Calcitonin receptor in human placental syncytiotrophoblast brush border and basal plasma membranes

~Uot’eccular and Cellular Endocrinology, 99 (1994) 285-292 0 1994 Elsevier Science Ireland Ltd. 0303-7207/94/$07.00

285

MCE 03171

Calcitonin receptor in human placental syncytiotrophoblast plasma membranes

brush border and basal

J. Lafond *7a,L. Simoneau a, R. Savard a and D. Lajeunesse b a iinivetsite’ du Qukbec ci Montr6al, Dr?partementdes Sciences Biologiques, CJ? 8888, succ. A, Montrt!aI, Qulbec, Canada H3C 3PS; ’ Hopitai Maisonneuve-Rosemont, &en@ede recherche, Montreal, Canada (Received 2 September 1993; accepted 3 November 1993)

Key words: Placenta; Calcitonin; Receptor; lnositol phosphate; Brush border membrane; Basal plasma membrane

Summary The physiolo~ of calcium transport through the placenta has not been studied thoroughly. In particular, the effect of caicaemic hormones on this process has never been reported. In this paper we questioned if cafcitonin, a hypocalcaemic hormone, is also implicated in the regulation of calcium transport by one of the placental syncytiotrophoblast bipolar membranes. In order to investigate the implication of calcitonin on calcium transport, we first studied whether this hormone binds to any of these bipolar membranes, i.e. purified syncytiotrophoblast brush border (facing the mother) and basal plasma membranes (facing the fetus). The initiation of binding of human [1251]calcitonin to the two types of membranes was rapid and reached a steady state after 10 min of incubation at 37°C. The number of binding sites and the affinity of these receptors for the hormone were studied for each type of membrane, with concentrations of [‘251]calcitonin varying from 0.01 to 1.8 nM. Scatchard analysis revealed a single affinity binding site for human calcitonin with Z&s of 0.83 f 0.09 nM and 0.67 + 0.26 nM for brush border and basal plasma membranes respectively. The maximal number of receptors was significantly different (p < 0.001) in the two membranes: B,, of 66.64 t- 9.15 fmol/mg protein for brush border membranes and 19.66 f 2.80 fmolfmg protein for basal plasma membranes. Competitive displacement of [‘~IJcal~itonin with other ligands showed the following potencies between human calcitonin > salmon calcitonin > calcitonin gene-related peptides and segments analogues but no competition with some human calcitonin gene-related peptides fragments. Half-maximal displacement concentration for human calcitonin was reached at approximatively 1 nM for BBM and 0.1 nM for BPM. Calcitonin stimulated inositol phosphate production in both membranes by 175% and 330% in BBM and BPM, respectively. We conclude that calcitonin receptors are present in the two polar syncytiotrophoblast membranes, but are more abundant in brush border membranes, facing the mother. These results also suggest that in the placenta, both maternal and fetal calcitonin might intervene in ion transport and particularly calcium transport or on the intracellular metabolism of the placental syncytiotrophoblast cells.

Introduction The syncytiotrophoblast cell constitutes the main barrier to maternal-fetal transport and exchange of minerals and nutrients between the mother and the fetus (Sideri et al., 1983). Fetal skeletal mineralization requires large quantities of calcium (Ca”> and phosphate, which are actively transported from mother to the fetal circulation against a concentration gradient

* Corresponding author. Fax: (514) 987-4647. SSDI 0303-7207(93)E0281-X

(Garel, 1983; Pitkin, 1985; Lafond et al., 1991). The physiology of calcium transport through the placenta is not well defined. Hormones, like parathyroid hormone (PTH), are known to bind at specific receptors on syncytiotrophoblast brush border (BBM; facing to the mother) and basal plasma membranes (BPM; facing to the fetus) (Lafond et al., 1988). In the placenta, PTH regulates phosphate exchange (Brunette et al., 1989) whereas it is a well-known regulate of calcium reabsorption (Morel and Doucet, 1986) and calcium channel (Bacskai and Freidman, 1990) in the kidney. Another hormone known to act on phosphatemia and

