Release Of Human Placental Lactogen Research Articles

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants.

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.

Apolipoprotein A-I stimulates human placental lactogen release by activation of MAP kinase

Apolipoprotein A-I (apo A-I) stimulates human placental lactogen (hPL) release via protein kinase C (PKC)-dependent pathways. Since PKC has been shown to activate the MAP kinase cascade in other cell types, we examined the effect of two inhibitors of the MAP kinase cascade on apo A-I-induced hPL secretion and the effect of apo A-I on MAP kinase activity in human trophoblast cells. Apigenin (10 μM) and PD98059 (100 μM) inhibited apo A-I-induced hPL release by 94 and 73%, respectively. Moreover, apo A-I activated MAP kinase in a time- and dose-dependent manner. Activation of PKC by phorbol myristate acetate (PMA) stimulated MAP kinase activity, and down-regulation of PKC completely prevented apo A-I-stimulation of MAP kinase activity. Taken together, these results strongly suggest that activation of MAP kinase is involved in the intracellular mechanism of apo A-I-induced hPL release.

Apolipoprotein A-I stimulates placental lactogen expression by human trophoblast cells.

Earlier studies from our laboratory indicated that apolipoprotein A-I (Apo A-I) stimulates the acute release of human placental lactogen (hPL) from trophoblast cells in culture. We have now demonstrated that Apo A-I also causes a secondary increase in hPL release, beginning about 6 h after exposure to Apo A-I, that is blocked by cycloheximide and actinomycin D. Apo A-I also stimulated a dose-dependent increase in hPL promoter activity in JAR cells transfected with a 1.1-kilobase (-1078/2) fragment of the hPL3 promoter coupled to a chloramphenicol acetyltransferase (CAT) reporter gene. Maximal stimulation, 5.2-fold above basal levels, occurred at an Apo A-I concentration of 1.5 mg/ml, which is within the physiological concentration of Apo A-I during pregnancy. 37pA, a synthetic amphipathic peptide that mimics the secondary structure of Apo A-I and stimulates the synthesis and release of hPL, also stimulated a dose-dependent increase in CAT activity, with maximal stimulation comparable to that caused by Apo A-I. In addition, Apo A-I stimulated a modest increase in CAT activity in BeWo choriocarcinoma cells, Chinese hamster ovary cells, and HeLa cells. However, the maximal stimulation of hPL promoter activity in the Chinese hamster ovary and HeLa cells (approximately 2.5-fold above basal levels) was less than that in choriocarcinoma cells, suggesting that trophoblast cell nuclear factors may be necessary for maximal expression of the promoter in response to Apo A-I. Taken together, these results indicate that Apo A-I stimulates hPL gene expression, and that DNA elements in the first 1.1 kilobase of the promoter are sufficient for transactivation by Apo A-I.

Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants

Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants

Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants

Kinetics of 45Ca outflow and human placental lactogen (hPL) release were characterized in human placental explants. Measurements of the rate of 45Ca and [ 3H]-sucrose (extracellular space marker) outflow from preloaded explants showed that, after a 40 min washout period, the 45Ca effluent radioactivity presumably originated from an intracellular compartment. This view was further supported by the La 3+ and temperature sensitivity of the 45Ca outflow. Moreover, the addition of ionomycin as well as an increase in the extracellular Ca 2+ or Ba 2+ concentration provoked a dose-dependent rise in both the 45Ca outflow and hPL release. There were no systematic temporal analogies between the pattern of 45Ca outflow and hPL release. Taken together, these observations suggest that the stimulation of 45Ca outflow reflects an increased rate of 40Ca entry. The present data also extend previous observations indicating that hPL release can be stimulated by Ca 2+ entry. Lastly, the ‘in vitro’ method described herein allows one to compare rapid changes in hPL release with associated ionic events and can be used to further document the relationships between placental secretory responses and cell calcium metabolism.

Retinoic acid and thyroid hormone regulate placental lactogen expression in human trophoblast cells.

