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

N. Cirelli1,,3, P. Lebrun2, C. Gueuning1, J. Delogne-Desnoeck1, A.-M. Vanbellinghen1, G. Graff1 and S. Meuris1

1 Research Laboratory on Reproduction and 2 Laboratory of Pharmacology, Université Libre de Bruxelles, B-1070 Brussels, Belgium


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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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°C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 µmol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca2+ or in the presence of 1 or 10 mmol/l Ca2+ 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.

Key words: albumin/hormone secretion/trophoblast


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Throughout pregnancy, the trophoblast is the site of exchange for nutrients, gases and ions between the maternal and fetal circulations. Moreover, the human trophoblast synthesizes and secretes numerous polypeptide hormones directly into the maternal circulation. Among these, human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) are structurally and functionally related to hormones produced by the pituitary gland (Chard, 1986Go; Talamantes and Ogren, 1988Go). The release of both HCG and HPL from placental explants has been shown to be stimulated by the influx of calcium ions (Polliotti et al., 1990Go, 1992Go, 1994Go; Meuris et al., 1994Go; Petit and Belisle, 1995Go) in a manner concordant with the `stimulus-secretion coupling' concept (Douglas, 1968Go). However, in contrast to their pituitary homologues for which the secretory responses are modulated by definite external stimuli, little is known about the physiological control of the placental HCG and HPL releases (Ringler and Strauss, 1990Go; Handwerger, 1991Go).

In-vivo, the trophoblast is in direct contact with albumin, the most abundant plasma protein (3.5–5.5 g/dl blood), involved in numerous biological processes. This protein contributes to the maintenance of colloidal osmotic pressure, fluid distribution and acid-base equilibrium (Doweiko and Nompleggi, 1991Go). Albumin, because of its unique binding properties, is also involved in the transport of circulating calcium (Kragh Hansen and Vorum, 1993Go), fatty acids, amino acids (Kragh Hansen, 1990Go), steroid and thyroid hormones (Mendel et al., 1990Go). Albumin further participates in the transplacental transfer of free fatty acids (Stephenson et al., 1993Go), steroids (Dancis et al., 1980Go), digoxin (Schmolling et al., 1996Go) or cocaine (Krishna et al., 1993Go) and increases the secretion of the lysosomal beta-hexosaminidase from human placental villi (Douglas, 1981Go). Moreover, human albumin is reported to bind to placental villi (Takami et al., 1988Go) and to be internalized by syncytiotrophoblastic brush-border membranes (Douglas et al., 1998Go). Finally, this serum protein is commonly used to prevent adsorption of hormones to glass and plastic surfaces (Bitar et al., 1978Go; Fried et al., 1983Go; Whitehouse et al., 1986Go).

The present in-vitro study aimed to investigate whether albumin at physiological concentrations not commonly used for experimental purposes, could influence HCG and HPL releases from human term placental explants.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Term human placentae (n = 30) were obtained after vaginal delivery following normal pregnancy (37–41 weeks gestation) and immediately transferred to the laboratory. The protocol of this investigation was approved by the Ethical Committee of the Faculty of Medicine of the Université Libre de Bruxelles.

Villous tissue free of visible infarct, calcification, or haematoma, was sampled from at least five cotyledons, midway between the chorionic and basal plates. These central parts of cotyledons were cut into multiple fragments (± 0.5 g) which were thoroughly rinsed in cold (4°C) Hanks' medium (pH 7.4) containing (in mmol/l): NaCl 137, KCl 5, CaCl2 1, MgSO4 1, Na2HPO4 0.3, KH2PO4 0.4 and NaHCO3 4. Fragments (n >= 10) were preserved overnight at 4°C, as previously described (Cirelli et al., 2000Go). The preservation medium was composed of a HEPES-buffered physiological salt solution (pH 7.4) with the following composition (in mmol/l): HEPES 10, NaCl 139, KCl 5, MgCl2 1, glucose 4.2, supplemented with dialysed albumin 0.5% (w/v), penicillin 50 IU/ml and streptomycin 50 µg/ml (Gibco BRL, Gaithersburg, MD, USA). All reagents were of analytical grade and purchased from Sigma Chemical Co (St Louis, MO, USA), except when specified.