calcaemia is calcitonin (CT) (Garel et al., 1974). In particulary, fetal calcemia is in linear relationship with CT secretion and CT infusion into the fetus. However, the exact role of fetal and maternal CT in modulating ion transport is unknown, although Barlet (1974) demonstrated that endogeneous CT prevents excessive demineralization of the skeleton in pregnant female. Many tissues implicated in calcium physiology contain CT receptors, such as the kidney (Marx et al., 1972, 1973) and osteoclasts (Warshawsky et al., 1980; Nicholson et a1.,1986). Low specific binding activities are also detected in brain (Rizzo and Goltzman, 1981), testis (Chausmer et al., 1980) and cell lines obtained from a large range of neoplasias (Marx et al., 1974; Hunt et al., 1977; Martin et al., 1980; Eilon et al., 1983). In addition, Nicholson et al. (1988) demonstrated the presence of CT receptors in human placenta. Unfortunately, these authors used a total human placental membrane fraction thus not differentiating the maternal and fetal side. In the present paper, we investigated the binding of human CT (hCT) to the two polar membranes of the placental syncytiotrophoblast: BBM and BPM. For this study we used hCT, which has been previously demonstrated to possess full biological activity and bind with high capacity to CT receptors (Nicholson et al., 1988). Our results demonstrate that both syncytiotrophoblast membranes (BBM and BPM) contain binding sites for CT, but the number of receptors and the affinity are different between the two membranes. Materials

and methods

Purification of placental membranes

Fresh human placentas were obtained from fullterm normal vagina1 deliveries. After perfusion with saline (0.9% NaCl) solution through the umbilical artery ramifications, 120-150 g, placenta1 tissue were obtained from the central part of the placenta. The amnion, chorion, and decidua were removed. The tissue was cut into 2 to 5 mm fragments and placed in 270 mM mannitol and 10 mM Tris-Hepes, pH 7.4, for 30 min at 4°C with magnetic stirring. After this incubation, the BBM were prepared using the technique of Smith et al. (1974, 1977) as modified and described in Lafond et al. (1988). Briefly, the membrane suspension was filtered through six layers of cotton gauze. The filtrate was centrifuged at 100000 X g for 60 min. The whitish pellet was separated from the red blood cells, suspended in 40 ml Tris-mannitol medium, and homogenized with a Potter Teflon homogenizer at 2000 rpm. MgClz was added to a final concentration of 10 mM, and the suspension was stirred for 20 min at 4°C and then centrifuged at 2500 Xg for 20 min. The BPM were prepared from the pellet, and the BBM from the supernatant.

BBM preparation

The supernatant of the 25000 Xg centrifugation was collected and centrifuged at 25 000 X g for 20 min at 4°C. The pellet was suspended in Tris-mannitol buffer, pH 7.4, washed, and resuspended to obtain a final protein concentration of 20-25 kg/PI. BPM preparation

The pellet (obtained after the 2500 X g centrifugation) was suspended in 10 mM Tris-Hepes, pH 7.4 (100 vol) and frozen immediately at -80°C for 60 min. The frozen pellet was thawed in ice, homogenized with a Potter Teflon homogenizer at 2000 rpm, and centrifuged at 90 000 X g for 30 min. The pellet was suspended with 2 ml of 10 mM Tris-Hepes, pH 7.4, homogenized 10 strokes with a Potter Teflon homogenizer at 2000 rpm for 10 strokes, and then loaded on top of a step gradient of Ficoll (4% and 10%). The gradient was centrifuged at 90 x thinsp;OOOx g for 60 min. The membranes at the 4-10% interface were collected, washed twice in Tris-mannitol buffer, pH 7.4, and resuspended to obtain a final protein concentration of 15-20 Fg/pl. The membranes were kept at -80°C for up to 1 month without loss of activity. Protein determination

Protein concentrations were determined by the method of Lowry et al. (1951), using BSA as standard. Assays for membrane markers

The purity of the membranes was monitored by measuring the alkaline phosphatase (BBM marker) activity determined by the measurement of p-nitrophenol (Kelly and Hamilton, 1970) and Na+/K+-ATPase (BPM marker enzyme) activity according to the technique of Post and Sen (1967). Binding of the calcitonin to the receptor