In this study, we have demonstrated that retinoic acid (RA) and thyroid hormone (T3) stimulate the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically, dispersed trophoblast cells from term placentas exposed continuously to RA (0.5 microM) and T3 (0.1 microM) for 5 days released significantly more hPL than control cells after 3 days of exposure (P < 0.001 in each instance). On days 4 and 5, the amounts of hPL released by cells exposed to RA and T3 were approximately 3- and 5-fold higher than those in control cells, respectively. The stimulation by both RA and T3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. RA and T3 caused 3.5- and 5.6-fold increases, respectively, in chloramphenicol acetyltransferase activity in BeWo choriocarcinoma cells transfected transiently with a 2.3-kilobase (kb) fragment of the hPL promoter (-2300 to 2 basepairs) coupled to a chloramphenicol acetyltransferase reporter gene. Deletion construct analysis of the hPL promoter (2.3, 1.2, and 0.5 kb) indicated that the T3- and RA-responsive elements are localized -0.5 to -1.2 kb up-stream from the transcriptional start site (+1), where several consensus RA- and T3-responsive element sites are present. These results indicate that RA and T3 stimulate the synthesis and release of hPL by a mechanism involving hPL gene transcription and further support a role for these steroids in placental function.

Regulation of human placental lactogen expression by 1,25- dihydroxyvitamin D3

The human placenta synthesizes 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and expresses the vitamin D receptor (VDR), but the roles of 1,25-(OH)2D3 and the VDR in placental physiology are poorly understood. In this study, we have demonstrated that 1,25-(OH)2D3 stimulates the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically dispersed trophoblast cells from term placentas exposed continuously to 1,25-(OH)2D3 (0.1, 6, and 37 microM) for 5 days released significantly more hPL than control cells after the third day of exposure. On days 4 and 5, the amounts of hPL released by cells exposed to 1,25-(OH)2D3 were 2.54- and 4.14-fold that of control cells (P < 0.001 in each instance). The stimulation by 1,25-(OH)2D3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. Transient transfection studies of BeWo choriocarcinoma cells transfected with hPL promoter constructs coupled to the chloramphenicol acetyltransferase reporter gene indicated that the stimulation of hPL expression is due at least in part to stimulation of hPL gene expression. Deletion analysis studies of the hPL promoter indicated that a region between -500 to -1200 basepairs is necessary for 1,25-(OH)2D3 responsiveness. Analysis of this region shows a consensus vitamin D response element (VDRE) DNA-binding site of a direct repeat motif separated by three bases. Ligation of this placental VDRE site into a heterologous chloramphenicol acetyltransferase vector caused 1,25-(OH)2D3 responsiveness. Moreover, mobility shift assays demonstrated binding of VDR to placental VDRE. These results indicate that 1,25-(OH)2D3 stimulates the synthesis and release of hPL by a mechanism involving hPL gene transcription and support a role for vitamin D and the VDR in placental function.

Regulation of human placental lactogen expression by 1,25-dihydroxyvitamin D3.

The human placenta synthesizes 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and expresses the vitamin D receptor (VDR), but the roles of 1,25-(OH)2D3 and the VDR in placental physiology are poorly understood. In this study, we have demonstrated that 1,25-(OH)2D3 stimulates the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically dispersed trophoblast cells from term placentas exposed continuously to 1,25-(OH)2D3 (0.1, 6, and 37 microM) for 5 days released significantly more hPL than control cells after the third day of exposure. On days 4 and 5, the amounts of hPL released by cells exposed to 1,25-(OH)2D3 were 2.54- and 4.14-fold that of control cells (P < 0.001 in each instance). The stimulation by 1,25-(OH)2D3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. Transient transfection studies of BeWo choriocarcinoma cells transfected with hPL promoter constructs coupled to the chloramphenicol acetyltransferase reporter gene indicated that the stimulation of hPL expression is due at least in part to stimulation of hPL gene expression. Deletion analysis studies of the hPL promoter indicated that a region between -500 to -1200 basepairs is necessary for 1,25-(OH)2D3 responsiveness. Analysis of this region shows a consensus vitamin D response element (VDRE) DNA-binding site of a direct repeat motif separated by three bases. Ligation of this placental VDRE site into a heterologous chloramphenicol acetyltransferase vector caused 1,25-(OH)2D3 responsiveness. Moreover, mobility shift assays demonstrated binding of VDR to placental VDRE. These results indicate that 1,25-(OH)2D3 stimulates the synthesis and release of hPL by a mechanism involving hPL gene transcription and support a role for vitamin D and the VDR in placental function.

