Serotoninergic (5-HT) neurons of adult recipients provide a much denser innervation of striatal than ventral mesencephalic grafts implanted into the neostriatum of the rat. Moreover, grafts from both brain regions are more innervated by host 5-HT axons after implantation in neonatal than adult hosts. To test the hypothesis that differences in glial scarring or expression of the growth inhibitory molecules, chondroitin sulfate proteoglycans (CSPG), be responsible for these differences in 5-HT innervation of neural grafts, we examined the 5-HT innervation, the astroglial reaction and the expression of CSPG in ventral mesencephalic grafts implanted into newborn (1-5 days old), juvenile (15 days old), or adult rats and in striatal grafts implanted in adult rats, using immunohistochemistry against 5-HT, glial fibrillary acidic protein (GFAP) and CSPG. Immunostaining for GFAP showed a stronger initial gliosis (1-10 days after grafting) in neonatal than adult recipients of mesencephalic grafts, but this gliosis subsided gradually at later time points. Nevertheless, a glial scar formed at the graft-host interface in both neonatal and adult recipients, 5-10 days after transplantation, although it decreased over a longer time course--up to 60 days--in adults. Immunostained astrocytes appeared first in the host brain tissue around the graft and then immunoreactive processes and perikarya gradually invaded the graft. Immunoreactivity for CSPG was similar in neonatal and adult hosts: it was strongly expressed inside the graft early after transplantation, and almost completely down-regulated at 60 days. The reaction of adult hosts to striatal and mesencephalic grafts was similar, although GFAP was more heterogeneously distributed and CSPG immunoreactivity remained in patches inside striatal grafts, even after 60 days. The 5-HT innervation of mesencephalic grafts was much denser after implantation in newborns than in adults. It was also stronger in striatal than in mesencephalic grafts implanted in adults. Thus, the presence of a glial scar or the expression of CSPG cannot totally account for the different degrees of 5-HT innervation in the various types of neural grafts.

Previous studies have demonstrated the presence of D2-dopamine binding sites in the human placenta, and that dopamine (DA), via these D2-like receptors, inhibits both basal- and hormone-stimulated secretion of human placental lactogen (hPL) from trophoblastic cells. However, nothing is known about the ontogenesis of this placental D2-dopamine receptor (DZR) during pregnancy. Therefore, the aim of this study was to analyse the expression of these receptors throughout gestation in placentae from normal as well as abnormal pregnancies. Western and Northern blot analysis were performed on membrane protein and messenger RNA (mRNA) preparations of human placentae from various weeks of gestation as well as from pregnancies complicated by pre-eclampsia of hydatidiform mole. The autoradiographs of both proteins and rnRNA showed differential expression of placental D2R during normal pregnancy. When the relative levels of D2R proteins were analysed throughout pregnancy, there was a significant but transient decrease of ∼ 23 per cent of D2R content at 9–16 weeks of gestation with a return to baseline levels at 17–18 weeks. An increase in rMRNA levels began at week 19 of gestation and reached a maximum value at term. During the first half of gestation, the relative levels of DZR mRNA (2.5 kb) showed an inverse pattern of expression when compared to D2R protein content. Specifically, the levels of D2R mRNA increased by ∼ 26 per cent between weeks 9 and 16 of pregnancy in comparison with the values observed at 7–8 weeks, and returned to baseline levels at 17–18 weeks of gestation. The D2R relative protein levels subsequently increased from 19 to 30 weeks of gestation, and then remained stable. The autoradiographs of both proteins and mRNA showed significantly decreased expressions in placentae from both pre-eclamptic (∼45 per cent inhibition) and molar (∼0–70 per cent inhibition) pregnancies. Moreover, there was important variability in the expression of placental D2R from hydatidiform moles. Using immunological and molecular biology techniques, the present study confirms the presence of D2R in human placenta. The variations of placental D2R expression during normal and abnormal pregnancies argue for an important role of DA in human placental function, although this remains to be investigated further.