Calcitonin binding to the receptor was studied using the following microtechnique. Brush border membranes or BPM suspensions (5 pg) were incubated with ‘251-hCT (1000-50000 cpm) in 20 ~1 of a medium containing 50 mM Tris-HCl, 1 mM EDTA, 0.5% BSA and 250 mM sucrose, pH 7.4 at 4°C. The incubations, in quadruplicate, were performed for 10 min at 37°C unless otherwise noted. The membrane-bound 12’1-hCT was separated from unbound radioligand using a rapid filtration using Multi-Screen Filtration System (Millipore, Canada) through 0.65 pm PVDF Durapore membrane filters. Each filter was washed three times with 0.3 ml ice-cold incubation medium. The filters were dried and then counted in a gamma-counter for the assay of “‘1 . Binding to the filters in the absence of BBM or BPM was less than 1% of the ‘251-hCT and,

287

for each experiment, blank values were substracted from each corresponding binding value. For displacement experiments we first used a maximal amount of ‘251-hCT (1.5 nM) that was incubated with appropriate membranes for 10 min. The fixed ‘251-hCT was displaced with purified human caicitonin gene-related peptide (hCGRP II), human calcitonin gene-related peptide segment 8-37 (hCGRP 8-371, human cyclic calcitonin gene-related peptide (hcCGRP), rat calcitonin c-terminal adjacent peptide (CAP or CCP>, human calcitonin (hCT), salmon calcitonin (sCT), katacalcin, human parathyroid hormone l-34 (hPTH 1-34) and human parathyroid related peptide l-34 (hPTI-I-rp l-34) were added to the incubation medium at increasing concentrations from 10c16-10-4 M. Human CGRP 8-37 was used to verify the importance of the N-terminal or C-terminal portion in the binding.

of inositol phosphate release by the me~ranes The day before the experiment, the placental homogenate was preincubated for 18 h at 4”C, in the presence of 2 pCi/ml of [3H]myo-inositol (20 Ci/ mmol). The tissue was then centrifuged, the radioactive supernatant discarded and the membranes were purified as described in the ‘purification of membranes’ section. Fresh membranes were suspended and incubated for 30 min in 1 ml of a phosphate-glucose buffer to which 10 mM LiCl was added. This medium contained: 1% gfucose, 0.44 mM CaCl,, 3.5 mM KCl, 0.5 mM MgCI,, 138 mM NaCl, 8.1 mM Na,HPO, and 1.5 mM KH, PO, (pH 7.4). To this medium, 10e7 M CT or the vehicle was included. The incubation (30 min at 20°C) was stopped by removing the media and adding 1 ml of 5% perchloric acid to which 0.2 ml of bovine serum albumin (2%) was added to facilitate precipitation. This suspension was centrifuged and the supernatant kept for the separation of the inositol phosphate. Three types of inositol phosphate were extracted using chromatographic columns containing 1.5 ml of Dowex AG l-X8 anion exchange resin. The fractions were eluted with 15 ml H,O followed by 15 ml of 60 mM ammonium formate and 5 mM sodium tetraborate for inositol, 15 ml of I50 mM ammonium formate and 5 mM sodium tetraborate for inositoImonophosphate (IP), 15 ml of 400 mM ammonium formate and 100 mM formic acid for inositolbiphosphate UP,) and 15 ml of 1 M ammonium formate and 100 mM formic acid for inositoltriphosphate (IP,) (Berridge et al., 1983; Bone et al., 1984). The radioactivity present in 1 ml of each sample was assayed in a formula P-963 scintillation liquid cocktail and the fraction factor was correlated for all samples. Results are presented as absolute values rather than as percentages of the total lipids as

Dete~i~t~n

often reported in the literature, in an attempt to detect a quantitative effect of the CT. Corrections were made according to the recoveries of internal standards run in parallel. Drugs and materials

Purified human calcitonin gene-related peptide
Alkaline phosphtase (gmol/mg prot./ 15 min)

Na+/K’ ATPase (~mol/mg prot./ 15 min)

Homogenate BBM BPM

10.91 f 0.60 = 248 i 13 (X25) a,b 52 f 3 (X5)

0.36kO.02 1.2 + 0.2 (X3) b 8.0 2 0.6 (X22)

a Values are expressed as the mean*SEM of 15 placentas. The magnitude of enrichment is indicated in parentheses. b p < 0.001 vs BPM.