Labelling of D2-dopaminergic and 5-HT2-serotonergic binding sites in human trophoblastic 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.

A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells.

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.

Steroid regulation of oncofetal fibronectin expression in human cytotrophoblasts

Oncofetal fibronectin (onfFN) is a uniquely glycosylated form of FN suggested to play a critical role in uterine/placental adherence during pregnancy. In the present study we have examined steroid regulation of onfFN in highly purified preparations (⩾95%) of cytotrophoblasts isolated from human term placentas. Based on immunoassays, relative to controls, treatment of cytotrophoblasts with 10 −6M medroxyprogesterone acetate (MPA) down-regulated media levels of onfFN 25,53,59, and 62% on days 1,2,3 and 4,respectively. The pattern of steroid regulation and levels of total FN were nearly identical to that of onfFN suggesting that chronic steroid treatment regulates synthesis of FN and not its oncofetal glycosylation. MPA treatment induced a 2-fold stimulation in media levels of hCG indicating that increased placental function was associated with steroid-mediated changes in FN expression. Steroid specificity experiments demonstrated that MPA, cortisol, and dexamethasone were potent inhibitors of onfFN expression whereas estradiol (E2), deoxycorticosterone, testosterone, progesterone, and the synthetic progestin OD-14, were not. This suggested that glucocorticoids and not progestins may be the physiologic regulators of placental FN expression and that MPA may mediate its matrix-modifying activity through a glucocorticoid-like mechanism. Treatment of cells with dexamethasone (10 -7M) did not affect the levels of total protein synthesis or the release of human placental lactogen to the culture medium. This indicated that steroid-mediated down-regulation of onfFN expression in cytotrophoblasts did not result from a general reduction of protein synthesis. Based on densitometric scanning of Western blots, MPA and dexamethasone treatments down-regulated media levels of onfFN 70% relative to control levels. Northern blotting revealed that MPA and dexamethasone mediated a 60–90% reduction in steady state levels of FN mRNA in the presence or absence of E2. Our in vitro model may provide a unique system to evaluate steroidal effects on extracellular matrix (ECM) protein expression. In addition, we suggest that steroids may critically regulate placental ECM protein synthesis, and thus affect trophoblast/uterine adherence throughout pregnancy and expulsion of the placenta and membranes following delivery of the fetus.

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 Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. 45Ca 2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca 2+ influx by ethylketocyclazocine (EKC) with an EC 50 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-Ala 2,NMe-Phe 4,Gly-ol 5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca 2+ influx (128 ± 5% of control, p > 0.05) whereas D-Ser 2,Leu, Thr 6)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca 2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca 2+, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca 2+. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.

High density lipoproteins stimulate molecular weight 80K protein phosphorylation in human trophoblast cells: evidence for a protein kinase-C-dependent pathway in human placental lactogen release.

