Human syncytiotrophoblast NPY receptors are located on BBM and
activate PLC-to-PKC axis
Jacques
Robidoux1,
Lucie
Simoneau2,
Serge
St-Pierre3,
Hafid
Ech-Chadli2, and
Julie
Lafond1,2
1 Département
d'Obstétrique-Gynécologie, Faculté de
Médecine, Université de Montréal, Montreal H3C
3J7; and Départements des
2 Sciences Biologiques and de
3 Chimie, Université du
Québec à Montréal, Montréal, Québec,
Canada H3C 3P8
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ABSTRACT |
Neuropeptide Y (NPY) is abundant in plasma and
amniotic fluid of women throughout pregnancy, during which its
involvement in placental hormonogenesis has been proposed. In
accordance with its putative role, the aim of this study was to
characterize the human placental syncytiotrophoblast receptivity to
NPY. Thus we performed this study on brush-border membranes (BBM) and
basal plasma membranes (BPM). Specific
125I-labeled NPY
(125I-NPY) binding to BBM was
rapid (20 min), saturable, with a maximum binding capacity of 604 ± 100 fmol/mg protein, and of high affinity, with a dissociation constant
of 11 ± 3 nM. No saturable binding could be
shown in BPM. The rank order of affinity of NPY and related peptides to
compete for 125I-NPY binding sites
was peptide YY (PYY) > NPY = [Leu31,Pro34]NPY > 13-36NPY >>
pancreatic polypeptide (PP). It is noteworthy that PYY displaced only
45% of the binding sites. In BBM, both NPY and PYY were potent
phospholipase C (PLC) stimulators, leading to a four- to fivefold
increase of control phosphodiesterase activity. The latter effect could
be prevented by preincubation of membranes with 5 µM U-73122, a known
inhibitor of G protein-linked receptor activation of
PLC-
. Furthermore, 5 µM BIBP-3226, a
Y1-receptor antagonist, shifted
both dose-response curves to the right in a similar fashion for both
peptides. In accordance with the PLC stimulation, both peptides also
induced stimulation of protein kinase C (PKC) activity, which could be
partially but additively prevented by U-73122 and LY-294002, a
selective inhibitor of phosphatidylinositol-3 kinase (PI3K). Taken
together, these data suggest that placental and blood-derived NPY binds
to a mixed population of receptors composed of
Y1 and
Y3 subtypes on the maternal side
of the syncytiotrophoblast, where it can mediate its physiological
purposes via PLC-
and PI3K activation, both of which lead to PKC
activation. However, because BIBP-3226 antagonized both effects, the
physiological relevance of the apparent
Y3 fraction is still unsolved.
placenta; neuropeptide Y; phospholipase C-
; protein kinase
C
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INTRODUCTION |
HUMAN PLACENTA, fetal membranes (amnion
and chorion), and maternal decidua play an important role in the
maintenance of pregnancy because of their ability to produce a large
variety of bioactive peptides (28). Among these,
neuropeptide tyrosine (neuropeptide Y; NPY), first isolated from brain
(40), is particularly abundant in plasma and amniotic fluid of women
throughout pregnancy (27). The importance of this peptide in the
gestational process is still unclear, but its synthesis by
cytotrophoblastic cells, amnion, chorion, and decidua favors an
important physiological contribution (25, 26). Binding sites for NPY
are present in all peripheral cells of terminal villi, which are
composed of the outer syncytiotrophoblastic layer and of the inner
cytotrophoblastic cells. In those cells, NPY has been implicated in the
control of corticotropin-releasing factor (CRF) and inhibin release
(29, 30).
There is evidence that NPY and its related peptides, i.e., peptide YY
(PYY) and pancreatic polypeptide (PP), perform their physiological
actions through interaction with at least nine receptor subtypes. These
subtypes include the cloned Y1,
Y2,
Y4/PP1,
Y5JBC, and
Y5NAT (3, 14, 19, 34, 45), the
pharmacologically well-characterized
Y3, the PYY-preferring and
nonselective (2, 7, 43), and the recently added feeding receptor (24).
