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1 Department of Child Health
and School of Biological Sciences, This study examined the effect of
hyposmotic solutions on the syncytiotrophoblast microvillous membrane
potential (Em)
in mature intermediate villi isolated from term human placentas. When
villi were exposed to a control solution (280 mosmol/kgH2O; 116 mM NaCl) and
then to either a 138-hyposmotic (138 mosmol/kgH2O; 37 mM NaCl) or
170-hyposmotic (170 mosmol/kgH2O;
55 mM NaCl) solution, there was a significant hyperpolarization of
Em (
potassium; chloride; conductance; placenta; volume regulation
THE PLACENTA plays a central role in the transfer of
solutes and water 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. The
capillary circulation of the villus is supplied via the umbilical cord
of the fetus. During the last two-thirds of pregnancy, the barrier that
separates maternal blood in the intervillous space from fetal blood in
the villus capillaries comprises two cell layers: the syncytiotrophoblast, in immediate contact with maternal blood, and the
fetal capillary endothelium. The syncytiotrophoblast is thought to be
the transporting epithelium of the human placenta (24). It is a true
syncytium that extends over the entire surface of the villous tree. The
plasma membrane of the syncytiotrophoblast facing the intervillous
space is covered in microvilli [the microvillous membrane
(MVM)] (22).
In epithelia the control of cellular hydration is challenged by
changing solute transport rates (19). If solute uptake exceeds solute
exit, accumulation of osmolytes in the cytoplasm leads to intracellular
hypertonicity, water entry, and cell swelling (18). Under these
circumstances, volume regulatory mechanisms are initiated to return
cell volume to its original value [regulatory volume decrease
(RVD)] (19, 20, 34). When cell swelling is induced experimentally
by exposure to a hyposmotic solution, RVD is achieved by the loss from
the cell cytoplasm of inorganic osmolytes, predominantly
K+ and
Cl In fulfilling its role in effecting maternofetal solute exchange, the
syncytiotrophoblast is exposed to conditions that challenge cell volume
homeostasis. Indeed, there is evidence to suggest that the placenta, in
common with other epithelia, has the ability to regulate its volume.
For example, fragments of placental tissue and cytotrophoblast cells
isolated from human placenta respond to a hyposmotic stimulus by
releasing 86Rb, used as a marker
for K+ (8, 15, 16);
125I, used as a marker for
Cl There are several mechanisms by which
K+ and
Cl The primary aim of this study was to determine whether ions are lost
over the microvillous membrane of the syncytiotrophoblast in response
to swelling. This was achieved by measuring the response of the
microvillous membrane potential
(Em) to a
hyposmotic stimulus in isolated intact placental villi and examining
the effect of ion channel blockers. The studies constitute the first
step toward characterizing the role of ion conductances in volume
regulation by this unique human syncytial epithelium. Preliminary
findings were published in abstract form (5).
Tissue Collection
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
5.1 ± 1.5 mV, P < 0.01 and
5.0 ± 0.5 mV, P < 0.001, respectively; n = 10), which was
reversible on removal of the hyposmotic stimulus. Low-NaCl (37 and 55 mM) solutions made isosmotic with control (i.e., 280 mosmol/kgH2O) by addition of
raffinose did not significantly alter Em, suggesting
that reducing NaCl concentration per se had no effect on
Em. Exposure to 170-hyposmotic
solution in the presence of 5 mM
BaCl2 depolarized
Em by +4.1 ± 0.7 mV (P < 0.001, n = 6);
BaCl2 similarly depolarized
Em when added in
control solution (+5.6 ± 1.1 mV, n = 5). Exposure to 170-hyposmotic solution containing 1 mM DIDS
hyperpolarized Em
by 9.0 ± 1.7 mV (P < 0.001, n = 5). This degree of
hyperpolarization was significantly greater than that observed in
hyposmotic solution alone (P < 0.01)
but was not different from the hyperpolarization when DIDS was added to control solution (7.4 ± 0.2 mV,
n = 6). We conclude
1) that
Ba2+-sensitive
K+ conductances and DIDS-sensitive
anion conductances contribute to the resting potential of the
syncytiotrophoblast microvillous membrane and
2) that the syncytiotrophoblast
microvillous membrane responds to a hyposmotic stimulus by activating
both Ba2+-sensitive
K+ and DIDS-sensitive anion conductances.
