MOLECULAR REPRODUCTION AND DEVELOPMENT 65:283–288 (2003)

Expression and Role of Calcium-ATPase Pumpand Sodium-Calcium Exchanger in DifferentiatedTrophoblasts From Human Term PlacentaROBERT MOREAU,1 GEORGES DAOUD,1,2 ANDRE MASSE,3 LUCIE SIMONEAU,1,2

AND JULIE LAFOND1,2,3*1Departement des Sciences Biologiques, Universite du Quebec a Montreal, Quebec, Canada2Laboratoire de Physiologie materno-foetale, Universite du Quebec a Montreal, Quebec, Canada3Departement d’obstetrique-gynecologie de l’Universite de Montreal, Montreal, Quebec, Canada

ABSTRACT Although placental transfer ofmaternal calcium (Ca2þ) is a crucial process for fetaldevelopment, the biochemical mechanisms are notcompletely elucidated. Especially, mechanisms ofsyncytiotrophoblast Ca2þ extrusion into fetal circula-tion remain to be established. In the current study wehave investigated the characteristics of Ca2þ efflux insyncytiotrophoblast-like structure originating from thedifferentiation of cultured trophoblasts isolated fromhuman term placenta. Time-courses of Ca2þ uptake bydifferentiated human trophoblasts displayed rapidinitial entry (initial velocity (Vi) of 8.82�0.86 nmol/mg protein/min) and subsequent establishment of aplateau. Ca2þ efflux studies with 45Ca2þ-loaded cellsalso showed rapid decline of cell-associated 45Ca2þ

with a Vi of efflux (Vie) of 8.90�0.96 nmol/mg protein/min. Expression of membrane systems responsible forintracellular Ca2þ extrusion from differentiated humantrophoblast were investigated by RT-PCR. MessengerRNAs of four known isoforms of PMCA (PMCA 1–4)were detected. Messenger RNAs of two cloned humanNCX isoforms (NCX1 and NCX3) were also revealed.More specifically, both splice variants NCX1.3 andNCX1.4 were amplified by PCR with total RNA of dif-ferentiated human trophoblast cells. Ca2þ flux studiesin Na-free incubation medium indicated that NCXplayed a minimal role in the cell Ca2þ fluxes. However,erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated 45Ca2þ suggest-ing a principal role of plasma membrane Ca2þ-ATPase(PMCA) in the intracellular Ca2þ extrusion of syncytio-trophoblast-like structure originating from the differ-entiation of cultured trophoblast cells isolated fromhuman term placenta. Mol. Reprod. Dev. 65: 283–288, 2003. � 2003 Wiley-Liss, Inc.

Key Words: placenta; trophoblast; plasma mem-brane Ca2þ-ATPase (PMCA); Naþ/Ca2þ exchanger(NCX); calcium

INTRODUCTION

Mammalian fetal nutrition is assured during gesta-tion by the placental transfer of maternal nutrients (Hill

and Longo, 1980; Munro et al., 1983; Boyd, 1987;Shennan and Boyd, 1987). Of nutrients exchanged, allthe Ca2þ ions acquired by the fetus throughout gestationcome from the maternal circulation. It is activelytransported across the placenta from the maternal tothe fetal circulation, and consequently the total Ca2þ

concentration in the fetal blood over a wide range ofspecies of mammals exceeds that in the mother circula-tion especially during the late part of the gestation(Schauberger and Pitkin, 1979; Pitkin, 1985). Thisactive Ca2þ transfer must therefore occur against aconcentration gradient and is carried out in vivo bythe placental syncytiotrophoblast layer (Dearden andOckleford, 1983). Cytotrophoblasts isolated from humanterm placenta undergo spontaneous syncytiotropho-blastic-like morphological and biochemical differen-tiation in vitro, and are thought to resemble in vivosyncytiotrophoblast. Although the cell isolation pro-cedure of Kliman et al. (1986) has greatly improvedthe investigation of placental nutrient exchanges, themechanisms of placental Ca2þ transfer remain to beestablished. Particularly, the membrane gates forthe differentiated trophoblast Ca2þ extrusion are nottotally resolved. Primary culture of trophoblasts iso-lated from human term placenta represents a model toinvestigate this aspect.