The Y1,
Y2,
Y4/PP1,
Y5,
Y6, PYY-preferring, and
nonselective subtypes are all linked to the inhibition of stimulated
adenylate cyclase (2, 3, 7, 14, 19, 34, 45), whereas the
Y1,
Y2, Y3, and
Y4/PP1
subtypes are linked to the rise of intracellular calcium concentration
(3, 19, 34, 43). The latter effect has been shown in some (10, 37) but
not all (20, 22) studies to be dependent on phospholipase C (PLC)
activation. Recently, Nakamura et al. (23) showed that the
Y1 subtype is also linked to
phosphatidylinositol-3 kinase (PI3K) and subsequently to
mitogen-activated protein kinase activation. Until now, the linking of
the feeding receptor described by O'Shea et al. (24) to a second
messenger system has not been explored. Moreover, the vast majority of
the above effects have been suggested to be mediated via pertussis toxin-sensitive G proteins (20, 22, 47).
In light of the putative role of NPY in hormonogenesis control and the
polarized nature of the syncytiotrophoblast (46), the aim of this study
was to investigate both human syncytiotrophoblastic brush-border (BBM)
and basal plasma (BPM) membrane receptivity to NPY and its related
peptides and to evaluate the possible modulation by NPY and PYY of
adenylate cyclase and PLC activities, both known triggers of hormone
release via an increase of subplasmalemmal calcium
concentration.
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MATERIALS AND METHODS |
Preparation of syncytiotrophoblastic BBM and BPM.
Membranes were purified from placental tissue collected according to
established institutional ethical guidelines from St-Luc Hospital
(Montreal, PQ), mainly as described by Lafond et al. (17) with some
modifications. After the amnion, chorion, and decidual layer were
removed, the tissue was minced and stirred for 45 min in 10 mM
tris(hydroxymethyl)aminomethane
(Tris)-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4) containing 270 mM mannitol, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml benzamidine, 10 µM
leupeptin [except for PLC and protein kinase C (PKC)
experiments], and 0.5 µg/ml aprotinin (Tris-HEPES-mannitol
buffer). This homogenate was filtered through cotton gauze, the
filtrate was centrifuged for 15 min at 2,900 g, and the supernatant, from which BBM
were prepared, was centrifuged at 150,000 g for 60 min. The placental tissue, from which BPM were prepared, was washed and stirred for an additional 45 min with the Tris-HEPES-mannitol buffer in the presence of 10 mM
EDTA and was processed as the first homogenate. Both crude membrane
preparations were separately suspended in 2 mM Tris-HEPES-mannitol buffer (pH 7.0) containing the same antiproteases and stirred for 20 min after the addition of 10 mM
MgCl2. Both mixtures were centrifuged for 20 min, at 2,900 g for
BPM and 3,600 g for BBM. For BBM,
supernatant was centrifuged twice at 35,000 g for 30 min in Tris-HEPES-mannitol
buffer, and the purified membranes were stored at
80°C until
use (except for PLC and PKC experiments, in which membranes were used
fresh).
For BPM, the pellet was diluted in 10 mM Tris-HEPES buffer (pH 7.4) and
stored at
80°C for 30 min. The thawed BPM were centrifuged for 30 min at 90,000 g, and the pellet
was suspended in Tris-HEPES buffer and layered on top of a 4%-10%
discontinuous Ficoll gradient and centrifuged for 60 min at 60,000 g. The interface was collected and
centrifuged twice at 35,000 g, and the
purified membranes were stored at
80°C until use.
Membrane purity was monitored by measurement of alkaline phosphatase
activity (BBM marker) as already described (17) and by measurement of
Na+-K+-adenosinetriphosphatase
(ATPase) activity (BPM marker) using the technique of Post and Sen
(31).
Binding of 125I-labeled NPY to
syncytiotrophoblast membranes.
Binding experiments were performed as previously described (18) with
minor modifications. Briefly, membranes were washed and suspended in a
binding buffer consisting of 20 mM HEPES (pH 7.4), 250 mM sucrose, 1%
bovine serum albumin (BSA), 1 mM
MgCl2, 1 mM
CaCl2, 0.1 mM PMSF, 1 mg/ml
bacitracin, 1 mg/ml benzamidine, 10 µM leupeptin, 20 µg/ml
antipain, 1 µg/ml pepstatin, and 0.5 µg/ml aprotinin. Membranes (10 µg) were incubated in presence of increasing concentrations of
125I-labeled NPY
(125I-NPY; 0.08-10 nM) in a
final volume of 50 µl using 96-well polyvinylidene fluoride (PVDF)
Durapore filter (0.65 µm) plates from Millipore (Nepean, ON)
presoaked with 4% BSA. Under these conditions,
nonspecific binding to the PVDF membrane is relatively low, ranging
consistently between 0.73 and 0.85% of the total count with no further
reduction on addition of 0.1% polyethylenimine.