288

Contamination by intracellular organelles was very low y9;~) not shown) as previously reported (Lafond et al., A

80

Calcitonin receptors in BBM and BPM

Total binding of the radioligand 1251-hCT to purify syncytiotrophoblast BBM and BPM represented lo15% of the added ligand. Specific binding corresponded approximatively to 80-90% of the total binding. The specific binding of 12’I-hCT was time-dependent, increasing to a maximum after 10 min at 37°C for BBM receptors (Fig. 1). Similar results were obtained for BPM (data not shown). Total binding (without unlabeled hCT) and non-specific binding were also maximal after 10 min of incubation (Fig. 1). Thus, for the following experiments BBM and BPM were always incubated for 10 min at 37°C. Fig. 2 shows the specific binding of ‘251-hCT to BBM and BPM receptors. At 0.5 x lo-” M ‘251-hCT specific binding values were significantly different (b < 0.001) between BBM and BPM receptors. Scatchard analysis of the binding data obtained with purified BBM and BPM using radioactive ligand i2’I-hCT are presented in Fig. 3. Scatchard analysis of the specific binding data was linear, indicating a single class of high affinity sites for hCT for both types of membranes of syncytiotrophoblast cells (K,‘s of 0.83 _t 0.09 nM and 0.67 t_ 0.26 nM for BBM and BPM respectively). The number of receptors was signif100 0

Non

specific

a0

l-

ao :: : ,;L

0

60

40

20

0

0.00

0.50

20 TIME

30

(min)

Fig. 1. Time course of ‘Z51calcitonin (1.5 nM) binding to human syncytiotrophoblast BBM. The experiment was performed with 5 pg protein of BBM or BPM at 37°C. Non-specific and specific binding were carried out in the presence and absence of 10eh M unlabeled human CT. Results are expressed as the mean + SEM obtained in three separate experiments in which quadruplicate determinations

were done.

1.50

2.00

CLT (nM) Fig. 2. Binding of increasing concentration of [‘Z51]calcitonin (O.Ol1.8 nM) to 5 pg protein of human syncytiotrophoblast BBM or BPM. Specific binding is derivated by substracting non specific from total binding, determined after 10 min of incubation at 37°C. All values are expressed as the mean& SEM for three separate experiments in which quadruplicate determinations were done.

icantly different (p < 0.001) in the two membranes with B,, of 66.64 f 9.15 fmol/mg protein for BBM and 19.66 + 2.80 fmol/mg protein for BPM. Hill plot analysis of the data yield straight lines with Hill coefficients of 0.84 and 0.99 for syncytiotrophoblast BBM and BPM respectively (r2 are 0.99 and 0.93), suggesting an absence of cooperative behavior (Fig. 4). Specificity of human receptors

10

1.00

CT binding to BBM and BPM

To investigate the specificity of the binding of ‘*‘ICT to syncytiotrophoblast membranes (BBM and BPM), displacement binding studies were performed with various human and salmon CT, CT analogue (katacalcine, rat calcitonin c-terminal adjacent peptide (CAP), human CGRP type II (hCGRP II), hCGRP segment 8-37 (hCGRP 8-37), human cyclic CGRP (hCCGRP) and others peptides known to act also on calceamia (PTH and PTH-rp) (Figs. 5 and 6). The displacement of hCT and of salmon CT was similar for BBM (Fig. 5) and BPM (Fig. 6) but was more effective for BBM. The concentration of hormones inhibiting the maximal binding of radiohgand by 50% (IC,,) was 1 nM for human CT and 0.1 PM for salmon CT in BBM and 0.1 nM and 0.01 nM in BPM, respectively. The IC,, values

289

Bm8x-

0

17.4 lmol/mg

5

-16 ii

0

10

20

30

40

50

60

70

50

BO 100

Bound ( fmollmg protein ) Fig. 3. Representative Scatchard plots of steady-state binding to syncytiotrophoblast BBM and BPM membranes (5 pg protein). Free [“*‘I]calcitonin was taken as the difference between added and bound ‘“I CT. B/F bound to free ratio. Similar results were obtained for three additional experiments and values are expressed as the means * SEM. Each performed in quadruplicate.