Prior to the experiments, fragments from the placental core were cut into small explants (approximately 20 mg wet weight) which were collected in a Petri dish containing the cold Hanks' medium. Explants were randomly distributed in glass vials (3/vial) containing the incubation medium composed of a HEPES-buffered physiological salt solution (pH 7.4) having the following composition (in mmol/l): HEPES 10, NaCl 139, KCl 5, CaCl2 1, MgCl2 1, glucose 4.2, 0.5% (w/v) dialysed bovine serum albumin (BSA, V fraction) and no added serum. The 270 min incubation of placental explants, in a shaking water bath (35 cycles/min) at 37°C, started with a 3x60 min equilibration period in order to reach a steady basal HCG and HPL release (Polliotti et al., 1990Go; Cirelli et al., 2000Go) and followed by an 18x5 min experimental period. Placental explants were transferred, at each time interval, through a series of glass vials containing 1 ml of incubation medium gassed with 100% O2. Incubation media collected at each time interval were stored separately at –20°C until assayed.

The exposure to high BSA or Ca2+ concentrations, the addition of human serum albumin (HSA; fraction V, highly purified globulin-free grade) or the temperature lowering were performed between 30 and 59 min of the experimental period. Albumin used in experiments referred to BSA except when specified. Albumin (0.5%) was always present throughout control experiments, before modifying BSA or HSA concentration as well as during Ca2+-stimulated experiments. In some experiments, a second rise in albumin concentration was performed between 90 and 119 min after the equilibration period. In other experiments, the exposure to 5% albumin was processed during 30 min (min 60–89) after increasing Ca2+ concentration to 10 mmol/l between 30 and 89 min. When the albumin concentration was raised to 5% in the incubation medium, pH was adjusted to 7.2–7.4. Osmolarity was verified but did not significantly differ between media containing 0.5 or 5% albumin (295 ± 3.6 mOsmol and 285 ± 6.7 mOsmol, respectively, n = 9). When the Ca2+ concentration was modified in the incubation medium, the NaCl concentration was adjusted accordingly to keep the osmolarity constant. In some experiments, cycloheximide (100 µg/ml, 0.36 mmol/l), colchicine (1 mmol/l), cytochalasin B (40 µmol/l) or EGTA (2 mmol/l) were added to the incubation medium during the whole experiment (270 min). When EGTA was present, the incubation medium was prepared without added calcium.

Total HCG and HPL concentrations were determined in the incubation media using homologous double antibody radioimmunoassays with primary polyclonal anti-hormone rabbit sera performed as previously described (Robyn et al., 1971Go; Polliotti et al., 1990Go). Their sensitivities were 0.6 µg HPL/ml and 1.5 mIU HCG/ml (2nd International Standard distributed by the World Health Organization), respectively. Tracer hormones were radiolabelled with 125I (Amersham, Bucks, UK) using the chloramine-T method (Greenwood et al., 1963; modified by Robyn et al., 1971). Human and BSA did not interfere with the assays. All samples from the same placenta were measured within the same assay. Intra- and inter-assay coefficients of variation were respectively 6 and 8% for HCG and 8 and 12% for HPL. At 180 min, the amounts of hormone released from explants issuing from different placentae ranged between 6 and 48 mIU/vial for HCG and 200 and 920 µg/vial for HPL. The large variations between hormone release from different placentae led us to express, for each individual experiment, changes in hormone release during the stimulation period, as a percentage of a baseline value (100%) defined as the mean amount of hormone released by each group of explants during the first 30 min of the experimental period.

For each experimental condition, mean ± SE refer to experiments repeated with five groups of explants from three different placentae. The significance of the effect of increasing albumin concentrations on hormone releases was assessed using an analysis of variance followed by a Dunnett's post-hoc test. Statistical differences between hormone secretory amplitudes during the 6x5 min stimulation period were assessed using multivariate analysis of variance for repeated measures. Statistical differences between mean total hormone amounts released per placenta during the whole 30 min stimulation periods were assessed using paired Student's t test. A two-tail P value of < 0.05 was considered significant.