Incubations were done at 37°C for 20 min (time necessary to reach
equilibrium; data not shown), in the absence (total binding) or
presence (nonspecific binding) of 5 µM unlabeled NPY and were stopped
by rapid filtration followed by four washes with 250 µl of ice-cold
binding buffer using a Multi-Screen system from Millipore. The
radioactivity retained on the filters was measured in a
-scintillation counter (Cobra II: Auto-gamma, Canberrra Packard,
Montreal). Membrane protein content was assayed by the method of
Bradford (6) using Bio-Rad protein assay reagent (Mississauga, ON), and
BSA was used as standard.
Characterization of NPY receptor subtypes on BBM.
Competition binding experiments were performed as described in
Binding of
125I-NPY to syncytiotrophoblast
membranes, except that the incubation time was raised
to 30 min (time necessary to reach equilibrium state with 2 competing
ligands; data not shown). Membranes were incubated in
presence of a fixed concentration of
125I-NPY (1 nM) alone or with
increasing concentrations
(10
12-10
5
M) of unlabeled NPY, PYY,
[Leu31,Pro34]NPY,
13-36NPY, or PP.
D-Myo-inositol
1,4,5-trisphosphate assay.
BBM (50 µg/10 µl) were added to a reaction mixture consisting of 20 mM HEPES (pH 7.45), 100 mM NaCl, 25 mM
NaHCO3, 20 mM KCl, 2 mM
MgSO4, 1 mM
NaH2PO4,
100 µM CdCl2, 100 nM
CaCl2, 100 µM PMSF, 1 mg/ml
bacitracin, 1 mg/ml benzamidine, 20 µg/ml antipain, 1 µg/ml
pepstatin, 0.5 µg/ml aprotinin, and 0.05% BSA in a final volume of
50 µl. Membranes were incubated at 37°C under shaking (90 cycles/min) in the presence of increasing concentrations
(10
11-10
6
M) of NPY or PYY, and the reaction was stopped 1 min later by the
addition of perchloric acid (5% final concn) and albumin (0.2% final
concn). In one set of experiments, membranes were preincubated for 5 min with 5 µM
1-(6-[17
-3-methoxyestra-1,3,5-(10)triene-17-yl]amino/hexyl)1H-pyrroledione (U-73122) before being exposed to
10
7 M NPY or PYY for 1 min.
In another set of experiments, membranes were preincubated for 5 min
with 5 µM BIBP-3226 and stimulated with increasing concentrations
(10
11-10
6
M) of NPY or PYY. Membrane
D-myo-inositol
1,4,5-trisphosphate [Ins(1,4,5)P3]
production derived from the endogenous phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2] pool
was measured by the protein binding method of Challiss et al. (9) using
the Ins(1,4,5)P3 [3H] assay system from
Amersham Canada.
PKC assay.
Membrane-associated PKC activity was measured mainly as previously
described by Chakravarthy et al. (8), using myristoylated Ala-rich C
kinase substrate (MARCKS) as a selective PKC substrate. Briefly,
membranes (20 µg/4 µl) were added to a reaction mixture containing
100 µM MARCKS in a final volume of 16 µl consisting of 40 mM HEPES
(pH 7.45), 25 mM NaHCO3, 2 mM
MgSO4, 1 mM
NaH2PO4, 1 mM NaF, 100 µM Na vanadate, 100 µM Na pyrophosphate, 100 nM CaCl2, 10 µM
[
-32P]ATP
(~10-15 µCi), 100 µM PMSF, 1 mg/ml benzamidine, 1 µg/ml pepstatin, 0.5 µg/ml aprotinin, and 0.05% BSA. The reaction was carried out under shaking (90 cycles/min) over 3 min at 37°C in the
presence of increasing concentrations
(10
11-10
7
M) of NPY or PYY, was stopped by the addition of sodium dodecyl sulfate
(SDS) sample buffer [final concn 4% (wt/vol) SDS, 0.01 M EDTA,
0.25 M sucrose, 0.083 M dithiothreitol, 0.08% (wt/vol) bromophenol
blue], and was boiled for 5 min. In one set of experiments, membranes were preincubated for 5 min with 5 µM U-73122, 100 nM calphostin C (specific PKC inhibitor at this concentration), or 10 µM