The experimental approaches for analyzing humanplacental Ca2þ transport are mostly limited to eitherwhole organ perfusion or uptake by cell-free membranevesicles (Bissonnette, 1982; Lafond et al., 1991; Kamathet al., 1994). On the fetal side, which corresponds tosyncytiotrophoblast basal membranes, a membrane

� 2003 WILEY-LISS, INC.

Grant sponsor: March of Dimes, Saving Babies Together (USA)(to J.L.).

*Correspondence to: Julie Lafond, PhD, Departement des SciencesBiologiques, Laboratoire de Physiologie materno-fætale, Universitedu Quebec a Montreal, C.P. 8888, Succursale ‘‘Centre-Ville’’, Montreal,Quebec, Canada, H3C 3P8. E-mail: lafond.julie@uqam.ca

Received 15 January 2003; Accepted 13 February 2003Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/mrd.10303

Ca2þ pump (Ca2þ-ATPase) has been identified as con-tributor to the cell Ca2þ extrusion on the human fetalcirculation (Lafond et al., 1991; Kamath et al., 1994;Strid and Powell, 2000). The biochemical function ofthe plasma membrane Ca2þ ATPases (PMCAs) is theextrusion of cytosolic Ca2þ from the cell. PMCA is a highaffinity low capacity Ca2þ pump present in virtually alleukaryotic cells. There are four genes in mammalsgiving rise to PMCA isoforms 1–4, the main differencebetween them is in the N- and C-termini (Stauffer et al.,1993; Strehler and Zacharias, 2001). A second systeminvolved in intracellular Ca2þ extrusion is the Na/Caexchanger (NCX). In mammals, plasma membraneNCX is a low-affinity, high capacity Ca2þ translocatingmolecule assumed to play an important role, in parallelwith the PMCA, in the maintenance of the intracellularCa2þ homeostasis (Philipson and Nicoll, 1993; Blausteinand Lederer, 1999). Mammal NCXs are encoded by threegenes, NCX1 (Nicoll et al., 1990; Shieh et al., 1992),NCX2 (Li et al., 1994), and NCX3 (Nicoll et al., 1996).However, human homologous NCX2 has not been cloneduntil now. The NCX1 gene product is widely distribut-ed in tissues and cells, and a number of tissue-specificvariants of the NCX1 transcript have been identified(Schulze et al., 1996). In contrast, the NCX2 and NCX3gene products have thus far been found only in brain andskeletal muscle (Li et al., 1994; Nicoll et al., 1996).

In the current study, we investigated the pathwaysof Ca2þ exit in differentiated trophoblast cells in func-tion of the expression of plasma membrane systemsresponsible for cell Ca2þ extrusion, namely PMCAs andNCXs.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle medium (DMEM), new-born calf serum, and TriZol were purchased fromInvitrogen (Burlington, Ontario, Canada). Hank’sbalanced salt solution (HBSS), trypsin, DNase, Percoll,and erythrosin B were from Sigma (Oakville, Ontario,Canada). The 24-well plates were obtained fromSarstedt (Montreal, Quebec, Canada). Fetal bovineserum (FBS) and enzyme-linked immunosorbent assay(ELISA) for human chorionic gonadotropin (hCG) werefrom Medicorp (Montreal, Quebec, Canada). Bovineserum albumin (BSA), [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid (HEPES), and the randomhexamer primers were from Roche Molecular Bioche-micals (Laval, Quebec, Canada). Radiolabeled Ca2þ

(45CaCl2, specific activity of 8–25 mCi/mg) was fromICN Biomedicals (Irvine, CA). The bicinchoninic acid(BCA) reagent was purchased from Pierce (Brockville,Ontario, Canada). OmniscriptTM RT and Taq PCR corekits were from Qiagen (Mississauga, Ontario, Canada).The thermal cycler GeneAmp PCR system 2400 wasfrom Perkin Elmer (Markham, Ontario, Canada). Allother products were obtained from Sigma and were ofanalytical grade.