In order to quantify hormone sticking on glass surfaces, radioiodinated hormones were diluted (160 to 4000 cpm/ml) in the HEPES incubation medium containing various BSA concentrations up to 5% (w/v). These solutions were incubated in glass vials during 5 to 120 min at 4, 20 or 37°C. Experiments were ended by transferring the radioactive solution to another glass vial. Radioactivity was counted separately in emptied vials, in vials containing the transferred solutions and in vials containing the tips used to transfer the radioactive solution. Recoverable radioactivity corresponding to summed counts per min (cpm) in transferred solutions and on transfer tips was expressed as a percentage of the initial cpm amount. For each experimental condition, mean ± SE refer to experiments repeated five times within three different procedures. Statistical differences between recovery percentages of the two experimental groups were assessed using the Kruskall–Wallis test.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
In the absence of tissue, the incubation of radiolabelled hormones with increasing BSA concentrations resulted in a dose-dependent recovery of the initial radioiodinated HCG and HPL amounts (Table IGo). For both 125I-HCG and 125I-HPL, the recovery percentages were maximal in the presence of 0.5% albumin in the radioactive solution (Table IGo). These recovery percentages were comparable whatever the temperature (4, 20 or 37°C), the exposure time (5, 15, 30, 60 or 120 min) or the initial radiolabelled hormone amounts (160, 800 or 4000 cpm/ml) (Table IGo). Thus, 0.5% albumin is required and sufficient to recover the maximal amounts of HCG and HPL from glass vials. All further incubation experiments were conducted in the presence of 0.5% albumin in the basal media.


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Table I. Percentages of radioiodinated hormone recovered from glass vials under different experimental conditions
 
Incubation of placental explants with increasing albumin (BSA) concentrations during 6x5 min resulted in a dose-dependent increase in total amounts of released HCG and HPL (Figure 1Go). When 5% HSA was tested, total HCG and HPL amounts released during 30 min (178 ± 16.8% and 206 ± 24.0% respectively) did not differ from those secreted in the presence of 5% BSA (185 ± 15.2% and 223 ± 15.3% respectively; NS). A second rise in albumin (BSA) concentration from 0.5 to 5%, after a 30min interval, provoked a similar increase in total HCG and HPL releases (169 ± 19.0% and 205 ± 17.3% respectively; NS). Whether HSA (Figure 2Go) or BSA (Figure 3AGo) was used at a 5% concentration, the increases in HCG and HPL releases were rapid but not sustained during the whole stimulation period. By contrast, a rise in the extracellular Ca2+ concentration from 1 to 10 mmol/l caused a marked and sustained increase in both HCG and HPL releases (Figure 3BGo). In the presence of 10 mmol/l Ca2+ throughout, a 5% albumin rise further stimulated both hormone releases. The transient secretory effects of 5% albumin were of similar amplitude whether the incubation medium contained 1 or 10 mmol/l Ca2+. When the elevations of the albumin (Figure 3AGo) or Ca2+ (Figure 3BGo) concentrations were carried out at 4°C, their secretory effects were totally abolished.



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Figure 1. Dose-dependent effect of increasing extracellular albumin (bovine serum albumin; BSA) concentrations on human chorionic gonadotrophin (HCG) (open bars) and human placental lactogen (HPL) (stippled bars) releases from placental explants. Mean values (± SE) refer to experiments repeated with five groups of explants from three different placentae. Changes in hormone released during the stimulation period are expressed as a percentage of the previous 30 min period (100%) carried out in media supplemented with 0.5% BSA. Mean total releases were significantly different (*P< 0.05) from those observed at the 0.5% albumin concentration using analysis of variance (ANOVA) followed by a Dunnett's test.