Preeclampsia (gestational hypertension) is accompanied by decreased hPL and increased hCG levels in maternal serum. The expression of these peptides as well as the endocrine mechanisms responsible for their regulation in preeclampsia are unknown. We have demonstrated that regulatory GTP-binding proteins (G proteins) are implicated in the modulation of hPL production by placentas from normal pregnancies. In order to extend our knowledge on placental endocrinology, we analyzed in this study the expression of hPL and β-hCG mRNAs as well as placental G protein α-subunits in pregnancies complicated by gestational hypertension. Western and Northern blot analyses were respectively performed on membrane protein and total mRNA preparations from human placentas of preeclamptic (n = 7) and normal pregnancies (n = 4). The levels of hPL and β-hCG mRNAs were respectively 108% and 105% of those from normal placentas, suggesting that the altered circulating levels of hPL and β-hCG are not related to dysfunctional mRNA expression of these peptides. The autoradiographs for G proteins and their mRNAs showed no difference in G protein expression between preeclamptic and normal tissues. Specifically, Gαi2, Gαi3, Gαo, Gαs, and Gαq/11 levels reached 87%, 81%, 91%, 99%, and 103% respectively of those from normal placentas. In parallel with the protein levels, their mRNAs expression were respectively 93%, 89%, 113%, 104%, and 94% of normal values for Gαi2, Gαi3, Gαo, Gαs, and Gαq/11. These results suggest that neither a change in hPL and β-hCG expression nor a change in signal transduction machinery is implicated in the altered circulating levels of hPL and β-hCG in preeclampsia.

We have demonstrated the presence in human placenta of D2 dopamine receptors (D2R) which inhibit human placental lactogen (hPL) release. This inhibitory effect of dopamine (DA) was sensitive to pertussis toxin (PTX) indicating that it may be mediated by the Gi/Go family of G proteins. However, nothing is known on this G proteins/D2R interaction in human placenta. In this study, we demonstrate that DA (10−4 M) inhibits by 39 % the ADP-ribosylation by PTX of two G proteins of 40 and 41 kDa. This inhibition is receptor specific since it is reversed by spiperone, a D2R antagonist. Moreover we show that bromocriptine, a D2 agonist, inhibited the labeling of these two proteins in a dose-dependent manner with a maximal inhibition of 37 % at a concentration of 10−6 M. In order to understand the role of D2R in placental endocrinology, we have analyzed the interactions of these two PTX-sensitive G proteins with D2R in normal and abnormal pregnancies. The autoradiographs of both PTX ADP-ribosylated placental proteins of 40 and 41 kDa showed differential labeling during normal pregnancy. Thus, the relative levels of ADP-ribosylation by PTX of both proteins were 2.5 and 3.0 fold lower at term than those observed during fisrt and second trimester whereas no difference was observed between the first and second trimester. Also, no significant change in the level of inhibition by DA was observed between 7–9 weeks and 18–40 weeks of pregnancies (35–45 % inhibition). However, we observed a maximal inhibition between 10 to 17 weeks of pregnancy (64 % inhibition). In placentas from preeclamptic pregnancies, the levels of ADP-ribosylation were similar to those observed in normal pregnancy, while the DA inhibition was increased by 24 %. The levels of ADP-ribosylation in molar placentas reached 20 % of normal values, while no difference in DA inhibition was observed. This study demonstrates that two distinct PTX-sensitive G proteins are coupled to human placental D2R. The physiological significance of the variations in these ADP-ribosylated-G proteins/D2R interaction during normal and preeclamptic pregnancies remains to be investigated.

Hydatidiform mole is a conceptus, usually devoid of an intact fetus, with variable proliferation of trophoblast and altered placental protein synthesis, including high human chorionic gonadotropin (hCG) and low human placental lactogen (hPL) production. Little is known about the control of the production of these two placental proteins in molar pregnancies. Regulatory guanine 5′ triphosphate (GTP)-binding proteins (G proteins) play key roles in the endocrine control of peptide production by the placenta. The present authors recently demonstrated that Gig, Gi3 Go, and Gs α-subunits were expressed in normal human placenta throughout pregnancy. This study analysed the expression of placental G protein α-subunits in molar pregnancies. Western and Northern blot analyses were performed on membrane protein and total mRNA preparations of human placentae, respectively, from hydatidiform mole (n=5) and normal pregnancies (n=4). The levels of hPL and β-hCG mRNAs were 60 and 237 per cent respectively, of those from normal placentae. The autoradiographs for G proteins and their mRNAs showed decreased expression in molar placentae in comparison with normal tissues. Specifically, Gαi2, Gαi3, Gαo, and Gαs levels reached 39, 4, 42, and 89 per cent, respectively, of those from normal placentae. In parallel with the protein levels, their mRNAs expression were 8, 3, 54 and 65 per cent of normal values for Gαi2, Gαi3, Gαo, and Gαs, respectively. The results demonstrate important changes in placental G protein expression in hydatidiform moles suggesting alterations in the signal transduction machinery within the molar trophoblast.