INTRODUCTION
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METHODS
RESULTS
DISCUSSION
REFERENCES
(13, 19, 20, 34) and
organic solutes, for example amino acids, methylamines, and polyols
(19, 35).
(31, 37); and amino
acids, including taurine, glutamic acid, methylaminoisobutyric acid,
and aspartate (27, 32, 33). However, as yet there is little information
concerning the mechanisms of swelling-induced osmolyte loss.
Furthermore, it is not known whether, in the intact placental villus,
the solutes lost during RVD pass over the basal membrane of the
syncytiotrophoblast into the fetal circulation or cross over the MVM
into maternal blood.
are lost from the
cytoplasm in response to cell swelling, including the activation of
K+ and
Cl channels, KCl
cotransport, and coupled
K+/H+
and
Cl/HCO3
transporters (19, 20). Activation of
K+ and
Cl channels is probably the
predominant mechanism effecting K+
and Cl loss in epithelial
cells (18). The activation of K+
and Cl conductances in
response to swelling has been shown to affect the potential across the
plasma membrane of cells in culture [e.g., liver (12, 13),
proximal tubule (11), colonic carcinoma (3), and Madin Darby canine
kidney cells (26, 29)] and across the basolateral membrane of
cells in intact tissues such as the intestine and gallbladder (30, 36).
METHODS
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RESULTS
DISCUSSION
REFERENCES
3-Earle's solution (in mM; 116 NaCl, 5.4 KCl, 1.8 CaCl2, 0.4 MgSO4, 1.0 NaH2PO4,
5.5 glucose, 26 NaHCO3; 280 mosmol/kgH2O), gassed with 95%
O2-5%
CO2 (to pH 7.4), and maintained at
room temperature.
Measurement of Em
Em was measured in villi isolated from term placentas via a previously described method (6, 14). Individual mature intermediate villi (22) were removed, placed in a thermostatically regulated heated tissue perfusion chamber (Intracel, Royston, UK), and immobilized with glass suction pipettes in such a way as to isolate the villous core from the bath at the cut end of the villus. The villi were continuously superfused (1.5 ml/min) with HCO3-Earle's solution (control
solution) at 37°C and gassed with 95%
O2-5% CO2. The perfusion chamber had
microenvironmental control, which allowed a stream of gas (95%
O2-5%
CO2) to pass across the surface of solution in the perfusion chamber, maintaining a pH of 7.4. The
perfusion chamber was placed on the stage of an inverted microscope (Nikon Diaphot), and the electrical measurements were made using an
AxoClamp 2B amplifier (Axon Instruments, Foster City, CA). After being
mounted, the villus was allowed to equilibrate with the control
solution for a minimum of 10 min.
Em was then
measured between a recording microelectrode (A) tapped into the
outermost layer of the tissue (syncytiotrophoblast) and a similar
electrode (B) placed in the bathing fluid. Both electrodes were pulled
with a horizontal puller (Brown and Flaming model P-87; Sutter
Instruments, Novato, CA) from 1.2-mm (outer diameter) borosilicate
glass containing a filament (Clark Electromedical Instruments, Reading,
UK). 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; ground) positioned directly in the bath fluid.
Em was recorded
as
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Effect of Hyposmotic and Isosmotic Solutions on Em
Effect of hyposmotic solution on Em. Em was measured after villi were exposed to solutions made hyposmotic by removal of NaCl from the control solution. Two hyposmotic challenges were used: a 138-hyposmotic solution (138 mosmol/kgH2O; 37 mM NaCl; concentrations of other solutes as for control solution) and a 170-hyposmotic solution (170 mosmol/kgH2O; 55 mM NaCl). Osmolalities were measured by freezing point depression (Roebling MOD 100; Camlab, Cambridge, UK).After equilibration in control solution, each villus was impaled with a recording microelectrode (as described in Measurement of Em). If a stable impalement was achieved (meeting criteria 1 and 2), a control Em was recorded for a minimum of 2 min. Then, with the electrode still positioned in the tissue, the inflow to the bath was exchanged for either 138-hyposmotic or 170-hyposmotic solution. Em was measured during a 13-min exposure to hyposmotic challenge, after which the bath fluid was changed to control solution, and Em was recorded during a 5-min (minimum) recovery period. 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.