-14

-t2 W

-10

-8

/

-8

/

-4

IW

Fig. 5. Competitive displacement of receptor ‘251-hCT (1.5 nM) from human ~n~iotrophobl~t BBM (5 fig protein) binding sites by various hormones at 3T%. Binding is expressed as a percentage of maxima1 binding; 100% represents Izl-hC1: binding in the absence of unlabeled hormone. Each point is the mean of quadruplicate determination and three additional separate experiments were done. Human a (a ), salmon CI’ ( q 1, human CGRP CO),human CGRP II ( A 1,human CGRP 8-37 (XI, human c-terminal CGRP( A 1.

for the displacement of ‘Z51-hCT were similar for sff, hCGRP and hCGRP II in BBM. In contrast, these values were much higher for hCGRP and hCGRP II in BPM whereas for SC?‘, the IC,, was lower than for hC3’ (Table 2). Human CGRP segments were poorly effective to compete with hCT in displacement studies performed with BBM (Fig. 5) and BPM (Fig. 6). On the other hand, unrelated hormones like human PTH, human PTH-rp and katacalcin, were ineffective to displace ‘*‘I-hCT in both membranes at concentrations ranging from lo-* to 10V4 M (data not shown).

1.0

0.5

TABLE 2 SUMMARY OF HALF MAXIMAL, CONCENTRATIONS OF INHIBITOR FROM DISPLACEMENT STUDIES OF “?-human CT BY CT AND RELAZD HORMO~S IN BBM AND BPM OF NORMAL TERM HUMAN PLACENTA =

-2.0

-1.5

-1.0

-0.5

0.0

0,s

1.0

Log Froo Fig. 4. Hill plot of steady state binding data with BBM and BPM

membranes presented

in

Fig. 3.

hCT sCT hCGRP hCGRP II

BBM

BPM

10-g” 10-7 10-7 10-7

0.1 x 10-9 b 0.01 x 10-9 5x10-6 5x10-6

a Half-maximal inhibitory values were obtained from data with BBM and BPM displacement curves in Fig. 5 and Fig. 6, respectively. b Values are expressed as molar and were obtained for three additional experiments. Each performed in quadruplicate.

290

increase in IP liberation. Table 3 presents the [“HIlabelled IP, IP,, IP, released by the BBM and BPM incubated with CT or the vehicle. Calcitonin enhanced IP formation in both membranes, thus confirming our hypothesis that an hormonal effect via the phospholipid route as a second messenger is present in both BBM and BPM. The effect of CT was maximal in BPM with a 330% increase in IP production in response to lo-’ M CT whereas the increase was only 174% in BBM (Table 3).

100

;

z

80

s a

Discussion

0

/

I,

r! 0

I

(

I

-14

-18

,

-12 WI

I

(

-10

/

,

-8

I

,

-8

I,

1

-4

WI

Fig. 6. Competitive displacement of receptor ‘251-hCI (1.5 nM)from human syncytiotrophoblast BPM (5 pg protein) binding sites by various hormones at 37°C. Binding is expressed as a percentage of 1251-hff binding in the absence maximal binding; 100% represents of unlabeled hormone. Each point is the mean of quadruplicate determination and three additional separate experiments were done. Human CT (m), salmon CT (01, human CGRP CO),human CGRP II ( A 1,human CGRP 8-37 (Xl, human c-terminal CGRP (A 1.