OBJECTIVE:Regulatory guanine nucleotide-binding proteins (G proteins) play key roles in the stimulus-response coupling of many important biological systems. Recent studies from our laboratory suggest a functional role for many G proteins in the human placenta. However, the expression of these proteins has not yet been reported. Therefore, the aims of this investigation were to identify the expression of placental G protein alpha subunits in human placenta, and to study their level of variation during pregnancy.METHODS:Western and Northern blot analyses were performed on membrane protein and mRNA preparations, respectively, of human placentas from the first (7-11 weeks), second (16-19 weeks), and third (term) trimesters.RESULTS:The autoradiographs of both proteins and mRNA showed differential expression of placental G proteins during pregnancy. Thus, the relative levels of G alpha i2 and G alpha i3 subunits were highest during the first trimester, whereas no differences were observed between second-trimester and term placentas for both subunits. The levels of placental G alpha o and G alpha s subunits stayed relatively stable during pregnancy.CONCLUSION:The demonstration of human placental G alpha protein expression during pregnancy provides new insight into the components of the signal transduction machinery within trophoblast. However, the physiologic significance of the variations of placental G alpha i protein expression during pregnancy remains to be investigated.

We previously reported that dopamine (DA) acted via D2-dopamine receptors in human trophoblastic cells to inhibit All-stimulated inositol phosphate (InsP) accumulation. However, the mechanism by which DA inhibited All-stimulated InsP accumulation is still unknown except that this inhibitory effect was sensitive to Pertussis toxin (PTX). In this study, we characterize this DA-mediated inhibition of All-stimulated InsP production in human placenta. Freshly isolated human term placental cells were prelabeled with myo-[2-3H]inositol and incubated with various stimuli in the presence of 10 mM LiCI. All (10-6 M) stimulated 1.5 times the human trophoblastic cell InsP production whereas DA (10−4 M) inhibited this All-stimulated InsP production by 54 ± 7 %. This inhibitory effect was mimicked by bromocriptine (53 ± 3 % of inhibition), a D2-dopamine agonist. We recently reported that bromocriptine inhibited human placental adenosine 3′,5′-cyclic monophosphate (cAMP) production. Increasing the intracellular concentration of cAMP levels by adding forskolin did not modify the effect of DA and bromocriptine on InsP accumulation. On the other hand, the effect of DA and bromocriptine on All-stimulated InsP production were greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the D2-dopaminergic mediated inhibition was prevented by treatment of cells with the Ca2+ ionophore ionomycin (10−5 M) and was mimicked either by removal of Ca2+ from incubation medium (53 ± 6 %) or by blockage of voltage-gated Ca2+ channels with nifedipine (51 ± 7 %). Our data indicate that the inhibitory effect of D2-dopamine agonists on All-stimulated InsP production is an indirect event probably due to the DA-inhibition of calcium influx.