LY-294002 before being exposed to
10
7 M NPY or PYY for 3 min.
In another set of experiments, membranes were preincubated for 5 min
with 5 µM BIBP-3226 and stimulated with increasing concentrations
(10
11-10
7
M) of NPY or PYY. Samples were loaded on an alkaline
tricine-SDS-polyacrylamide gel electrophoresis system consisting of a
4% acrylamide stacking gel and a 12% acrylamide-glycerol separating
gel, as described by Schägger and von Jagow (36). After
migration, gels were fixed in 50% (vol/vol) methanol and 10%
(vol/vol) acetic acid for 30 min, stained in 10% acetic acid, 0.25%
(wt/vol) Coomassie blue R-250 for 15 min, and washed by three 5-min
washes with 25% methanol, 10% acetic acid. The phosphorylated peptide
was then detected on the polyacrylamide gels by autoradiography
performed at 4°C and quantified with the Personal Densitometer from
Molecular Dynamics and ImageQuant software (Sunnyvale, CA).
Statistics and curve analysis.
Statistical analysis was performed using Student's
t-test. Differences were considered
significant when P values were
<0.05. Binding experiment data and concentration-response data were
analyzed using computerized nonlinear regression analysis with PRISM
(version 1.02) from GraphPad Software (San Diego, CA).
Reagents.
Human NPY,
[Leu31,Pro34]NPY,
and 13-36NPY were synthesized
as previously described (12), as were porcine PYY and PP.
125I-NPY was purchased from
Amersham (Oakville, ON, Canada). Leupeptin, antipain, pepstatin, and
aprotinin were purchased from Boehringer Mannheim (Laval, PQ, Canada).
Bacitracin, PMSF, benzamidine, BSA fraction V, and ATP were purchased
from Sigma Chemical (St. Louis, MO). U-73122 was purchased from RBI
(Natick, MA),
[
-32P]ATP from ICN
Pharmaceuticals (Montreal, QC, Canada), and MARCKS-protein phosphorylated site domain (psd) from BIOMOL (Plymouth Meeting, PA).
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RESULTS |
Characterization of membrane fractions.
The BBM used in this study were of high quality because they were
enriched in alkaline phosphatase, a BBM marker, 36 ± 4-fold compared with the homogenate of the corresponding placental tissue. The
cross-contamination with BPM was low because
Na+-K+-ATPase
activity, a marker for BPM, was in these BBM enriched only by 4.0 ± 0.5-fold. The purity of the BPM used in this study was good, as
determined by
Na+-K+-ATPase
activity, because their enrichment was 24 ± 2-fold and cross-contamination was in the published range with 6.0 ± 0.5-fold (1, 18).
Characterization of 125I-NPY binding to
syncytiotrophoblast membranes.
The specific binding of 125I-NPY
to syncytiotrophoblastic BBM of human term placenta was rapid and
reached apparent equilibrium conditions within 20 min (data not shown).
Isotherm saturation binding under these conditions demonstrated a
saturable high-affinity 125I-NPY
binding with dissociation constant and maximum binding capacity values
of 11 ± 3 nM and 604 ± 100 fmol/mg of membrane proteins, respectively (Fig. 1A). Specific
binding to BPM (caused by much higher nonspecific binding) was
negligible and nonsaturating (Fig. 1B).

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Fig. 1.
Saturation curves of 125I-labeled
neuropeptide Y (125I-NPY) binding
to brush-border (BBM; A) and basal
plasma (BPM; B) membranes of
syncytiotrophoblast from human term placenta at 37°C for 20 min.
Data are means ± SE from 3 experiments done in triplicate.
Nonlinear regression analysis was performed using PRISM version 1.02 from GraphPad.
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Characterization of NPY receptor subtypes.
To characterize the pharmacology of the receptor subtypes present
in BBM of human term syncytiotrophoblast, we performed competition experiments using increasing concentrations of NPY, PYY,
[Leu31,Pro34]NPY,
13-36NPY, and PP.