Effect of isosmotic solution on Em. In the experiments described above, the solutions were made hyposmotic by removal of NaCl. To determine whether the response observed with hyposmotic challenge was a result of the reduction in osmolality of the solution or the reduction in NaCl concentration or ionic strength, the protocol described in Effect of hyposmotic solution on Em was repeated with low-NaCl (i.e., 138-hyposmotic and 170-hyposmotic) solutions made isosmotic to the control solution (i.e., 280 mosmol/kgH2O) by addition of either mannitol or raffinose.
Effect of Inhibitors on Em Response to Hyposmotic Solutions and on Resting Em
Cell swelling and subsequent volume recovery during exposure to low-NaCl hyposmotic solution will be accompanied by dynamic and unpredictable changes in intracellular ion content. The concurrent change in Em will thus reflect both activation of membrane ion conductances and changes in intracellular ion activities. The role of K+ and Cl conductances in the
hyperpolarization of syncytiotrophoblast Em in response to
hyposmotic challenge was investigated by measuring Em in hyposmotic
or control solution in the presence of the
K+ channel blocker barium chloride
(Ba2+) or the anion transport
inhibitor DIDS. Ba2+ forms an
insoluble product in the presence of sulfate; therefore, in these
inhibitor experiments 0.4 mM MgSO4
was replaced in control and hyposmotic solutions with 0.4 mM
MgCl2.
As before, the isolated villus was equilibrated by superfusing with control solution, a stable impalement was achieved (meeting criteria 1 and 2), and the Em was recorded. Then, with the electrode still positioned in the tissue, the inflow to the bath was exchanged for either 170-hyposmotic solution containing 5 mM Ba2+ or 1 mM DIDS or control solution containing 5 mM Ba2+ or 1 mM DIDS. Em was measured during a 10-min exposure to solutions containing Ba2+ and during a 14-min application of solutions containing DIDS. 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 again acted as its own control.
Chemicals
All reagents were analytic grade from standard suppliers. Ba2+ was purchased from Sigma (Poole, UK), and DIDS was purchased from Calbiochem-Novabiochem (Beeston, UK).Statistics
The effects of hyposmotic challenge, isosmotic solution, Ba2+, and DIDS (under control and hyposmotic conditions) on MVM Em were assessed statistically with a repeated-measures ANOVA followed by a Bonferroni multiple comparisons test.A two-way ANOVA was used to determine whether the change in Em observed in 138-hyposmotic and equivalent isosmotic solutions differed significantly from the change in Em observed with 170-hyposmotic and equivalent isosmotic solutions. Any significant differences identified by this analysis were examined further with a Mann-Whitney test (nonparametric statistics were used for this comparison because the 138-hyposmotic and equivalent isosmotic data did not follow a normal distribution; Bartlett's test for homogeneity).
The maximum change in Em measured in 138-hyposmotic and equivalent isosmotic solutions (containing mannitol or raffinose) was compared statistically using a Kruskal-Wallis nonparametric ANOVA followed by a Mann-Whitney test.
An ANOVA followed by a Bonferroni multiple comparisons test was used to compare 1) the maximum change in Em measured in 170-hyposmotic and equivalent isosmotic solutions (containing mannitol or raffinose) and 2) the change in Em measured in 170-hyposmotic and control solutions containing either Ba2+ or DIDS.
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DISCUSSION
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Effect of Hyposmotic and Isosmotic Solutions on Em
Effect of hyposmotic solutions on Em. The effects of 138-hyposmotic and 170-hyposmotic solutions on isolated placental villus Em are shown in Figs. 1A and 2A, respectively; an example trace from an individual experiment is shown together with the mean data. Exposure to both 138-hyposmotic and 170-hyposmotic solutions induced a significant hyperpolarization of Em, which was reversible on removal of the hyposmotic stimulus (P < 0.01 and P < 0.001 vs. control recovery for 138-hyposmotic and 170-hyposmotic solutions, respectively; repeated-measures ANOVA plus Bonferroni). Despite the difference in the degree of osmotic challenge, the mean change in Em was similar with both 138-hyposmotic and 170-hyposmotic solutions (Fig. 3).