 


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Figure 2. Effect of raising albumin concentrations on HCG (left panel) and HPL (right panel) releases by placental explants. Effect of 5% human serum albumin (—•—) compared with control experiments (···{circ}···) carried out in media containing 0.5% BSA. Mean values (± SE) refer to experiments repeated with five groups of explants from three different placentae. Hormone secretory amplitudes during the 6x5 min stimulation period were significantly different (P < 0.05) using multivariate analysis of variance for repeated measures. Changes in hormone release during the stimulation period are expressed as a percentage of the mean hormone amounts released during the first 30 min of the experimental period (100%) carried out, in all cases, at 37°C and in media supplemented with 0.5% albumin and 1 mmol/l Ca2+.

 


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Figure 3. Effect of the temperature on the albumin or calcium stimulations of HCG (left panels) and HPL (right panels) releases by placental explants. Effect of 5% BSA (A panels) and of 10 mmol/l Ca2+ (B panels) at 37°C (—•—) and 4°C (···{circ}···). Mean values (± SE) refer to experiments repeated with five groups of explants from three different placentae. Hormone secretory amplitudes during the 6x5 min stimulation period were significantly different (P < 0.05) using multivariate analysis of variance for repeated measures. Changes in hormone release during the stimulation period are expressed as a percentage of the mean hormone amounts released during the first 30 min of the experimental period (100%) carried out in media supplemented with 0.5% albumin and 1 mmol/l Ca2+ and maintained at 37 or 4°C respectively.

 
In order to further characterize the albumin-mediated release process, placental explants were incubated in the presence of drugs known to interfere with cellular mechanisms underlying hormone secretion.

Addition of cycloheximide (0.36 mmol/l), a ribosomal peptidyl transferase inhibitor, to the incubation medium resulted in a significant decrease in the amplitude of albumin- or Ca2+-induced hormone releases (Figures 4A and 5AGoGo). Total HCG and HPL amounts released during the albumin- stimulation periods were reduced respectively to 61.8 ± 3.27% (P = 0.047) and 57.1 ± 6.69% (P = 0.039) of the amounts released by explants incubated in the absence of cycloheximide. By comparison, when extracellular Ca2+ was used as a secretagogue, total HCG amounts released in the presence of cycloheximide were lowered to 74.0 ± 3.88% (P = 0.035) while total HPL amounts were lowered to 65.8 ± 4.63% (P = 0.027).



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Figure 4. Effect of raising albumin (0.5 to 5% BSA; left panels) or calcium (1 to 10 mmol/l; right panels) concentrations on HCG releases from explants incubated in the absence (—•—) or in the presence of cycloheximide 0.36 mmol/l (···{circ}···; A panels), colchicine 1 mmol/l (···{circ}···; B panels) or cytochalasin B 40 µmol/l (···{circ}···; C panels) throughout. Mean values (± SE) refer to experiments repeated with five groups of explants from three different placentae. Hormone secretory amplitudes during the 6x5 min stimulation period are significantly different (P < 0.05) using multivariate analysis of variance for repeated measures. Changes in hormone release during the stimulation period are expressed as a percentage of the mean hormone amounts released during the first 30 min of the experimental period (100%) carried out in media supplemented with 0.5% albumin and 1 mmol/l Ca2+.

 


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Figure 5. Effect of raising albumin (0.5 to 5% BSA; left panels) or calcium (1 to 10 mmol/l; right panels) concentrations on HPL releases from explants incubated in the absence (—•—) or in the presence of cycloheximide 0.36 mmol/l (···{circ}···; A panels), colchicine 1 mmol/l (···{circ}···; B panels) or cytochalasin B 40 µmol/l (···{circ}···; C panels) throughout. Mean values (± SE) refer to experiments repeated with five groups of explants from three different placentae. Hormone secretory amplitudes during the 6x5 min stimulation period are significantly different (P < 0.05) using multivariate analysis of variance for repeated measures. Changes in hormone release during the stimulation period are expressed as a percentage of the mean hormone amounts released during the first 30 min of the experimental period (100%) carried out in media supplemented with 0.5% albumin and 1 mmol/l Ca2+.