All peptides used caused a progressive displacement of
125I-NPY binding to BBM (Fig.
2). NPY and
[Leu31,Pro34]NPY
were the most potent competitors of
125I-NPY binding sites; PYY
displaced with high affinity ~45% of the binding sites but was
unable to displace the other portion up to
10
5 M. Moreover,
13-36NPY displaced
125I-NPY (with ~7-fold less
affinity than NPY), whereas PP displaced 125I-NPY binding with 150-fold
less affinity (Table 1).

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Fig. 2.
Ligand competition curves for
125I-NPY binding to BBM of
syncytiotrophoblast from human term placenta.
125I-NPY concentration was 1 nM,
and incubation time was 30 min at 37°C. Data are means ± SE
from 3 experiments done in triplicate. Competition analysis was
performed by PRISM version 1.02 from GraphPad. PP, pancreatic
polypeptide; PYY, peptide YY.
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Table 1.
NPY and related peptide affinities for 125I-NPY binding
sites in brush-border membranes of syncytiotrophoblast from human
term placenta
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Modulation of Ins(1,4,5)P3 production by
NPY and PYY.
After a 1-min stimulation with increasing concentrations of NPY or PYY,
formation of Ins(1,4,5)P3, the
direct product of the breakdown of
PtdInsP2 catalyzed by PLC, was
raised in a concentration-dependent manner (Fig.
3). Moreover, this figure shows that the
peptides possess similar sensitivity, with a
pEC50 of 10.17 ± 0.27 and 10.06 ± 0.38 for NPY and PYY, respectively, and similar efficiency, with a maximal effect being reached at 100 nM for both peptides. To
determine whether the PLC activity associated with NPY or PYY stimulation was of the PLC-
type, BBM were preincubated 5 min with
or without 5 µM U-73122 (inhibitor of G protein-linked mediated PLC-
activation). As shown in Fig. 4, an
incubation of BBM for 1 min in presence of 100 nM NPY or PYY resulted
in a highly significant increase in
Ins(1,4,5)P3 production compared
with the basal values (P < 0.005 and
0.01, respectively), whereas in the presence of 5 µM U-73122, the
increase in Ins(1,4,5)P3
production was completely abolished (stimulated vs. basal,
P > 0.5 and 0.3, respectively) for
NPY and PYY. In an attempt to define which binding sites could be
attributed to the PLC-
stimulating effect, we used BIBP-3226, a
highly potent and selective nonpeptide
Y1 receptor antagonist. Surprisingly, BIBP-3226 displayed a similar antagonistic activity on
both NPY (Fig. 5) and PYY (data not shown)
effects, with a rightward shift of ~2 log.

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Fig. 3.
D-Myo-inositol
1,4,5-trisphosphate
[Ins(1,4,5)P3]
production in BBM of syncytiotrophoblast in presence of increasing
concentration of NPY or PYY. Membranes were incubated at 37°C for 1 min, and Ins(1,4,5)P3 production
derived from endogenous phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2] was
assayed using Ins(1,4,5)P3
[3H]assay system from
Amersham. Data are means ± SE from 4 experiments done in
triplicate.
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Fig. 4.
Effects of U-73122 on Ins(1,4,5)P3
production stimulated by NPY or PYY in BBM of syncytiotrophoblast.
Membranes were preincubated 5 min with 5 µM U-73122, and reaction was
initiated by addition of NPY or PYY. Membrane Ins(1,4,5)P3
production derived from endogenous
PtdIns(4,5)P2 was measured using
Ins(1,4,5)P3
[3H]assay system from
Amersham. Data are expressed as percentage of respective control and
are means ± SE from 3 experiments done in triplicate. PLC,
phospholipase C.
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Fig. 5.
Ins(1,4,5)P3 production in BBM of
syncytiotrophoblast in presence of increasing concentration of NPY or
PYY after 5-min preincubation with 5 µM BIBP-3226 or solvent.
Membranes were incubated at 37°C for 1 min, and
Ins(1,4,5)P3 production derived
from endogenous PtdIns(4,5)P2 was
assayed using Ins(1,4,5)P3
[3H]assay system from
Amersham. Data are means ± SE from 4 experiments done in
triplicate.
