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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta . 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-beta 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-beta ; protein kinase C

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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-[17beta -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 [gamma -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), [gamma -32P]ATP from ICN Pharmaceuticals (Montreal, QC, Canada), and MARCKS-protein phosphorylated site domain (psd) from BIOMOL (Plymouth Meeting, PA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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

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-beta type, BBM were preincubated 5 min with or without 5 µM U-73122 (inhibitor of G protein-linked mediated PLC-beta 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-beta 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.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha o- and alpha 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 beta gamma -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 beta -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-beta . 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-beta . Moreover, the antagonist does not antagonize effects attributed to the Y5NAT receptor described (14). Nevertheless, even if both peptides activate PLC-beta 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 epsilon  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 zeta - and lambda -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Endocrinol Metab 274(3):E502-E509
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