Previous studies from our laboratory have demonstrated that high density lipoprotein (HDL) HDL and apolipoprotein-AI (apoAI) stimulate human placental lactogen (hPL) release from human trophoblast cells. To determine whether protein kinase-C (PKC) activation is involved in the mechanism of HDL- and apoAI-mediated hPL release, we examined the effects of these factors on the phosphorylation of cytosolic proteins known to be phosphorylated in response to PKC activation by phorbol myristate acetate (PMA). HDL and apoAI each caused a dose- and time-dependent increase in phosphorylation of a PMA-inducible 80K mol wt acidic cytosolic protein in a manner similar to that observed in many other cell types. Stimulation of 80K protein phosphorylation was apparent 5 min after the addition of HDL, apoAI, or PMA and was maximal at 15 min. Maximal 80K protein phosphorylation in cells exposed to PMA (1.6 microM), HDL (1500 micrograms/ml), and apoAI (600 micrograms/ml) was 284%, 206%, and 239% that in untreated cells, respectively. The increase in both 80K protein phosphorylation and hPL release in response to apoAI was prevented by pretreatment of the cells with the PKC inhibitor staurosporine (10 microM) or by down-regulation of PKC after extended preincubation of the cells with 16 microM PMA. (Bu)2cAMP and the adenylate cyclase activator forskolin, which stimulate hPL release, had no effect on 80K protein phosphorylation. These results strongly suggest that HDL- and apoAI-stimulated hPL release involves a PKC-dependent pathway. Since earlier studies also implicate a cAMP-mediated pathway in the stimulation of hPL release by these agents, it appears that multiple intracellular pathways are involved in the stimulation of hPL release.

High density lipoproteins stimulate molecular weight 80K protein phosphorylation in human trophoblast cells: evidence for a protein kinase-C-dependent pathway in human placental lactogen release

Previous studies from our laboratory have demonstrated that high density lipoprotein (HDL) HDL and apolipoprotein-AI (apoAI) stimulate human placental lactogen (hPL) release from human trophoblast cells. To determine whether protein kinase-C (PKC) activation is involved in the mechanism of HDL- and apoAI-mediated hPL release, we examined the effects of these factors on the phosphorylation of cytosolic proteins known to be phosphorylated in response to PKC activation by phorbol myristate acetate (PMA). HDL and apoAI each caused a dose- and time-dependent increase in phosphorylation of a PMA-inducible 80K mol wt acidic cytosolic protein in a manner similar to that observed in many other cell types. Stimulation of 80K protein phosphorylation was apparent 5 min after the addition of HDL, apoAI, or PMA and was maximal at 15 min. Maximal 80K protein phosphorylation in cells exposed to PMA (1.6 microM), HDL (1500 micrograms/ml), and apoAI (600 micrograms/ml) was 284%, 206%, and 239% that in untreated cells, respectively. The increase in both 80K protein phosphorylation and hPL release in response to apoAI was prevented by pretreatment of the cells with the PKC inhibitor staurosporine (10 microM) or by down-regulation of PKC after extended preincubation of the cells with 16 microM PMA. (Bu)2cAMP and the adenylate cyclase activator forskolin, which stimulate hPL release, had no effect on 80K protein phosphorylation. These results strongly suggest that HDL- and apoAI-stimulated hPL release involves a PKC-dependent pathway. Since earlier studies also implicate a cAMP-mediated pathway in the stimulation of hPL release by these agents, it appears that multiple intracellular pathways are involved in the stimulation of hPL release.

Novel regulation of the synthesis and release of human placental lactogen by high density lipoproteins

Novel regulation of the synthesis and release of human placental lactogen by high density lipoproteins

Free alpha molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy.

Free alpha molecules isolated from pregnancy, as well as highly purified reference preparations of hCG alpha subunit (CR119 or CR123), stimulated the release of prolactin from human decidual cells in culture. The amount of prolactin secreted during a 24 h incubation was concentration-dependent over a range of increasing doses of alpha from 0.2 to 20 ng/ml with an ED50 of about 1.6 ng/ml. These concentrations are well within the physiologic maternal serum free alpha levels which average 350 ng/ml during the third trimester of pregnancy. Incubation of decidual cells with a reference preparation of intact hCG (CR123) at a concentration of 260 ng/ml resulted in stimulated secretion of prolactin, however, the observed stimulation could be attributed to contamination of the preparation with free alpha or dissociated hCG alpha subunit. Purified hCG beta subunit had no stimulatory activity on the decidual cell culture. The effect of alpha subunit on the stimulated release of prolactin was not due to a generalized stimulation of protein synthesis and secretion since no increase was observed in the release of 35S-labeled proteins compared to controls. In addition, the observed increase in prolactin secretion was not due to a toxic effect of the alpha subunit since there was no visible effect on cell viability, and the cellular enzymes, LDH and alkaline phosphatase, were not detected in the culture medium. Addition of exogenous hCG alpha subunit to primary cultures of human trophoblast cultures did not result in stimulated release of human placental lactogen. We conclude that free alpha molecules of pregnancy stimulate release of prolactin from human decidual cells in culture. These results suggest a novel role for free alpha in the paracrine regulation of decidual prolactin secretion.