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Effect of isosmotic solution on Em. In the hyposmotic experiments described in Effect of hyposmotic solutions on Em, the osmolality of the solutions was reduced by removal of NaCl. The hyperpolarization of Em during exposure to these hyposmotic solutions may therefore be caused by the reduction in osmolality or the reduction in NaCl concentration or ionic strength of the solutions. To distinguish between these possibilities, the villi were exposed to solutions with low NaCl concentration (i.e., 138-hyposmotic or 170-hyposmotic solutions), whose osmolality was increased to 280 mosmol/kgH2O (isosmotic with control solution) by addition of mannitol or raffinose.
The effects of 138-hyposmotic and 170-hyposmotic solutions made isosmotic by the addition of mannitol [hereafter termed 138-isosmotic (mannitol) and 170-isosmotic (mannitol)] on placental villus Em are shown in Figs. 1B and 2B, respectively. An example trace from an individual experiment is shown together with the mean data. Application of either 138-isosmotic (mannitol) or 170-isosmotic (mannitol) solution induced a significant hyperpolarization of Em, which was reversible on removal of the isosmotic (mannitol) solution [P < 0.01 vs. control, P < 0.001 vs. recovery for 138-isosmotic (mannitol), and P < 0.001 vs. control and recovery for 170-isosmotic (mannitol); repeated-measures ANOVA plus Bonferroni]. The degree of hyperpolarization induced by 138-isosmotic (mannitol) solution did not differ significantly from that observed during exposure to 170-isosmotic (mannitol) solution. Both 138-isosmotic (mannitol) and 138-hyposmotic solutions caused a similar change in Em. However, the hyperpolarization of Em observed during application of 170-isosmotic (mannitol) solution was smaller than that initiated during 170-hyposmotic challenge (Fig. 3).
The Em responses to 138-hyposmotic and 170-hyposmotic solutions made isosmotic by the addition of raffinose [hereafter termed 138-isosmotic (raffinose) and 170-isosmotic (raffinose)] are shown in Figs. 1C and 2C, respectively. Again, an example trace from an individual experiment is shown together with the mean data. Neither 138-isosmotic (raffinose) nor 170-isosmotic (raffinose) solution had any significant effect on Em (repeated-measures ANOVA plus Bonferroni). The change in Em observed with 170-isosmotic (raffinose) solution was significantly smaller than that induced by both 170-hyposmotic solution (P < 0.001; ANOVA plus Bonferroni) and 170-isosmotic (mannitol) solution (P < 0.01; ANOVA plus Bonferroni). The change in Em in 138-isosmotic (raffinose) solution was not significantly different at the 5% level (Kruskal-Wallis nonparametric ANOVA) from that observed with 138-hyposmotic and 138-isosmotic (mannitol) solutions.
Effect of Inhibitors on Resting Em and Em Response to Hyposmotic Solutions
Effect of Ba2+. The Em responses to 170-hyposmotic solution containing 5 mM Ba2+ and control solution containing 5 mM Ba2+ are shown in Fig. 4, B and C, respectively. The Em response to 170-hyposmotic solution previously presented in Fig. 2A is shown again in Fig. 4A to allow direct comparison of the data. Ba2+ applied in control solution induced a depolarization of resting Em (P < 0.001 at 4 and 10 min vs. control; repeated-measures ANOVA plus Bonferroni). The maximum depolarization induced in the presence of Ba2+ was similar when the inhibitor was applied in either 170-hyposmotic or control solution (Fig. 6). The hyperpolarization of Em observed previously (Fig. 4A) during exposure to hyposmotic challenge did not occur in the presence of Ba2+ (P < 0.001, 170-hyposmotic Ba2+ vs. 170-hyposmotic; ANOVA plus Bonferroni; Fig. 6). Indeed, exposure to 170-hyposmotic solution in the presence of Ba2+ significantly depolarized Em (P < 0.01 at 4 min and P < 0.001 at 10 min vs. control; repeated-measures ANOVA plus Bonferroni; Fig. 4B).
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Effect of DIDS. The
Em responses to
170-hyposmotic solution containing 1 mM DIDS and control solution
containing 1 mM DIDS are shown in Fig. 5,
B and
C, respectively. The
Em response to 170-hyposmotic solution previously presented in Fig.