Human Placental Cytotrophoblast Isolation

Cytotrophoblasts were isolated from human termplacentas according to the procedure of Kliman et al.(1986). The placentas were obtained from normal deli-veries at the Pavilion St-Luc of the Centre HospitalierUniversitaire de Montreal (CHUM) (Quebec, Canada)and were immersed in DMEM for no more than 1 hrafter delivery. Briefly, fetal membranes and maternaldecidua were removed and villous tissue was cut intoapproximately 1 inch cubes and washed extensivelywith saline in order to remove blood. The tissue was thenincubated three times in HBSS containing 1.5–1.6 mg/ml trypsin and 0.2 mg/ml DNase at 378C for 30 min.After each incubation, the supernatant was removedand replaced by fresh digestion media. The supernatantwas layered on to newborn calf serum and centrifugedat 1,215 g for 15 min. Pellets were resuspended inDMEM, deposited on top of a discontinuous 5–70%Percoll gradient, and centrifuged at 507 g for 20 min.Cytotrophoblast layers were removed and washed inDMEM. Cells were seeded at approximately 1.7�106

cells/well in 24-well plates. The medium was refresheddaily with DMEM containing 10% FBS.

Secretion of hCG by primary culture of tropho-blast was evaluated by ELISA following manufacturerinstructions. For daily analysis of hCG secretion,incubation media werecollected, centrifuged, and super-natants were frozen at �208C until measurements.

Calcium Uptake Studies

Calcium uptake studies were performed on 4-dayscultured trophoblasts. Briefly, cells were washed twicewith the Ca2þ uptake buffer (HBSS containing 1.26 mMCaCl2, 10 mM HEPES, and 0.1% BSA) and allowed toequilibrate in the same buffer for 10 min. Thereafter,cells were incubated at 378C for different intervals oftime following the addition of 500 ml of uptake buffercontaining 45CaCl2 (2–4 mCi/well). The incubation wasstopped by aspiration of the uptake buffer. The cellswere rapidly (less than 30 sec that exclude intracel-lular Ca2þ chelating) washed three times with 1 ml ofice cold PBS containing 4 mM EGTA to eliminate thenon-specific component of the uptake as we reportedpreviously (Belkacemi et al., 2003), and then solubilizedin 500 ml of 0.5 M NaOH. The cell-associated radioacti-vity was measured by a b-scintillation 1400TM Wallaccounter (Turky, Finland).

The cellular protein content of each well was eval-uated by spectrophotometric quantification using theBCA reagent with BSA as standard. The Ca2þ uptake isexpressed as nmole of Ca2þ (from specific activity) permg of cellular proteins.

Ca2þ Efflux Studies

Cells were washed twice and equilibrated with theCa2þ uptake buffer. Thereafter, cells were incubatedat 378C in 500 ml of uptake buffer containing 45CaCl2 (2–4 mCi/well) for 15 min. After two washing with HBSS(without radiolabel), cells were further incubated in500 ml HBSS or free-Na HBSS (LiCl was used to replace

284 R. MOREAU ET AL.

Na instead of choline chloride for erythrosine B stability)for different intervals of time. Cells were then washedthree times with 1 ml of ice cold PBS containing 4 mMEGTA, and solubilized in 500 ml of 0.5 M NaOH.