Inositol phosphate

production

Because both BBM and BPM had high specific CT binding, we questioned which second messengers were implicated in their hormonal action. Thus, inositol phosphate (IP) production was measured in both membranes in the presence or absence of hCT lo-‘M. In these experiments, the placental tissue was preincubated the day before the experiment with [3H]myoinositol (2 pCi/ml) for 18 h at 4°C. Since Li (LiCl) inhibits the hydrolysis of IP, any acceleration in the metabolism of phosphatidylinositol should result in an

TABLE

3

INOSITOL PHOSPHATE PRODUCTION IN THE PRESENCE OR ABSENCE OF CALCITONIN BY BBM AND BPM OF NORMAL TERM HUMAN PLACENTA

IP, IP,

BBM

BBM + CT lo-’ M

BPM

BPM + CT lo-’ M

3.22 _+0.50 2.41+ 0.44

2.39 + 0.48 h 2.63 f 0.45

8.42+ 1.69 7.14* 1.12

6.86k 1.01 7.58 f 2.15

a Values are the mean f SEM of 3 separate placental preparations in quadruplicate experiments and are expressed in nmol/mg protein. ’ p < 0.001 vs control (BBM or BPM respectively). ’ p < 0.001 vs treated BPM.

In our study, we have shown the presence of specific CT receptors in human placental syncytiotrophoblast BBM and BPM using ‘251-hCT. The continuous layer of the syncytiotrophoblast cells constitute the main barrier to maternal-fetal transport representing a small proportion of the total parenchyma (Shennan and Boyd, 1987). Syncytiotrophoblast cells represent the majority of cells present in full term placenta (Sherman and Boyd, 1987). The physiological and biochemical functions associated with this specific binding of CT remains to be investigated. In our preparation, amnion, chorion and decidua were removed before the purification of BBM and BPM from syncytiotrophoblast bipolar cells. Each enrichment values of alkaline phosphatase (Kelly and Hamilton, 1970) and Na+/K+ATPase (Post and Sen, 1967) was determined by the ratio of purified membranes to crude homogenate (data from Table 1, and as described in Lafond et al. (1988, 1991)). Our values of enrichment in BBM and BPM suggest a high degree of purification of our membrane preparations. The membrane purity is the first important step for the interpretation of the binding data. Our results show a significant difference in the number of specific CT receptors between BBM and BPM (B,,, = 66.64 + 9.15 and 19.66 _+2.8 fmol/ mg protein, respectively). Our results also show a significant difference in binding affinity. These data in BBM and BPM are comparable to the K,‘s reported in total placental membranes (Nicholson et al., 19881, osteoclasts (Nicholson et al., 1986) and renal membranes (Marx et al., 1973) varying from 0.1 to 2 nM. In the same tissues, the number of receptorial sites or maximal specific binding activities have been in the range of 48 fmol/mg protein in the total placental homogenate (Nicholson et al., 1988) to 190 fmol/mg protein in the hypothalamus (Nakamuta et al., 1981). The difference in the B,,, and K, values obtained from our BBM and BPM purified membranes could be explained by the differential role of each bipolar syncytiotrophoblast membranes in human placenta, while we cannot exclude the possibility that the binding in BPM, representing approximatively 25% of the binding to BBM, may be solely due to BBM contamination of these