2A is shown again in Fig.
5A to allow direct comparison of the
data. Exposure to both control solution containing DIDS and
170-hyposmotic solution containing DIDS significantly hyperpolarized
Em
(P < 0.001 at 4 and 10 min vs.
control and P < 0.001 vs. control,
respectively; repeated-measures ANOVA plus Bonferroni). The
hyperpolarization of
Em usually
observed with 170-hyposmotic solution alone was potentiated when this
hyposmotic solution was applied in the presence of DIDS (Fig.
6). The degree of hyperpolarization
observed during exposure to control solution containing DIDS or to
170-hyposmotic solution containing DIDS did not differ significantly
(Fig. 6). In these experiments DIDS was initially dissolved in DMSO.
This vehicle alone (at concentration used in DIDS-containing solution) has no significant effect on placental villus Em
(control Em 18.9 ± 1.0 mV, after
10-min exposure to DMSO 18.3 ± 1.0 mV;
n = 6, repeated-measures ANOVA plus
Bonferroni).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Hyposmotic and Isosmotic Solutions on Em
Exposure of placental villi to 138-hyposmotic or 170-hyposmotic solutions induced a sustained hyperpolarization of Em, which was reversible on removal of the hyposmotic stimulus. These data suggest that ion conductances are activated in the MVM of the syncytiotrophoblast during hyposmotic challenge. The observation that Em hyperpolarized when villi were bathed in hyposmotic solution suggests that there is a net loss of positive ions from the syncytiotrophoblast in response to this stimulus.In these experiments the solutions were made hyposmotic by removal of NaCl. The hyperpolarization of Em observed during exposure to these hyposmotic solutions might therefore be caused by the reduction in NaCl concentration or ionic strength per se, rather than the reduction in osmolality of the solution. To distinguish between these two possibilities, the villi were exposed to solutions with low NaCl concentration (i.e., 138-hyposmotic or 170-hyposmotic solutions) made isosmotic to control solution by addition of mannitol or raffinose. Solutions made isosmotic by the addition of mannitol induced a significant hyperpolarization of Em; however, those made isosmotic with raffinose had no effect on Em.
The ionic composition of the 138-isosmotic (mannitol) and 138-isosmotic (raffinose) solutions were identical, as were the compositions of the 170-isosmotic (mannitol) and 170-isosmotic (raffinose) solutions. Therefore, the differing Em responses observed with isosmotic (mannitol) and isosmotic (raffinose) solutions cannot have occurred as a consequence of the reduction in NaCl concentration. The probable explanation for the different Em responses to isosmotic (mannitol) and isosmotic (raffinose) solutions is differential permeability of the syncytiotrophoblast MVM to the two saccharides. Jansson et al. (21) found that the MVM of the syncytiotrophoblast is more permeable to the monosaccharide mannitol than to the trisaccharide raffinose, whose molecular weight and molecular radius are substantially greater. It is likely that, in our experiments, mannitol enters the syncytiotrophoblast across the MVM down its concentration gradient, inducing intracellular hypertonicity, which in turn causes cell swelling and activation of volume regulatory mechanisms resulting in hyperpolarization of Em. The lack of effect of isosmotic (raffinose) solutions on Em supports the theory that hyperpolarization during exposure to hyposmotic solutions was a response to the reduction in osmolality per se, not specifically to a reduction in NaCl concentration.
Previous observations in other tissues suggest that the magnitude of
ion loss during RVD increases with decreasing extracellular fluid
osmolality. For example, the magnitude of the whole cell chloride
conductance increases in several cell types as extracellular osmolality is decreased (1, 2, 25). However, because the degree of
hyperpolarization induced in isolated placental villi in response to
138-hyposmotic and 170-hyposmotic solutions is similar, there does not
appear to be a differential loss of positive or negative ions from the
syncytiotrophoblast as the hyposmotic challenge increases. In this
context it is interesting that
86Rb
(K+) efflux from villous
fragments in response to hyposmotic challenge is increased as solution
osmolality is decreased from 170 mosmol/kgH2O to 138 mosmol/kgH2O (S. L. Greenwood and
C. P. Sibley, unpublished observations). Thus it is
probable that, although quantitatively more
K+ and
Cl ions might be lost from
the syncytiotrophoblast on exposure to 138-hyposmotic than would be
lost on exposure to 170-hyposmotic solution, the relative loss of ions
is the same with both solutions.