 
Adding colchicine (1 mmol/l), an inhibitor of microtubule assembly, to the incubation medium also significantly reduced the amplitude of the HCG and HPL secretory responses to albumin and Ca2+ (Figures 4B and 5BGoGo). Total HCG amounts released during the albumin-stimulation were lowered to 77.9 ± 8.40% (P = 0.007) in the presence of colchicine while total HPL amounts were reduced to 81.7 ± 10.3% (P = 0.042). During the Ca2+-stimulation, total HCG and HPL amounts released were decreased respectively, to 70.9 ± 4.42% (P = 0.027) and 77.8 ± 6.95% (P = 0.040).

By contrast, the presence of cytochalasin B (40 µmol/l), an inhibitor of actin polymerization, in the medium potentiated HCG and HPL secretory responses to albumin and Ca2+ (Figures 4C and 5CGoGo). Total HCG and HPL amounts released during the albumin-stimulation were increased, respectively, to 124 ± 6.07% (P = 0.042) and 111 ± 3.37% (P = 0.044) of those released by explants incubated in the absence of cytochalasin B. During the Ca2+-stimulation performed in the presence of cytochalasin B, total HCG amounts increased to 126 ± 11.8% (P = 0.034) whereas total HPL increased to 148 ± 23.3% (P = 0.048) of the control without cytochalasin B.

In the last series of experiments, the putative dependence of the albumin stimulatory effect on the presence of extracellular Ca2+ was investigated. For such a purpose, explants were incubated in the presence of EGTA 2 mmol/l, a Ca2+ chelator. The secretory responses elicited by the rise of albumin concentration to 5% were not significantly different when EGTA was present in the medium throughout the 270 min incubation. The increases in HCG and HPL release amounted respectively to 181 ± 17.9% (NS) and to 198 ± 25.6% (NS) (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Throughout human pregnancy, the placental villous trophoblast, which is the specific site of HCG and HPL production, is directly bathed by maternal blood and, consequently, by albumin, the major plasma protein (3.5–5.5 g/dl blood). In the present in-vitro study, albumin was shown to stimulate HCG and HPL releases from human term placental explants at physiological concentrations which are not commonly used for experimental purposes.

Addition of albumin to solutions containing hormones (Bitar et al., 1978Go; Fried et al., 1983Go; Whitehouse et al., 1986Go) has been previously used to prevent the adsorption of these hormones onto glass and plastic surfaces. In this work, there is evidence that the presence of albumin in media containing radiolabelled HCG and HPL improved the recovery percentage from glass vials. This effect was optimal at an albumin concentration reaching 0.5% and did not depend on temperature, exposure time or initial amounts of labelled hormones. These findings confirm that 0.5% albumin is sufficient to reduce HCG and HPL adsorptions onto glass surfaces.

The data further revealed that the addition to the incubation medium of BSA at concentrations >0.5% provoked a dose-dependent increase in both HCG and HPL releases from placental explants. The HCG and HPL releases were also stimulated by the addition of 5% highly purified HSA to the incubation medium, indicating that the stimulatory effect was not species-dependent. The stimulation of hormone releases was completely abolished by lowering the temperature to 4°C. Similarly, the increases in HCG and HPL elicited by 10 mmol/l Ca2+ were completely abolished when experiments were conducted at 4°C. Together, such data indirectly indicate that a membrane- and/or a metabolism-dependent process (Atwater et al., 1984Go) might be involved in the secretory responses to albumin and Ca2+. However, as the albumin- and the Ca2+-stimulated releases were immediate, a direct membrane effect of albumin is more likely. It has also to be noticed that the secretory patterns to albumin were similar for both hormones. This could indicate that despite differences in their gestational profiles, biosynthesis and granule packaging (Morrish et al., 1988Go; Morrish and Marusyk, 1997Go), the cellular mechanisms underlying the exocytosis of HCG and HPL, in response to albumin or Ca2+, exhibit common features.