291

BPM. Indeed, although our membrane preparations were very good, alkaline phosphatase, the enzyme marker of BBM, represented a five-fold increase in BPM as compared to a 25-fold increase in BBM: thus BBM contamination of BPM could thereby explain, at least in part, the specific binding observed. However, in vivo results suggest that CT acts on placental BPM (Barlet, 1974; Garel et al., 1974; Nicholson et al., 1988; Rebut-Benneton et al., 1992). Thus our observation of specific binding on both membranes may support the hypothesis of a direct role for CT in BPM but also in BBM. In accordance with previous observations in total placenta (Nicholson et al., 1988) and other organs (Marx et al., 1972) our data showed that hCT is more potent than salmon CT to displace iZI-hCT from both membrane receptors. Related peptides (CGRP) and unrelated peptides (PTH and PTH-rp) have reduced or no capacity to displace the radioligand in both membranes, also in accordance with results obtained in other tissues expressing characteristic CT receptors with poor reversible binding. As shown in this study, CT was able to stimulate the synthesis of IP by both membranes. Thus the IP pathway seems to be, in part, implicated in the signal transduction with CT. The stimulator-y effect of CT was more evident in BPM although this membrane shows the lowest B,,,. This is not inconsistent with an action of CT on both membranes if, as we showed here, their binding characteristics were slightly different in response to the same hormonal competitors. Calcitonin may also activate other pathways in BBM not investigate in the present study, i.e. a production of diacylglycerol that is more important in BBM than BPM or the activation of specific G proteins not present in BPM. We previously reported that the two membranes produced IP in the presence of PTH (Lafond et al., 1993). In contrast, adenylate cyclase activity is exclusively located in BPM (Lafond et al., 1988). Nicholson et al. (1988) could not conclude whether CT receptors in total placental homogenate were coupled to the adenylate cyclase system, while Rebut-Benneton et al (1992) suggested that CT increases cyclic AMP production in cultured differentiated trophoblast cells. Taken together, these observations suggest that CT may activate the phosphoinositide pathway in both polar membranes whereas it would only activate cyclic AMP production in BPM since the adenylate cyclase system is absent from the BBM (Lafond et al., 1988). A likely hypothesis could thus be that CT, like PTH, after binding to the two placental membranes, activates phospholipase C in both membranes which in turn may activate certain ion transport via the phospholipid way. Thus, CT could regulate Ca*+ and/or PO, transports through the syncytiotrophoblast using cyclic AMP

and/or IP production in BPM, while in BBM, the entry of these ion could be regulated by IP. The role of CT in placental physiology is not yet defined and remains to be determined. Calcitonin, a hypocalceamic hormone, could be implicated in the control of Ca2+ transport between the mother and the fetus. The calceamia is higher in the fetus than in the mother (Garel et al., 1974; Garel, 1983). This is caused by an active transport of Ca2+ through BPM (Lafond et al., 1991) against a concentration gradient. Barlet (1974) has suggested that CT acts to prevent excessive demineralization of the skeleton in pregnant female but no mechanism has been suggested yet for this effect. Moreover, a role for CT in fetal metabolism also remains to be resolved. Calcitonin seems to be present in fetal thyroid tissues in the 14’h week of pregnancy (Leroyer-Alizon et al., 1980). A CT-like immunoreactivity has been detected in human placenta (Balabanova et al., 1987) whereas Rebut-Benneton et al. (1992) failed to demonstrate CT gene expression nor a mRNA production in the human placenta. These results combined with ours demonstrating the presence of CT receptors in syncytiotrophoblast BPM are in favor of a positive physiological role of CT produced by the fetus directly implicated in the inhibition of Ca*+ transport by BBM and/or BPM. This would prevent excessive demineralization on the maternal side by inhibiting maternal Ca2+ transport through BBM. In contrast, fetal CT would refrain Ca2+ exit on the fetal side by regulating Ca2+ efflux through BPM. Future studies on the regulation of CT receptors mRNA and protein targetting will enhance our comprehension of CT’s action in the placenta as well as the use of specific inhibitors of phospholiase C in our preparations. Acknowledgements

The authors wish to express their gratitude to Dr. Claude Duchesne and the departement of Obstetric and Gynecology at St-Luc Hospital for the donation of placentas. The technical assistance of Mrs Jacynthe Fortier, B.Sc. and the secretarial help of Mrs Christiane Binette are gratefully acknowledged. This study was presented in part at the 75’h Annual Meeting of the Endocrine Society, Las Vegas, Nevada, USA, June 1993. This work was supported by grants from the Medical Research Council of Canada (MT-117361 and Fonds de la Recherche en Sante du Quebec. References Bacskai, B.J. and Freidman, P.A. (1990) Nature 347, 388-392. Balabanova, S., Kruse, B. and Wolf, AS. (1987) Acta Obstet. Gynecol. &and. 66, 323-326. Barlet, J.P. (1974) Ann. Biol. Anim. Biochem. Biophys. 14, 447-457.