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Stimulation of membrane-associated PKC activity by NPY and PYY.
In conditions similar to those used for assessing PLC activity,
although the 1,2-diacylglycerol concomitantly generated with Ins(1,4,5)P3 is not the sole way
to activate membrane-associated PKC, we measured the ability of both
NPY and PYY to modulate membrane-associated PKC activity. Figure
6 shows that both peptides, in a
dose-dependent manner, stimulate MARCKS-psd phosphorylation, although
PYY was slightly less potent than NPY
(P values were <0.05 and <0.05
between PYY vs. control and vs. NPY treated, respectively).
Interestingly, the profiles of the dose-response curves were different
because the pEC50 were 11.26 ± 0.32 and 8.90 ± 0.23 for PYY and NPY, respectively. As for PLC
activity, MARCKS-psd phosphorylation was markedly influenced by
BIBP-3226; however, the rightward shift of the dose-response curves was
more pronounced in the phosphorylation assays (Fig. 7). Further studies were then conducted to
evaluate whether the inhibition of PLC was sufficient to inhibit the
subsequent stimulation of PKC activity (Fig.
8). In these experiments, 5 µM U-73122
was unable to counteract completely the PKC stimulation by both
peptides, suggesting an alternative way of stimulation. Under the same
conditions, 100 nM calphostin C, a nonselective subtype of PKC
inhibitor, abolished PKC activity. In another experiment, we wanted to
verify whether PI3K was one of the alternative ways. As shown in Fig. 9, 10 µM LY-294002 reduced PKC activity
significantly (P values were <0.05),
an effect that was additive to the effect of U-73122 (P < 0.01), because a combination of
both inhibitors leaves no residual activity.

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Fig. 6.
Protein kinase C (PKC) activation in BBM of syncytiotrophoblast in
presence of increasing concentration of NPY or PYY. Membranes were
incubated at 37°C for 3 min, and PKC activity was measured using
myristoylated Ala-rich C kinase substrate-protein phosphorylated site
domain (MARCKS-psd) peptide as phosphorylation substrate. After
separation on alkaline tricine-SDS-polyacrylamide gel electrophoresis
(PAGE) and autoradiography, phosphorylation was evaluated using
Personal Densitometer from Molecular Dynamics and ImageQuant software.
Data are expressed as percentage of control and are means ± SE from
3 experiments.
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Fig. 7.
PKC activation in BBM of syncytiotrophoblast in presence of increasing
concentration of NPY or PYY after 5-min preincubation with 5 µM
BIBP-3226 or solvent. Membranes were incubated at 37°C for 3 min,
and PKC activity was measured using MARCKS-psd peptide as
phosphorylation substrate. After separation on alkaline
tricine-SDS-PAGE and autoradiography, phosphorylation was evaluated
using Personal Densitometer from Molecular Dynamics and ImageQuant
software. Data are expressed as percentage of control and are means ± SE from 3 experiments.
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Fig. 8.
Effects of U-73122 or calphostin C on PKC activity in BBM of
syncytiotrophoblast incubated at 37°C for 1 min in presence of 100 nM NPY or PYY. Membrane-associated PKC activity was measured using
MARCKS-psd as phosphorylation substrate followed by separation on
alkaline tricine-SDS-PAGE and autoradiography. Phosphorylation was
evaluated using Personal Densitometer from Molecular Dynamics and
ImageQuant software. Data are expressed as percentage of control and
are means ± SE from 2 experiments.
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Fig. 9.
Effects of U-73122 (U), LY-294002 (LY), or both on PKC activity in BBM
of syncytiotrophoblast incubated at 37°C for 1 min in presence of
100 nM NPY or PYY. Membrane-associated PKC activity was measured using
MARCKS-psd as phosphorylation substrate followed by separation on
alkaline tricine-SDS-PAGE and autoradiography. Phosphorylation was
evaluated using Personal Densitometer from Molecular Dynamics and
ImageQuant software. Data are expressed as percentage of control and
are means ± SE from 2 experiments.