Expression of PMCA and NCX

Total RNA was isolated from cultured cells usingTriZolTM according to the manufacturer instructions.Isolated RNA was reversed transcribed using ran-dom hexamer primers and OmniscriptTM RT kit. Thepresence of transcripts for PMCA1 (accession numberNM_001682), PMCA2 (accession number L20977),PMCA3 (accession number U60414), PMCA4 (accessionnumber M25874), NCX1 (accession number AF108389),and NCX3 (accession number NM_033262) was evalu-ated in differentiated trophoblast cells by PCR usingspecific primers for PMCA1 (sense, 50-ACTGAGTCT-CTCTTGCTTCGGAAAC-30; antisense, 50-ACGAAATG-CATTCACCACTCG-30), PMCA2 (sense, 50-ACAGTGG-TACAGGCCTATGTCG-30; antisense, 50-CGAGCCGT-GTTGATATTGTCG-30), PMCA3 (sense, 50-CACACTG-GTCAAAGGGATTATCG-30; antisense, 50-AGAGCTG-CATCATGACGAACG-30), PMCA4 (sense, 50-GTTCTC-CATCATCCGAAACGG-30; antisense, 50-CAAGCATC-CAAGTGCCGTACTAG-30), NCX1 (sense, 50-TGTGGC-CCTTACCATTATCCG-30; antisense, 50-GGCTGCTT-GTCATCATATTCG-30), and NCX3 (sense, 50-CAGACG-GAGAGACTCGCAAG-30; antisense, 50-CATACGAGT-GGCTTGGATACGG-30). Antisense primers used todistinguish between splice variant 3 and 4 of NCX1(accession number X91614) were 50-TTCTCACTCAT-CTCCACCAGGC-30 (exon 3 for NCX1.4) and 50-TTCC-TCACGGTCAAATATTCTAATGG-30 (exon 4 for NCX1.3).PCRs were performed using Taq PCR core kit in athermal cycler GeneAmp PCR system 2400.

Statistical Analysis

Statistical analyses were performed using Student’st-test and ANOVA. Differences were considered signifi-cant when P values were <0.05. Results were expressedthe mean�SEM.

RESULTS

Differentiated Trophoblast Cellsand Ca2þ Flux Studies

In order to verify the differentiation of isolated cyto-trophoblast cells into functional syncytiotrophoblast, wemeasured over a period of 7 days of culture (n¼ 3–7different placentas) the secretion of hCG, a well-knownmarker of this process (Kliman et al., 1986). The hCGsecretion was very modest over the first 24 hr of culture(0.9� 0.4% compared to maximal secretion at day 4),then increased at days 2–3 (12.9� 4.5 and 61.6� 9.2%,respectively compared to 4-days cultured cells), reach-ing a peak at 4 days of culture (587.9� 140.5 mU/well/24 hr for a 40-week placenta), and declined thereafter(41.8� 16.9, 10.7�4.1, and 6.8�3.7% for days 5, 6, and7, respectively compared to secretion at day 4). Further-more, multinucleated cells were obviously apparent bymorphological observations further indicating tropho-

blast differentiation over the culture period. Thereforewe performed subsequent experiments with 4-dayscultured trophoblast cells, when differentiation hasoccurred.

Differentiated trophoblast cells exhibited uptakeof extracellular Ca2þ under experimental conditions(Fig. 1A). The Ca2þ uptake was rapid during the first2 min with an initial velocity (Vi) of 8.82�0.86 nmol/mg/min, and gradually reached a plateau at 14.16�0.64 nmol/mg protein. This plateau is generally knownto reflect both cell Ca2þ entry and exit. To confirm thatthe plateau corresponded to Ca2þ exit, efflux studieswere carried out with 45Ca2þ-loaded cells. Figure 1Bshows that cell-associated 45Ca2þ declined rapidly dur-ing the first 2 min with an initial velocity of efflux (Vei) of8.90�0.96 nmol/mg/min and again reached a plateau.

Membrane Gates for Ca2þ Exit inDifferentiated Human Trophoblasts

In view of our results indicating 45Ca2þ efflux fromdifferentiated human trophoblasts, we investigated the

Fig. 1. Time-course of Ca2þ flux by differentiated human tropho-blasts. A: The uptake of Ca2þ was performed for different intervals oftime.B: The efflux of Ca2þ was performed with cells loaded with 45Ca2þ

for 15 min. Loaded cells were then washed twice and incubated in HBSSfor different intervals of time. The results are the mean�SEM ofexperiments performed in duplicates on cell preparations from threeplacentas. When not apparent error bars are within the symbol.