Although the response of
Em to a
hyposmotic stimulus has been measured in several types of cell in
culture [e.g., liver (12, 13), rabbit proximal tubule (11), human
colonic carcinoma (3), and Madin Darby canine kidney (26, 29)],
there are fewer observations in intact tissues. A sustained
hyperpolarization, similar to that measured here in placental villi,
has also been observed in liver slices (12, 13) and in
Necturus small intestine and
gallbladder (30, 36). In the latter studies the changes in
Em with
hyposmotic swelling were measured at the basolateral (contraluminal)
membrane, suggesting that ions are lost from the cell cytoplasm to
plasma during RVD. It is interesting to note that the hyperpolarization
in response to the 138-hyposmotic and 170-hyposmotic solutions is
slower in onset than is the time for recovery of
Em on
reintroducing the control solution. We have not investigated the
mechanism of the repolarization, but it is similar to the time course
of the response reported in liver (12). One explanation is that
K+ (and
Cl) conductances are
inactivated more rapidly in response to shrinking than they are
activated during swelling.
Effect of Inhibitors on Response to Hyposmotic Challenge and on Resting Em
In epithelial cells the most common mechanism effecting RVD is the loss of K+ and Cl from the cytoplasm by
the activation of K+ and
Cl channels (18).
Preliminary evidence suggests that
K+ and
Cl channels are also
activated in placenta in response to swelling. In multinucleated
cytotrophoblast cells, RVD in response to a hyposmotic stimulus is
associated with an increase in
86Rb efflux, and both volume
recovery and 86Rb loss are
inhibited by the K+ channel
blocker Ba2+ (15, 17). Hyposmotic
swelling induces 86Rb efflux and
125I efflux from placental villous
fragments, and the latter is sensitive to the anion transport inhibitor
niflumic acid (8, 31). These data suggest
K+ and
Cl channel involvement in
RVD by the placenta, although the studies cannot discriminate between
the microvillous and basal membrane of the syncytiotrophoblast as the
site of channel activation. Therefore, we investigated the presence of
volume-sensitive K+ and
Cl conductances in the MVM
of the intact syncytiotrophoblast by evaluating the
Em response to a
hyposmotic solution in the presence of
Ba2+ and DIDS.
Effect of Ba2+. The hyperpolarization of Em usually observed in isolated placental villi in response to 170-hyposmotic solution did not occur in the presence of Ba2+; in fact, the Em depolarized to a value significantly less negative than the resting Em. The observation that Em depolarized during application of hyposmotic solution containing Ba2+ suggests that Ba2+ inhibits both a volume-activated K+ conductance and a K+ conductance that normally contributes to the resting Em. Indeed, application of Ba2+ in control solution resulted in a sustained depolarization of Em, confirming that a Ba2+-sensitive K+ conductance normally contributes to the resting Em of the syncytiotrophoblast. Thus in 170-hyposmotic solution, Ba2+ appears to inhibit both a volume-activated and a basal K+ conductance.
Ba2+-sensitive K+ conductance or 86Rb efflux has been shown to play an important role in volume regulation during hyposmotic challenge in a number of cells and tissues (e.g., Refs. 10, 12, 13, 23, 30). Ba2+ also inhibits volume-activated 86Rb loss and RVD in cytotrophoblast cells, which are the cytological precursors of the syncytiotrophoblast (15, 17). The observation in this study that Ba2+-sensitive conductances are activated in the MVM of the intact syncytiotrophoblast in response to hyposmotic solutions is consistent with the hypothesis that the syncytiotrophoblast achieves volume regulation, at least in part, by extrusion of K+ from the cytosol over the MVM into maternal blood.
Effect of DIDS. The hyperpolarization
of Em observed in
response to 170-hyposmotic solution was potentiated in the presence of
DIDS. These data suggest that a DIDS-sensitive
Cl conductance is activated
in the syncytiotrophoblast MVM in response to hyposmotic challenge.
When applied in control solutions, DIDS also hyperpolarized
Em, indicating
that a DIDS-sensitive Cl
conductance normally contributes to the resting
Em of the
syncytiotrophoblast. When applied in 170-hyposmotic solution, DIDS thus
appears to inhibit both a volume-activated and a basal
Cl conductance.