Insulin Stimulates Synthesis and Release of Human Chorionic Gonadotropin by Choriocarcinoma Cell Lines

Recent studies have shown that insulin regulates placental lactogen, progesterone, and estrogen production from human trophoblast cells. This study was performed to examine whether insulin also regulates the production of hCG by this type of cell. After 24-36 h of preincubation, JEG-3 and JAR cells (2-3 x 10(5) cells/ml.well) or human term trophoblast cells (1 x 10(6) cells/ml.well) were exposed to the test hormone in serum-free Dulbecco's Modified Eagle's Medium for 24-96 h. Secretion of hCG from JEG-3 cells was stimulated by human insulin, human proinsulin, or porcine insulin in a dose-dependent manner, with lowest effective doses of 6.7, 96, and 53 mg/L, respectively. Time-course studies showed that hCG secretion peaked at 72-96 h with insulin exposure; in contrast, no decernable peak was seen without insulin in serum-free media. Exposure of JEG-3 cells for 24 h to 209 mg/liter insulin stimulated hCG synthesis, with 40 +/- 3% more immunoreactive intracellular hCG (P less than 0.05). Cells grown in the presence of insulin and [35S]methionine had 47 +/- 21% more labeled intracellular hCG and 56 +/- 13% more immunoprecipitable [35S]methionine-hCG secreted into the medium than the control cultures (P less than 0.05). During this time period, human placental lactogen release and total trichloroacetice acid-precipitable [35S]methionine protein were not increased. The insulin-induced stimulation of hCG synthesis was inhibited by cycloheximide. Additionally, insulin did not significantly affect total intracellular protein during 24-96 h of incubation. Insulin also increased hCG release from JAR cells, but not from human term trophoblast cells. A mouse monoclonal antibody to the IGF-I receptor inhibited the stimulation of insulin in JEG-3 cells. We conclude that insulin stimulates the synthesis and secretion of hCG from JEG-3 cells and JAR cells, and that hCG regulation in choriocarcinoma cells differs from that in primary human placental trophoblast cells. The effect of insulin on JEG-3 cells may be mediated in part through the insulin-like growth factor-I receptor.

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 −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.

Melittin stimulates phosphoinositide hydrolysis and placental lactogen release: Arachidonic acid as a link between phospholipase A 2 and phospholipase C signal-transduction pathways

Previous investigations from this laboratory have implicated both phospholipase A 2 and phospholipase C in the regulation of human placental lactogen release from human trophablast. To study further the role of endogenous phospholipase A 2 and the relationship between phospholipase A 2 activation and phosphoinositide metabolism, we examined hPL and [ 3H]-inositol release from trophoblast cells in response to agents that stimulate or inhibit the endogenous enzyme. Melittin (0.5–2.0 μg/ml) stimulated rapid, dose-dependent, and reversible increases in the release of hPL, prostaglandin E, and [ 3H]-inositol. Mepacrine (0.1–0.25 mM) inhibited this stimulation. However, mepacrine had no effect on the stimulation of hPL and [ 3H]-inositol release by exogenous arachidonic acid (AA). These results indicate that the stimulation by melittin of phosphoinositide metabolism and hPL release is mediated by initial activation of phospholipase A 2. Furthermore, the results support the possibility that AA, released as a consequence of phospholipase A 2 activation, can act as a second messenger linking the two phospholipase pathways.

Angiotensin II Stimulates Both Inositol Phosphate Production and Human Placental Lactogen Release from Human Trophoblastic Cells

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.