25 µmol O2 · min1 · g
protein1 (25). One of the
main consumers of O2-derived
energy in most tissues is the
Na+-K+-ATPase.
Activity of this transporter accounts for more than 30% of the total
energy requirement of most cells (1). The observation that
O2 utilization in first trimester
placenta exceeds that of term therefore suggested that
Na+-K+-ATPase
activity might be greater in early than late gestation. We tested this
hypothesis directly by measuring
O2 consumption by first trimester
and term tissue in the presence and absence of ouabain. The experiments
showed that activity of the
Na+-K+-ATPase
accounted for ~22% of O2
consumed by first trimester placenta in vitro, but very little of the
term tissue O2 consumption. These data suggest that, in in vitro at least, this electrogenic transporter is significantly more active in placental tissue during early pregnancy
than at term. The proportion of O2
consumption inhibited by ouabain in placental tissue (a maximum of
22%) reported in the present study is, as with total
O2 consumption, low in comparison with other tissues. For example, ~50% of total
O2 utilization is inhibitable by
ouabain in the isolated rabbit proximal tubule (25). The low
ouabain-sensitive O2 consumption
rate observed in human placenta indicates that
Na+-K+-ATPase
activity is low in this tissue. This has recently been confirmed
directly as has the difference in activity between first trimester and
term placentas (26).
Microelectrode Studies
Measurement of Em. The primary objective of this study was to measure Em in villi from first trimester human placenta and to compare this to the Em measured at term. The median Em measured in first trimester placental villi was significantly more negative than that measured in term villi. This fall in Em between early and late gestation is in agreement with the findings of Carstensen et al. (9), who reported Em in human placentas between weeks 12 and 24 to be higher than that at term. In placentas from weeks 12 to 24 of pregnancy, 78% of Em values were between20 and 65 mV; at term, 91% of
Em values were
between 0 and 20 mV.
The Em was found to be significantly more negative in early than in late first trimester placental villi. Em values were compared before and after peak hCG secretion by the placenta (8), and the differences in Em suggest that there might be a link between hCG and membrane electrogenesis. hCG has been demonstrated to induce a 5-mV depolarization of Em in syncytialized human cytotrophoblast cells in culture (14), which was attributed to the activation of chloride currents, resulting in chloride efflux from the cell (13). It could be that changes in Em and hCG concentrations are independently associated with villus maturation (change in villus type and differentiation of the syncytiotrophoblast), which occurs throughout gestation. However, it seems most likely that both villus development (change in villus type from mesenchymal/immature intermediate in first trimester to mature intermediate/terminal villi at term) and the endocrine milieu will influence the characteristics of the MVM and lead to a change in Em.
The Em measured
in placental villi by us and others is relatively low compared with
intracellular potentials in other tissues (27). However, potentials of
a similar magnitude to those measured in human placenta have been
observed in some cells. A mean potential of 18 mV has been
reported in human hepatocytes, values ranging from 9 to
78 mV have been observed in rat hepatocytes, and in perfused rat
liver mean potentials ranging from 33 to 55 mV have been
recorded in vivo and 25 to 45 mV in vitro (27). Also,
membrane potentials of 20 to 80 mV have been reported in
Lettré cells (5).
Assuming similar electrical gradients exist in vivo, the effect of gestation on Em suggests that the electrical driving force for movement of ions from maternal blood into the syncytiotrophoblast across the MVM is different at term compared with early gestation; the electrical force favoring cation uptake will be diminished, whereas the force favoring anion uptake will be enhanced.
Factors contributing to the difference in
Em between first trimester and term
villi. In all cells, the major factors determining the
resting Em are the ionic permeability of the
plasma membrane and the activity of electrogenic transporters (29). In
most cells (e.g., nerve and muscle),
Em depends
primarily on the K+ conductance of
the membrane, such that
Ek 
Em and other ion conductances and electrogenic transporters such as the
Na+-K+-ATPase
make little or no contribution to the resting
Em (29). However,
in some cells, the
Na+-K+-ATPase
is an important contributor to resting
Em. In
hepatocytes that have a low
Em, of similar
magnitude to placental villi, the Na+-K+-ATPase
was found to be a major determinant of
Em (27). Bashford and Pasternak (5) demonstrated that at least 50% of the resting Em in Lettré cells could be generated by
the activity of electrogenic Na+-K+-ATPase
and the permeability of the membrane to potassium ions was less
important. These authors also demonstrated the
Em of human peripheral T lymphocytes to be
generated partially by the ionic diffusion gradient and partially by
electrogenic pumps (5).
1) ROLE OF NA+-k+-atpase in maintaining em. The contribution of the Na+-K+-ATPase to Em in first trimester and term placental villi was assessed directly by measuring Em before and after exposure to ouabain. These experiments demonstrated that, although Na+-K+-ATPase made little contribution to the Em in term placenta, it did make a small (3 mV) but significant contribution to first trimester villus Em. The lack of effect of ouabain on term tissue is unlikely to reflect an inability of ouabain to penetrate the tissue because the syncytiotrophoblast is thinner at term than in first trimester (22). That the contribution of the Na+-K+-ATPase to Em was greater in first trimester than term placental villi is in accordance with our observation (based on O2 consumption rates) and that of others (26) that the activity of this transporter is higher in first trimester than term placenta.