The present results also provide information about the putative mechanisms underlying the albumin-mediated secretory process. First, the stimulatory effect of albumin on both HCG and HPL releases was decreased in the presence of cycloheximide, a protein translation inhibitor (Lorberboum et al., 1984Go). This indicates that the albumin-mediated HCG and HPL releases require, at least in part, de-novo protein synthesis. Second, colchicine, an inhibitor of microtubule assembly (Jordan and Wilson, 1998Go), also inhibited albumin-mediated HCG and HPL releases. This finding confirms previous studies showing that microtubule integrity is necessary for placental hormone release (Maruo et al., 1987Go). Third, the addition of cytochalasin B, an inhibitor of actin polymerization (Jordan and Wilson, 1998Go), was associated with increased hormone releases during albumin-stimulation. This paradoxical enhancing effect of cytochalasin B on stimulated secretions was previously reported for various cell systems (Chertow et al., 1975Go; Gordon and Werb, 1976Go; Norgauer et al., 1992Go; da Costa et al., 1998Go). In the present study, Ca2+-stimulation of hormone releases was also found to be inhibited by cycloheximide or colchicine and potentiated by cytochalasin B. Taken together, these observations indicate that the cellular mechanisms of regulation underlying HCG and HPL responses to albumin and Ca2+ involve de-novo protein synthesis and cytoskeleton-mediated exocytosis.

Whatever the type of albumin used, the increases in hormone release were not sustained, as observed during Ca2+ stimulation. Moreover, the secretory responses elicited by albumin did not appear to be dependent on the extracellular Ca2+ concentration. Indeed, the current data indicate that the absence of Ca2+ or the presence of 1 or 10 mmol/l Ca2+ in the incubation medium did not modify the amplitude of the albumin-stimulated releases. All together, the observations in this study suggest that the cellular mechanism(s) and/or the hormonal pools involved in the albumin and the calcium-mediated stimulations might be partly different.

Trophoblastic cells are, as endothelial cells, in direct contact with blood and serum proteins. In endothelial cells, albumin has been reported to bind to glycoproteins present on membrane surfaces (Schnitzer et al., 1990Go; Siflinger-Birnboim et al., 1991Go). Such an albumin binding was shown to be associated with rises in cellular inositol triphosphate and cytosolic calcium, suggesting the activation of phospholipase C and the subsequent rapid Ca2+ mobilization from the endoplasmic reticulum (Nguyen et al., 1997Go; Tiruppathi et al., 1997Go). Because albumin binding to trophoblastic microvillous membranes has also been reported (Takami et al., 1988Go; Douglas et al., 1998Go), it is tempting to speculate that an identical cascade of events leading to the production of inositol triphosphate could underline the secretory capacity of albumin. In addition, albumin is known to serve as a transplacental transport vehicle for free fatty acids (Stephenson et al., 1993Go) and steroids (Dancis et al., 1980Go). Likewise, albumin addition to the incubation medium may also decrease the free biologically active fraction of steroids secreted by the placenta itself (Maslar et al., 1990Go). Thus, because steroids have been reported to modulate hormone releases (Queipo et al., 1998Go; Cronier et al., 1999Go), it can be suggested that the secretory response to albumin may be mediated, at least in part, by a modification in the extracellular concentration of free steroids.

In conclusion, the concentrations of albumin eliciting the HCG and HPL release in-vitro are at, or near, albumin concentrations in maternal plasma bathing the syncytiotrophoblast. The question of a physiological participation of albumin in the regulation of placental hormone release is therefore raised.


    Acknowledgments
 
We gratefully thank Mrs L.Lammers and the nursing staff from the Erasme Hospital (Brussels, Belgium) for providing placentae. This work was funded by the National Fund for Scientific Research (Belgium, FNRS) from which S.Meuris and P.Lebrun are Research Directors. N.Cirelli was supported by a grant in aid from the `Université Libre de Bruxelles'.


    Notes
 
3 To whom correspondence should be addressed. E-mail: meuris{at}ulb.ac.be Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on June 27, 2000; accepted on October 20, 2000.





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