292 Berridge, M.J., Dawson, R.M.C., Downes, C.P., Heslop, J.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482. Brunette, M.G., Auger, D. and Lafond, J., (1989) Pediatr. Res. 25, 15-18. Bone, E.A., Fretten, P., Palmer, S., Kirk, C.J. and Michell, R.M. (1984) Biochem. J. 221, 803-813. Chausmer, A., Stuart, C. and Stevens, M. (1980) J. Lab. Clin. Med. 26, 933-938. Eilon, G., Perkins, J. and Viola, M.V. (1983) Cancer Res. 43, 3763-3769. Garel, J.M., Care, A.D. and Barlet, J.P. (1974) J. Endocrinol. 62, 497-509. Garel, J.M. (1983) in M.F. Holick, T.K. Gray and C.S. Anast (Eds.), Perinatal Calcium and Phosphorus Metabolism, pp. 71-104, Elsevier, Amsterdam. Hunt, N.H., Ellison, M., Underwood, J.C.E. and Martin, T.J. (1977) Br. J. Cancer 35, 777-784. Kelly, M.H. and Hamilton, J.R. (1970) Clin. Biochem. 3, 33-43. Lafond, J., Auger, D., Fortier, J. and Brunette, M.G. (1988) Endocrinology 123, 2834-2840. Lafond, J., Leclerc, M. and Brunette, M.G. (1991) J. Cell. Physiol. 148, 17-23. Lafond, J., Ayotte, N. and Brunette, M.G. (1993) Mol. Cell. Endocrinol., 92, 207-214. Leroyer-AIizon, E., David, E. and Dubois, P.M. (1980) J. Clin. Endocrinol. Metab. 50, 316-321. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Martin, T.J., Findlay, D.M., MacIntyre, I., Eisman, J.A., Michelangeli, V.P., Moseley, J.M. and Partridge, N.C. (1980) B&hem. Biophys. Res. Commun. 96, 150-156.

Marx, S.J., Fedak, S.A. and Aurbach, G.D. (1972) J. Biol. Chem. 247, 6913-6918. Marx, S.J., Woodward, C., Aurbach, G.D., Glossmann, H. and Keutmann, H.T. (1973) J. Biol. Chem. 248, 4797-4802. Marx, S.J., Aurbach, G.D., Gavin, J.R. and Buell, D.W. (1974) J. Biol. Chem. 249, 6812-6816. Morel, F. and Doucet, A. (1986) Physiol. Rev. 66, 377-468. Nakamuta, H., Furukawa, J., Koida, M., Yajima, H., Orlowski, R.C. and Schlueter, R. (1981) Proc. Natl. Acad. Sci. (USA) 78, 39733977. Nicholson, G.C., Moseley, J.M., Sexton, P.M., Mendelsohn, F.A. and Martin, T.J. (1986) J. Clin. Invest. 78, 355-360. Nicholson, G.C., D’Santos, C.S., Evans, T., Moseley, J.M., Kemp, B.E., Michelangeli, V.P. and Martin, T.J. (1988) Biochem. J. 250, 877-882. Pitkin, R.M. (1985) Am. J. Obstet. Gynecol. 151, 99-107. Post, R.A. and Sen, A.K. (1967) Methods Enzymol. 10, 762-768. Rebut-Bonneton, C., Segond, N., Demingnon, J., Porquet, D. and Evain-Brion, D. (1992) Mol. Cell. Endocrinol. 85, 65-71. Rizzo, A.J. and Goltzman, D. (1981) Endocrinology 108, 1672-1677. Shennan, D.B. and Boyd, C.A.R. (1987) Biochem. Biophys. Acta 906, 437-487. Sideri, M., de Virgiliis, G., Rainoldi, R. and Remotti, G. (1983) Trophoblast Res. 1, 15-23. Smith, N.C., Brush, M.G. and Luskett, S. (1974) Nature 252,302-303. Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzuchi, S.M. and Kelly, L.K. (1977) Am. J. Obstet. Gynecol. 128,190-196. Warshawsky, H., Goltzman, D., Rouleau, M.F. and Bergeron, J.J. (1980) J. Cell. Biol. 85. 682-794.