Recent data suggest an important role for calcium (Ca2+) in human placental endocrinology. Thus, the regulation of Ca2+ influx seems to be implicated in the modulation of human placental lactogen and hCG release. A possible mechanism of influx regulation is through receptor-operated channels. One of the most characterized receptor gating Ca2+ channels, the ATP receptor, stimulates the intracellular calcium concentration ([Ca2+]i) in various tissues. The aim of this study was to determine whether ATP receptors gating Ca2+ channels are also present in placental cells. We thus determined the effect of ATP on [Ca2+]i in human term trophoblastic cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. ATP stimulated a 4.3 +/- 0.4 (+/- SE)-fold increase in [Ca2+]i, with a half-maximal effective concentration (EC50) of 1.5 mumol/L. The pharmacological activation profile suggests the presence of purinergic P2u receptors (nucleotide receptors), because uridine 5'-triphosphate (UTP) also stimulated [Ca2+]i (4.0-fold increase, with an EC50 of 10 mumol/L). The ATP-stimulated [Ca2+]i was partly sensitive to pertussis toxin; we observed a 58% inhibition of ATP-induced [Ca2+]i with the toxin without effect on basal [Ca2+]i. The ATP- and UTP-stimulated [Ca2+]i declined with time in the presence of ATP (or UTP). The rate of deactivation was rapid (t1/2, < 60 s with 10(-5) mol/L ATP) and concentration dependent. The deactivation occurring during one application of ATP or UTP resulted in a diminution of subsequent responses. The recovery was incomplete even with long waiting times (up to 30 min). ATP and UTP also stimulated inositol phosphate production with EC50 values of 11 and 15 mumol/L, respectively, but not human placental lactogen or hCG release in experiments in which known secretagogues were effective. The results suggest the presence in human term placental cells of P2u receptors pharmacologically similar to those observed in other tissues, especially in the pituitary and amnion. The physiological significance of this stimulation of [Ca2+]i by ATP and UTP in the human placenta remains to be investigated.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

We previously reported that dopamine (DA) acted via D2-dopamine receptors on human trophoblastic cells to inhibit basal and hormone-stimulated secretion of human placental lactogen (hPL). We also described that these DA effects were coupled with inhibition of calcium influx. The present study examines the interaction of placental D2-dopamine receptor with adenylate cyclase (AC). Incubations of isolated human term trophoblastic cells with R(-)-propylapomorphine (NPA), (+/-)-PPHT, and bromocriptine (3 different D2 agonists) led to time- and dose-dependent inhibitions of cAMP production as determined by measuring the conversion of [2-3H]-ATP into [2-3H]-cAMP. The maximal inhibition was reached after 15 min of incubation and was 33 +/- 1 (SE) %, 29 +/- 3% and 31 +/- 1% for bromocriptine (10(-5) M), NPA (10(-7) M) and (+/-)-PPHT (10(-8) M) respectively. However, the time- and dose-dependent curves were biphasic with NPA and (+/-)-PPHT and the inhibition of cAMP production was abolished at higher agonist concentrations or after time incubations longer than 15 min. These inhibitions were receptor specific since they were reversed by spiperone and haloperidol, two specific--dopamine antagonist, and by butaclamol (mix D2/D1-dopamine antagonists) but not by alpha- and beta-adrenergic, D1- and D4-dopaminergic, and 5-HT2-serotonergic antagonists. The results reported here suggest that human placental D2 receptors interact with AC to inhibit its activity. Also, bromocriptine seems a better agonist for the characterization of dopaminergic effects on human placenta.

We previously reported that angiotensin-II (AII) stimulated and dopamine (DA) inhibited the release of human placental lactogen (hPL) from trophoblastic cells. The mechanisms of action involved in these endocrine regulations are poorly known. In this study, we investigated the role of Ca2+ as a potential cellular mediator of the effects of AII and DA. Incubation of freshly isolated human term trophoblastic cells with DA led to a dose-dependent inhibition of 45Ca2+ influx, with a maximum of 55 +/- 5% and an EC50 of 10 +/- 3 mumol/L. This DA-inhibited Ca2+ influx was reversed by spiperone, a D2-dopamine receptor antagonist. Preincubation of cells with pertussis toxin completely blocked the inhibitory effect of DA on placental 45Ca2+ influx. Nifedipine (10(-5) mol/L), like DA, inhibited 45Ca2+ influx (41 +/- 3% inhibition). Moreover, nifedipine decreased hPL release (57 +/- 10%; EC50, 0.25 +/- 0.09 mumol/L). Coincubation of DA and nifedipine did not enhance the inhibitory effects of these agents on either 45Ca2+ influx or hPL release. The incubation of trophoblastic cells with [Sar1]AII, a potent agonist of AII, led to a dose-dependent stimulation of 45Ca2+ influx. The maximal stimulation was 221 +/- 37% of the control value, with an EC50 of 50 +/- 15 nmol/L. This stimulation was inhibited by coincubation with the AII antagonist [Sar1,Ala8]AII. [Sar1]AII-stimulated Ca2+ influx was blocked by preincubation with pertussis toxin. Bay K 8644 also stimulated 45Ca2+ influx (238 +/- 41% of the control). Moreover, Bay K 8644 stimulated hPL release. The maximal stimulation was 180 +/- 22% of the control value, with an EC50 of 0.40 +/- 0.30 mumol/L. Coincubation of Bay K 8644 and AII did not led to additional stimulation of either 45Ca2+ influx or hPL release. These results suggest that Ca2+ influx is one mechanism that mediates AII and DA regulation of hPL release in human term trophoblastic cells.