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DISCUSSION |
The syncytiotrophoblast is a single continuous structure covering the
entire outer surface of the villous tree and, as such, is strategically
devoted to a pivotal role in maternal-fetal communications. In
accordance with its putative endocrine role, the syncytiotrophoblast is
a major source of a large variety of peptide hormones (28) that include
inhibin (32) and CRF (33), two important factors in the maintenance and
termination of a successful pregnancy. Maternal decidua, chorion,
amnion, and cytotrophoblastic cells also release many peptide hormones
(28); among them, NPY may have an important endocrine, paracrine, or
autocrine role and has been, in that respect, associated with the
syncytiotrophoblastic release of CRF and inhibin (29, 30). Thus,
because the syncytiotrophoblast is polarized, its plasma membranes
being constituted of two distinct zones, a BBM facing the maternal side
and a BPM facing the fetal side, the first aim of the present study was
to characterize the NPY binding sites of both domains of the human term
syncytiotrophoblast. In contrast to BPM, the specific binding of
125I-NPY to BBM was rapid,
saturable, and of high affinity. This implies that NPY must come from
the maternal blood surrounding the villous tree to reach its binding
sites, because BBM face the intervillous space.
The recent cloning of Y5JBC and
Y5NAT receptor subtypes
complicates the pharmacological subtype classification of NPY/PYY receptors because the NPY analog
[Leu31,Pro34]NPY,
typically a Y1 agonist (13), shows
agonistic properties for Y1,
Y5JBC, and
Y5NAT receptor subtypes (14, 45).
However, because the primate ortholog of the
Y5JBC gene does not bind NPY, PYY,
or their analogs (21), it becomes unlikely that the NPY binding site
described in the present paper is the
Y5JBC subtype. Long
carboxy-terminal fragments such as
13-36NPY still preferentially bind to the Y2 receptor (44), and
PYY does not bind to the Y3 subtype (43). In accordance with the above nomenclature, we propose
that 125I-NPY binding sites on
syncytiotrophoblast BBM are constituted of a mixed-receptor population
constituted of Y1-like (~55%)
and Y3 (~45%) types. It is also
tempting to classify Y1-like
receptors present in BBM as Y1
receptors because Y5NAT receptors
are found primarily, if not exclusively, in discrete regions of the
brain (14). However, the known analogy between the
hypothalamo-pituitary axis and the
cytotrophoblastic-syncytiotrophoblastic axis renders this assumption
quite presumptuous. It has been shown that NPY and PYY, in cells
expressing Y1-like or
Y3 receptors, mobilize calcium
from intracellular stores or, in the case of
Y1-like subtype, also inhibit
adenylate cyclase activity (43). However, there is no consensus on the
ability of NPY and PYY to enhance turnover of inositol
lipids. Some investigators found that NPY induces the
stimulation of PLC (10, 37), but others did not (20, 22). However, all
these effects are mediated via pertussis toxin-sensitive heterotrimeric
Gi and
Go proteins (20, 22, 47). Because
o- and
i-subunits cannot directly
activate PLC, a mechanism involved in the pertussis toxin-sensitive
process has recently been proposed (15, 38). The ligand-receptor
interaction would lead to the activation of the pertussis
toxin-sensitive heterotrimeric G proteins and consequently to the
release of the 
-dimer, which then activates PLC. The activation
of PLC leads, in turn, to the phosphodiesteratic cleavage of
PtdInsP2, yielding
1,2-diacylglycerol production, which stimulates classical and new PKC
isotypes, and Ins(1,4,5)P3
production, which by binding to its receptors, mobilizes calcium from
intracellular stores to cytosol (4). To establish whether
the NPY binding sites described here represent a physiologically relevant receptor site in the syncytiotrophoblastic BBM, we attempted to study their relationship with the above-mentioned second messenger systems. Despite the presence of both
Go and
Gi proteins in the BBM of the
syncytiotrophoblast (11), the exclusive presence of adenylate cyclase
in BPM (17, 46) prompted us to explore the coupling of the NPY binding
sites to PLC modulation. The effect of NPY and PYY on the
Ins(1,4,5)P3 production shown in
this study on isolated BBM is in agreement with the studies of Daniels
et al. (10) and Shigeri et al. (37). The use of a highly selective Ins(1,4,5)P3 protein binding assay
in association with the addition of
CdCl2, a powerful inhibitor of
Ins(1,4,5)P3-5-phosphatase
activity (39), might explain the discrepancy with those groups who
could not find evidence of PLC activation although showing rise in
intracellular calcium concentration. The identical curves obtained for
these peptides also suggest that NPY and PYY stimulate PLC via the same receptors, even if we did not address the question directly in the
present study. To assess whether the PLC activity measured was of the
-type, we used U-73122, a new aminosteroid inhibitor of PLC
activation by G protein-linked receptors (41). The complete abolition
by U-73122 of NPY and PYY stimulation of
Ins(1,4,5)P3 accumulation suggests
that the effect of both peptides is mediated through activation of
PLC-
. Additionally, the magnitude of the rightward shift of the
dose-response curves in the presence of BIBP-3226 suggests that the
Y1 receptor is the primary binding site associated with the effect of both peptides on PLC-
. Moreover, the antagonist does not antagonize effects attributed to the
Y5NAT receptor described (14).