CALCIUM EFFLUX IN CULTURED HUMAN TROPHOBLASTS 285

expression of membrane gates involved in intracellularCa2þ extrusion. Figure 2 shows that mRNAs for PMCA1–4 were amplified from total RNA of differentiatedhuman trophoblasts. We have further pursued ouridentification of plasma membrane systems responsiblefor intracellular Ca2þ extrusion by PCR using specificprimers for two known human isoforms NCX1 andNCX3. Amplicons were generated by PCR reactionsusing total RNA that corresponded to both NCX1 andNCX3 (Fig. 3A). Previously, splice variants of NCX1have been reported (Schulze et al., 1996). To furtheridentify specific variant expressed in differentiatedtrophoblast cells, we have performed PCR reactionswith primers corresponding to exon 3 and 4 of NCX1.Figure 3B shows that nested PCR with new antisenseprimers using previously obtained PCR product alsoresulted in PCR amplification of expected size ampliconsfor splice variants NCX1.3 (exon 4) and NCX1.4 (exon 3).In addition, PCR products were observed with designedprimers for splice variants with exons 3 and 4 of NCX1with total RNA of differentiated trophoblasts (Fig. 3C).

Role of NCX and PMCA in the Ca2þ Fluxesof Differentiated Trophoblasts

In order to determine the role of NCX in Ca2þ fluxes ofdifferentiated human trophoblasts, we have carried outefflux studies with Na-free incubation media. Figure 4shows that the efflux of Ca2þ from 45Ca2þ-loaded cells inNa-free incubation medium was not different comparedto efflux in Na-containing medium.

In order to evaluate the role of PMCA in Ca2þ ex-extrusion of differentiated human trophoblasts, we

have performed uptake studies with increasing concen-trations of erythrosin B, an inhibitor of placental PMCA(Tuan and Kushner, 1987; Tuan and Bigioni, 1990).Figure 5 shows that erythrosin B time- and dose-dependently increased the 45Ca2þ-associated with cells.

DISCUSSION

Our contribution into knowledge of placental transfermechanisms is the demonstration of the expressionof multiple membrane systems for cell Ca2þ exit in

Fig. 2. Representative data of the expression of PMCA isoformsby differentiated human trophoblasts from two different placentae.Total RNA was used for RT-PCR with specific primers for (A)PMCA1and PMCA3 (lane 2 and 3: amplicons of 654 and 634 bp,respectively, (B) PMCA4 (lane 2: amplicon of 792 bp), and (C) PMCA2(lane 2: amplicon of 689 bp). Lane 1: 50-bp ladder.

Fig. 3. Representative data of NCX isoform expression by differ-entiated human trophoblasts from two different placentae. A: TotalRNA was used for RT-PCR with specific primers for NCX1 andNCX3 (lane 2 and 3, respectively). B: Nested PCR was performedwith PCR product of Figure 3A using sense primer for NCX1 andspecific antisense primers for splice variants 3 and 4 (lane 3 and 2,respectively). C, D: Total RNA was used for RT-PCR with specificprimers for splice variants NCX1.3 and NCX1.4. Lane 1: 50-bp ladder.

Fig. 4. Time-course of Ca2þ efflux by differentiated human tropho-blasts. Efflux studies were performed with 45Ca2þ-loaded cells andCa2þ exit was monitored for cells incubated in HBSS (*) or Na-freeHBSS (*). The results are the mean�SEM of experiments performedin duplicates on cell preparations from three placentas. When notapparent error bars are within the symbol.

286 R. MOREAU ET AL.

differentiated human trophoblasts isolated from termplacenta. All isoforms of the cloned PMCA (1–4) areexpressed in differentiated trophoblasts. In addition,both transcripts for NCX1 and NCX3 are also presentin 4-days cultured trophoblasts from human termplacenta. More specifically the splice variants NCX1.3and NCX1.4 have been revealed. Our results also sug-gest that NCXs play a minimal role in basal Ca2þ effluxof differentiated human trophoblasts, and that PMCAactivity more likely contributed to intracellular Ca2þ

extrusion in 4-days cultured trophoblasts isolated fromhuman term placenta.