DIDS-sensitive Cl currents
and
125I/36Cl
efflux contribute to hyposmotically induced RVD in many cell types (see
Ref. 25). In keeping with these observations, DIDS has been shown to
inhibit volume-activated 125I loss
and volume-activated whole cell chloride currents in cytotrophoblast cells (7, 37). Thus the activation of DIDS-sensitive conductances in
the MVM of the intact syncytiotrophoblast in response to hyposmotic solutions that was observed in this study suggests that the
syncytiotrophoblast loses
Cl from the cytosol over
the MVM to maternal blood to effect RVD.
In conclusion, the data from the present study imply that both
K+ and
Cl are lost from the
syncytiotrophoblast across the MVM to the maternal circulation during
(or as part of) a volume-regulatory response to hyposmotic challenge,
via Ba2+ and DIDS-sensitive
conductances, respectively. At present we are unable to assess
volume-regulatory changes in the syncytiotrophoblast of the intact
villus. However, multinucleated cytotrophoblast cells isolated from
term placenta and maintained in culture exhibited Ba2+-sensitive volume-regulatory
decreases over 15-30 min in response to 138 mosmol/kgH2O solution (17). These
data support our hypothesis that ion channel activation forms part of
the volume-regulatory response to hyposmotic challenge in placental
trophoblast. Our experiments also demonstrate that a
Ba2+-sensitive
K+ conductance and a
DIDS-sensitive Cl
conductance contribute to maintaining the resting
Em of the
syncytiotrophoblast; this has not previously been shown.
The lack of specific channel inhibitors complicates identification of
the channels involved in volume-activated and resting membrane
conductances. For example, DIDS, at the concentration used in this
study, not only inhibits Cl
channels but also other anion transport processes, e.g.,
HCO3/Cl
exchange; inhibition of this exchange will affect intracellular pH,
which might have secondary effects on
K+ and
Cl channels and
Em. Direct study
of the K+ and
Cl channels using the patch
clamp technique is therefore likely to be the most productive approach
in future studies to reveal the single-channel basis for both the
volume-activated and basal Ba2+
and DIDS-sensitive conductances.
Perspectives
Em is an important driving force for transport of solutes across the syncytiotrophoblast and thus for maternofetal exchange. However, measurement of Em in intact villi is technically very challenging (6), and to date there have been only four reports of placental villus Em in the literature (4, 6, 9, 14), none of which has addressed the contribution of Ba2+-sensitive K+ conductances and DIDS-sensitive Cl conductances to the
resting Em of the syncytiotrophoblast.
In this study we also show a hyperpolarization of the
syncytiotrophoblast
Em in isolated
placental villi exposed to hyposmotic solutions, apparently caused by a
differential loss of K+ vs.
Cl over the MVM. Hyposmotic
challenge is used as a reproducible means of altering cell volume and
has been used in this way in many studies in both placenta and other
cell types (17, 18). The physiological equivalent to this experimental
manipulation is likely to be accumulation of intracellular solute
resulting in intracellular hypertonicity, water influx, and cell
swelling (19). Indeed, exposure to hyposmotic solution or amino acids has been shown to induce similar volume-regulatory responses in cytotrophoblast cells (27, 28).
Clearly, understanding the interaction between solute transport and changes in electrical driving force will be important for a complete understanding of the mechanisms of maternofetal exchange.
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ACKNOWLEDGEMENTS |
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We thank the staff at St. Mary's Hospital for assistance in obtaining placentas.
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FOOTNOTES |
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This study was supported by The Wellcome Trust Grant 04023/2/95/Z/MP.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. J. Birdsey, Dept. of Obstetrics and Gynaecolgy, Univ. of Sheffield, Northern General Hospital, Herries Road, Sheffield, S5 7AU United Kingdom (E-mail: t.j.birdsey{at}sheffield.ac.uk).
Received 1 July 1998; accepted in final form 18 January 1999.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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P < 0.01 vs. 170-isosmotic (mannitol) (ANOVA plus Bonferroni multiple
comparisons test). Data for 138-hyposmotic and 138-isosmotic solutions
were analyzed with Kruskal-Wallis nonparametric ANOVA.