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 micrograms/ml streptomycin. 45Ca2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca2+ influx by ethylketocyclazocine (EKC) with an EC50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50,488 (kappa agonist) had the same stimulatory effect as EKC (221 +/- 25% of control). D-Ala2,NMe-Phe4,Gly-ol5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca2+ influx (128 +/- 5% of control, p > 0.05) whereas D-Ser2,Leu,Thr6)-enkephalin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 ± 5% of control value after 15 min exposure to 10−7M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 μg/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22,4 ng/ml and 46,5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.

We studied the binding of [3H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37 degrees C and was reversed (t1/2 approximately 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [3H]-spiperone (0-25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 +/- 2 nM and a maximal binding capacity (Bmax) of 222 +/- 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D2-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 +/- 2 and 56 +/- 22 nM respectively. Dopamine inhibited [3H]-spiperone binding with a Ki of 570 +/- 50 microM whereas Schering 23390 (D1 antagonist) and propranolol (beta-adrenergic antagonist) were without effect. The binding was also inhibited by 100 microM GTP gamma S (38 +/- 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D2-dopamine receptors in human placenta.

In isolated human trophoblastic cells, dopamine (DA) significantly inhibited the angiotensin-II (AII)-stimulated inositol phosphate (IP) accumulation by 44 +/- 8% (EC50, 0.5 +/- 0.2 microM) and human placental lactogen (hPL) release by 85 +/- 5% (EC50, 1.0 +/- 0.8 microM). These effects were blocked by sulpiride, a specific D2 antagonist. On the contrary, scherring 23390 (a specific D1 antagonist) and propranolol (a specific beta-adrenergic antagonist) were ineffective, suggesting that these DA effects are mediated through a DA receptor of the D2 subtype. The mechanism by which DA inhibited AII-stimulated inositol phosphate production implicates a GTP-binding protein sensitive to the islet-activating-protein (IAP), since DA's effects on IP accumulation and hPL release were blocked by this toxin. To further characterize this GTP-binding protein, particulate fractions of placental cells were incubated with [alpha-32P]NAD and IAP. Solubilized extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins of 40 and 41 kDa mol wt were specifically ADP ribosylated. They were probably involved in the DA inhibitory processes, since IAP treatment, known to suppress the effects of DA, also reduced the labeling of these two molecules by around 40%. The effects of AII and DA on hPL release appear to be insensitive to the external calcium concentration, since the results were not significantly different in normal (1.8 mM Ca2+) and low Ca2+ (10(-5) M Ca2+) concentrations. On the other hand, increasing the intracellular concentration of cAMP by adding forskolin did not modify the effect of DA on either IP accumulation or hPL release, suggesting that cAMP is not implicated in hPL release from freshly isolated human trophoblastic cells.