Both PMCA1 and 4 are considered to be housekeepingisoforms found in virtually all cells. Messenger RNAs ofPMCA1 and 4 have been previously shown in wholehuman placental tissue (Howard et al., 1992; Santiago-Garcia et al., 1996). Recently, localization of proteins

corresponding to PMCA1 and 4 has been performedin syncytiotrophoblast basal membranes (Strid andPowell, 2000). Therefore, our results are in agreementwith the expression of PMCA1 and 4 in human placentaltrophoblasts. Moreover, we demonstrated for the firsttime the expression of PMCA2 and 3 in differentiatedhuman trophoblasts. These cells also express both NCX1and 3. An amplicon of 748 bp would be expected for thesplice variant 7 of NCX1 (accession number AF108388)and of 788 bp for the splice variant 1 of NCX1 (accessionnumber NM_021097). According to the amplicon ob-tained (680 bp) with our primers and the very similararrangement of exons in the central portion of thehuman and rabbit gene (Kraev et al., 1996), differenti-ated human trophoblasts expressed either NCX1 splicevariants 3 and/or 4 (NCX1.3 and/or NCX1.4) (Van Eylenet al., 2001). The primers used were located in the exon 2(sense) and in the exon 9 (antisense) of NCX1, thereforebefore and after the multiple splice sites. With specificantisense primers for the exon 3 and 4 of NCX1, weobtained by nested PCR amplicons for both NCX1.3 andNCX1.4. Using total RNA of differentiated trophoblasts,we also revealed the expression of both splice variantsNCX1.3 and NCX1.4. Expression of NCX1 at the molec-ular level in whole human placental tissue has beenreported by Kofuji et al. (1992), who used NCX1 cDNA asprobe for Northern blot. The exchanger isoform NCX1 iswidely distributed in tissues and cells (Blaustein andLederer, 1999), and is apparently the dominant gene inmammals. NCX3 gene product has thus far been foundonly inbrainandskeletalmuscle (Shiehetal., 1992).Ourcurrent results demonstrate the expression of NCX3 in4-days cultured trophoblasts isolated from human termplacenta. In view of the presence of NCX3 in a restrictedtype of tissue, such trophoblast expression may point tospecific role of NCX3 in placental physiology.

Our efflux studies with 45Ca2þ-loaded differentiatedhuman trophoblasts indicated that the intracellularfree Ca2þ extrusion process was minimally affected byreplacing Na from the incubation medium in order toinhibit exchanger activity, suggesting that cell Ca2þ

efflux is not mainly accomplished by NCX under basalconditions. Accordingly, minimal role of NCX in thetransplacental movement of the Ca2þ from mother tofetus has been observed with perfusion studies ofplacental lobules (Williams et al., 1991). It is believedthat since the resting intracellular free Ca2þ concentra-tion is generally low, exchanger’s ion binding sites areunsaturated and therefore, exchanger net transport islow (Blaustein and Lederer, 1999). While identified invirtually every cell examined, NCX physiological rolesare more recognized in excitation-contraction (heart andsmooth muscle cells) and in neurotransmitter release(Blaustein and Lederer, 1999) where intracellular Ca2þ

levels are more prompted to large variation. Involve-ment of NCX in Ca2þ extrusion in epithelial cells is lessestablished and their role in trophoblasts may be morerelated to conditions where intracellular Ca2þ levelsshow large variation unlike basal conditions as in thecurrent study. Under basal conditions, it is more plau-

Fig. 5. Effect of erythrosine B on the Ca2þ uptake of differentiatedhuman trophoblasts. A: Cells were incubated in HBSS containing45Ca2þ in the absence or presence of increasing concentration oferythrosine B (0–1000 mM) for different intervals of time. B: Calciumuptake data at 30 and 45 min are shown in function of differentconcentrations of erythrosine B. P<0.001 by ANOVA for the effect oferythrosine. The results are the mean�SEM of experiments per-formed in duplicates on cell preparations from three placentas. Whennot apparent error bars are within the symbol.

