A. Petit

Labelling of D2-Dopaminergic and 5-HT2-Serotonergic Binding Sites in Human Trophoblatic Cells Using [3H]-Spiperone

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.

Expression of human placental D2-dopamine receptor during normal and abnormal pregnancies

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 (D2R) 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 mRNA 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 approximately 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 mRNA levels began at week 19 of gestation and reached a maximum value at term. During the first half of gestation, the relative levels of D2R mRNA (2.5 kb) showed an inverse pattern of expression when compared to D2R protein content. Specifically, the levels of D2R mRNA increased by approximately 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 (approximately 45 per cent inhibition) and molar (approximately 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.

D2-dopamine agonists inhibit adenosine 3′ :5′-cyclic monophosphate (cAMP) production in human term trophoblastic cells

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.

Interaction of D2-dopamine receptor with two pertussis toxin sensitive G proteins in human placenta

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 first 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.

The modulation of placental lactogen release by opioids: a role for extracellular calcium

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 Hams 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)

Expression of G proteins in human placentas from pregnancies complicated by gestational hypertension

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 beta-hCG mRNAs as well as placental G protein alpha-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 beta-hCG mRNAs were respectively 108% and 105% of those from normal placentas, suggesting that the altered circulating levels of hPL and beta-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, G alpha i2, G alpha i3, G alpha o, G alpha s, and G alpha q/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 G alpha i2, G alpha i3, G alpha o, G alpha s, and G alpha q/11. These results suggest that neither a change in hPL and beta-hCG expression nor a change in signal transduction machinery is implicated in the altered circulating levels of hPL and beta-hCG in preeclampsia.

Adenosine 3′:5′-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release

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(-7)M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 microgram/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.