The regulatory mechanism(s) for the production of human placental peptide hormones are poorly understood and two theories currently prevail: an autonomous production which varies according to the changing ratio of cyto- to syncytiotrophoblasts throughout pregnancy an autocrine-paracrine control mediated through receptor binding events. We have favored the latter hypothesis and postulated a functional analogy between the regulation of peptide hormone production by the pituitary with that by human placenta. Trophoblastic cells obtained by trypsin digestion of placentas from mid-term (13–18 weeks) and term (38–41 weeks) normal gestations were rested for 36–48 hours prior to study. A-HCG Studies. We observed the presence of specific, moderate affinity (Ka 108M−1 low capacity LHRH receptor sites which could be up- and down-regulated depending on the endocrine milieu in vitro. Photoaffinity labeling studies showed that the molecular weight of this placental receptor for LHRH was comparable to that observed in rat pituitary (60 kd) and structure-activity relationships indicated that it bound both agonists and antagonists of LHRH. The concentrations of these receptor sites decreased four-fold from mid-gestation to term which was paraleled by a decreased of LHRH-induced production of bioactive hCG. Ca2+ and cAMP, but not membrane lipid hydrolysis, mediated this LHRH-induced hCG production in human placentae. B-hPL Studies. We also found that its production by human placental cells was regulated by cellular events comparable to those responsible for prolactin production by the pituitary. Specific opioids and angiotensin II (AII) receptor sites were observed in human placentae with characteristics of high affinity (Ka 10−10M) and low capacity for ethylketocyclazocine (EKC) and AII. The concentrations of these receptors increased from midpregnancy to term and was manifested by an increased ability to release hPL which was stimulated 2-fold by 10−8 M AII and 2.2 fold by 10−7 M EKC. Dopamine (DA), naloxone, and AII antagonist (SAR-ALA II) completely inhibited this hPL production (>85%). AII stimulated inositol phosphate (IP) production whereas EKC had no effect. DA was without effect on basal IP production but inhibited the AII-induced accumulation of IP. The production of cAMP was inhibited by EKC (55%), stimulated by DA (600%), but unaffected by AII. Extracellular Ca2+ did not influence basal or AII stimulated hPL release but was essential for DA effects. We conclude that the placental production of hCG and hPL is under the control of paracrine influences.

Specific angiotensin II (AII) binding sites were identified and characterized in membranes from human term placenta. The binding of iodinated [125I](Sar1)AII was time-dependent and saturable; it could be totally reversed on addition of unlabelled (Sar1)AII and GTP + NaCl. Scatchard plot analysis of dose-dependent [125I](Sar1)AII binding indicated the presence of a single class of binding sites with an equilibrium dissociation constant of 0.27 +/- 0.06 nM and a maximum binding capacity of 38.4 +/- 4.3 fmol/mg protein. The affinity of five AII analogues for the placental receptor was determined in competitive binding assays; the order of inhibitory potency was: (Sar1)AII greater than (Sar1, Ile8)AII approximately (Sar1, Ala8)AII approximately AII greater than angiotensin I greater than (Des-Phe8)AII. (Sar1)AII did not cause any significant change in the basal or stimulated adenylate cyclase activity. In order to investigate the subunit molecular structure of the placenta AII receptor, membranes were covalently labelled with the photoaffinity ligand [125I](Sar1, (4N3Phe)8)AII. Sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by autoradiography showed that the labelling was specifically incorporated into a protein of Mr 92,000 in the presence or absence of dithiothreitol. It therefore appears that the AII receptor from human placenta has the same binding and pharmacological properties as other well-known AII receptors; by contrast, it is characterized by a significantly higher molecular weight, pointing out that structural differences in AII receptors may exist between species.

We postulated a role for lipid metabolism and Ca2+ in the LHRH-induced release of hCG by human placentas. Term placental cells in suspension prelabeled with [3H]myoinositol were stimulated without or with increasing concentrations of LHRH in the presence of 10 mM LiCl, and total inositol phosphate (IP) was measured by ion exchange chromatography; a nonsignificant 0.9 +/- 0.08-fold increase over the control value was observed. In contrast, placental cells stimulated with equimolar concentrations of angiotensin II (AII) induced a 4.6 +/- 0.9-fold increase in total IP (P less than 0.01), while rat pituitary cells showed 1.9 +/- 0.2- and a 2.4 +/- 0.07-fold increases in total IP production after stimulation with LHRH or AII, respectively (P less than 0.05). These increases were blocked by coincubation with specific LHRH and AII antagonists. When 1 x 10(6) placental cells were incubated with 45Ca2+ without and with increasing doses of LHRH for 0-75 s and then filtered under negative pressure, we observed significant incorporation of 45Ca2+. This influx was linear with incubation time, significantly more pronounced in cells exposed to LHRH than in control cells, and showed a dose-response curve to LHRH that reached maximal influx rates of 3.6 +/- 0.3 nM/min.1 x 10(6) cells with 10(-5) M LHRH. This response was completely blocked by coincubation with 10(-5) M LHRH antagonists; cobalt chloride and verapamil reduced it by 60% and 80%, respectively. Compared to placental cells stimulated with LHRH alone, those coincubated with LHRH and specific LHRH or Ca2+ antagonists released from 10-100% less hCG. We conclude that Ca2+ participates in the LHRH action in human placentas, but uncoupled to PI turnover.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.