Nevertheless, even if both peptides activate PLC-
in our membrane
preparations, it seems more likely that the physiological ligand of the
human term syncytiotrophoblast BBM is NPY, because its concentration is
preponderant during the course of pregnancy (27). Having in mind the
putative role of NPY in the release of CRF and inhibin (32, 33), it is
appropriate to monitor the activity of PKC (35). Additional experiments were performed to measure this activity directly in our membrane preparation. However, it must be kept in mind that the PKC activity showed corresponds to the activation via newly liberated activators of
a fraction of the enzyme already associated with the membrane preparation (1, 8) and cannot account for the possible induction of
translocation by the peptides. Nevertheless, both NPY and PYY were
potent inducers of PKC activity. PYY has, however, a higher potency
than NPY, which is consistent with its higher affinity as measured in
the competition experiment, and NPY is more efficient, which suggests
the stimulation of different pools of PKC or a more efficacious
coupling between NPY and the stimulation of PKC via other ways than
PLC. However, this hypothesis assumes that because BIBP-3226
antagonized both peptides, different transition states of the
Y1 receptor will be differentially
stabilized by NPY and PYY. One of these transition states could be
preferentially coupled to PLC and the other state to an alternative
effector. This phenomenon is well known in other systems (16) but to
our knowledge has not been published for NPY receptors. The incomplete inhibition of PKC activation by U-73122 for both peptides also suggests
that both peptides utilize an alternative way of PKC activation.
Although not excluding other potential ways, the results obtained after
PI3K inhibition favor this glycerophospholipid kinase as a major way by
which NPY and PYY activate PKC. Thus PtdIns(2,4,5)P3 resulting from the
3-kinase activity could substitute for the
PtdIns(4,5)P2, a poor activator of
PKC, in the activation of some PKC isoenzymes (5), preferentially the
isotype that is predominant in BBM of human syncytiotrophoblast
(1). Finally, the nature of the PKC stimulated by these peptides, even
if not addressed in the present study, should probably exclude atypical subtypes, because MARCKS does not seem to be a substrate for both
-
and
-PKC (42).
In conclusion, this study demonstrates that the syncytiotrophoblast
harbors NPY/PYY receptors and that in accordance with the polarized
nature of the syncytiotrophoblast, nonequivocal saturable binding could
be found on the maternal side only. Furthermore, the results provide
evidence that these receptors are linked to PLC and PI3K activation,
both of which lead to PKC activation. These observations are the first
steps in the understanding of the mechanism of NPY action already
described in human term syncytiotrophoblast.
 |
ACKNOWLEDGEMENTS |
The authors express their gratitude to Dr. André Masse (Chief
of Obstetrics Service), Thérèse Blackburn (Chief of
Nursing), and the staff of Department of Obstetrics and Gynecology for
the donation of placentas and acknowledge the skillful technical
assistance of Fatiha Moukdar and Mélanie
Laramée.
 |
FOOTNOTES |
This study was supported by grants from Université du
Québec à Montréal (J. Lafond). J. Robidoux is the
recipient of a Fonds de Recherche en Santé au Québec-Fonds
pour la Formation de Chercheurs et l'aide à la Recherche
Santé doctoral studentship.
This work was presented in part at the Endocrine Society meeting, June
1996, San Francisco, CA.
Address for reprint requests: J. Lafond, Université du
Québec à Montréal, Département des Sciences
Biologiques, C.P. 8888, Succursale "Centre-Ville,"
Montréal, Québec, Canada H3C 3P8.
Received 29 May 1997; accepted in final form 10 December 1997.
 |
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