In contrast with our findings, Bara et al. (4) reported that 0.1 mM ouabain has a small depolarizing effect on term placental Em after a 10-min exposure. The reasons for this discrepancy are not clear, although there are methodological differences between the two studies. Bara et al. (4) did not of course make any measurements on tissue from earlier in gestation. Whatever the reasons for the differences between the two studies, it is clear from our data that, although electrogenicity of the Na+-K+-ATPase does play a role in generation of Em in first trimester placenta in vitro, this alone cannot fully account for the difference in magnitude of Em between first trimester and term villi, and other factors must therefore be involved.
2) EFFECT OF ELEVATING EXTRACELLULAR KCL CONCENTRATION ON eM. In most cells, as discussed above, the conductance of the membrane to K+ is much greater than to other ions, and Em reflects the concentration ratio of K+ across the membrane.
Because the success rate of impaling placental villi and sustaining an
impalement during solution change is very low, in the present study we
decided to focus on the possibility of there being differences in the
relative
K+:Cl
conductance of the MVM between first trimester and term by comparing the effects of addition of KCl to the bath solution on
Em in first trimester and term villi. Increasing extracellular KCl concentration in
this way induced a significant depolarization of
Em at both gestations. In three villi from first trimester placentas, the induced
depolarization was greater than that observed in the other 21 first
trimester villi. The explanation for this difference in magnitude of
depolarization is not clear; these three villi were isolated from three
different placentas (gestational ages 9, 10, and 11 wk), and no clear
difference in morphological appearance was apparent. Interestingly, the
slope of the linear regression line fitted to these three data points
[17.46(logx)] was very similar to that fitted to the remaining 21 data points
[18.78(logx)], suggesting that the ion conductance of the MVM was similar over 22-114 mM KCl in all first trimester villi, but that the ion
conductance was different in the three outliers over the range
5-16 mM KCl. These outliers were excluded from all further
analyses. The magnitude of the depolarization induced by increasing
extracellular KCl concentrations was significantly greater in first
trimester than in term villi. Furthermore, the slopes of the regression
lines fitted to these data (Fig. 7) differed significantly between
first trimester and term, suggesting differences in the ion conductance of the MVM at the two gestations. The simplest explanation of these
data is that the K+ conductance of
the MVM is greater in the first trimester than at term.
However, as extracellular K+
concentrations were elevated by addition of KCl, extracellular
Cl concentration was also
elevated in these experiments. Thus the slopes of the two regression
lines cannot be assumed to represent the conductance of only
K+ across the MVM, but instead
reflects the relative
K+:Cl
conductance of this membrane. Under these circumstances, any hyperpolarizing effect of elevating extracellular
Cl would tend to oppose the
depolarizing effect of increasing extracellular K+ concentration, and thus we are
likely to be underestimating the magnitude of the
K+ conductance in the
syncytiotrophoblast MVM. A
Cl conductance has been
demonstrated in this membrane at term (20), but the ion channel(s)
responsible have not been identified. Whether chloride conductances in
the placental MVM change during gestation remains to be determined with
microelectrode experiments, but studies using MVM vesicles prepared
from human placenta have shown that
Cl conductance is similar
in first trimester and term (15). In both first trimester and term
villi, the net response to elevating extracellular KCl concentration
was a depolarization of
Em, which suggests that, as in most other tissues, the
K+ conductance of both first
trimester and term MVM exceeds that for
Cl. Therefore, these data
support the hypothesis that the higher Em
in the first trimester reflects a relatively higher
K+ conductance; the relative role
of
K+:Cl
in membrane electrogenesis at the different stages of gestation could
be determined in the future by examining the effect of
K+ and
Cl channel blockers and
single ion substitutions on
Em. However, microelectrode impalements of villi are technically difficult, and
direct study of K+ and
Cl channels with
patch-clamp techniques may be the most productive approach to reveal
differences in K+ and
Cl transport in first
trimester villi compared with term.
In summary, we report the first measurement of the syncytiotrophoblast microvillous Em in first trimester placental villi and find it to be more negative than at term. A difference in the relative K+ conductance of the MVM observed between first trimester and term villi is most likely to be a major contributor to the difference in the magnitude of Em.
Perspectives
The data reported here add to an increasing body of evidence that there are marked alterations in the transport physiology of the placenta over the course of pregnancy. The demonstration that membrane potential changes highlights the need for investigation of driving forces, as well as for, e.g., investigation of changes in expression of transport proteins. We previously reported that the activity of the system A amino acid transporter is lower in microvillous membrane vesicles isolated from first trimester compared with term placenta (28), but the more negative membrane potential in early pregnancy would tend to drive a greater amino acid influx on this Na+-dependent transporter than that which occurs toward term. The resultant effect of decreasing driving force but increasing expression could be that, in vivo, influx of amino acid across the microvillous membrane is kept constant. The data here, showing that microvillous membrane potential is different in early compared with late first trimester, also raise the possibility that this is a key time in the development of all aspects of placental function. There is clearly a continuing need for more information on all aspects of placental transport physiology in early pregnancy.| |
ACKNOWLEDGEMENTS |
|---|
We thank the staff at St. Mary's Hospital for their assistance.
| |
FOOTNOTES |
|---|
This study was supported by The Medical Research Council (Grant 99209554) and The Wellcome Trust (Grant 04023/2/95/Z/MP).
Address for reprint requests: T. J. Birdsey, Dept. of Child Health and School of Biological Sciences, Univ. of Manchester, St. Mary's Hospital, Hathersage Rd., Manchester M13 OJH, UK.
Received 6 March 1997; accepted in final form 10 June 1997.
| |
REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
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;
n = 46, villous samples taken from 7 placentas) or Dulbecco's modified Eagle's medium (DMEM; 
;
n = 53, villous samples taken from 9 placentas). Regression analysis was used to fit linear models to data
obtained with both 