CALCIUM EFFLUX IN CULTURED HUMAN TROPHOBLASTS 287

sible that PMCA is the main pathway of Ca2þ extrusionin differentiated human trophoblasts, which is in agre-ement with the active placental transfer of Ca2þ againsta concentration gradient.

Our results indicate that differentiated human tro-phoblast Ca2þ homeostasis was perturbed in thepresence of erythrosine B, leading to increased cell-associated 45Ca2þ. This effect was time- and dose-dependent. Tuan and Kushner (1987) and Tuan andBigioni (1990) have previously reported inhibition byerythrosin B of placental PMCA activity and Ca2þ

transport in placental basal membranes involved inCa2þ transfer to the fetal circulation. Our results are inagreement with PMCA inhibition by erythrosin B andsubsequent accumulation of intracellular Ca2þ suggest-ing an important role of PMCA in the Ca2þ homeostasisof differentiated human trophoblasts.

Cytosolic Ca2þ plays a key role in intracellular signal-ing in virtually all types of animal cells. Elevatedcytosolic free Ca2þ concentrations may be requiredfor only brief periods of time (Blaustein and Lederer,1999). In this view, tight regulation of intracellular freeCa2þ concentrations is mandatory. In epithelial tissueinvolved in Ca2þ transfer such as kidney, intestine,and placenta, transcellular transit of Ca2þ is performedwithout perturbation of cell signaling. The multipleisoforms of PMCA and NCX expressed in differentiatedhuman trophoblasts may together assure appropriatesignaling processes and transcellular transit of Ca2þ

through the placental epithelium. Further studies arewarranted in order to define the specific role of eachisoforms in the placental physiology.

In summary, we reported that differentiated humantrophoblasts isolated from term placenta express iso-forms 1–4 of PMCA and two isoforms of NCX, namelyNCX1 and NCX3. PMCA1 and 4, along with NCX1are generally considered as housekeeping isoforms ofCa2þ extrusion systems, which are found in virtually allcells. Of interest, differentiated human trophoblastsexpress PMCA2 and 3, in addition of NCX3, all iso-forms that show a more restricted tissue expression(brain and muscle) (Santiago-Garcia et al., 1996;Blaustein and Lederer, 1999). Reasons for the cellexpression of multiple isoforms of PMCA and NCX arenot resolved, although suggesting high regulation ofcell Ca2þ homeostasis.

REFERENCES

Belkacemi L, Gariepy G, Mounier C, Simoneau L, Lafrnd J. 2003.Expression of Calbindin-D28k (CaBP28K) in trophoblasts fromhuman termplacenta. Biol Reprod 2002 (in press).

Bissonnette JM. 1982. Membrane vesicles from trophoblast cells asmodels for placental exchange studies. Placenta 3:99–106.

Blaustein MP, Lederer WJ. 1999. Sodium/calcium exchanger: Itsphysiological implications. Physiol Rev 79:763–854.

Boyd RDH. 1987. Placental transport: Diversity and complexity.Arch Dis Child 62:1205–1206.

Dearden L, Ockleford C. 1983. Structure of human trophoblasts:Correlation with function. In: Loke Y, Whyte A, editors. Biology ofTrophoblast. Amsterdam: Elsevier. pp 69–110.

Hill EP, Longo LD. 1980. Dynamics of maternal-fetal nutrient transfer.Fed Proc Fed Am Socs Exp Biol 39:239–244.

Howard A, Legon S, Walters JRF. 1992. Plasma membrane calciumpump expression in human placenta and small intestine. BiochimBiophys Acta 183:499–505.

Kamath SG, Khaider N, Smith CH. 1994. ATP independent calciumtransport and binding by basal plasma membrane of humanplacenta. Placenta 15:147–155.

Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss III JF. 1986.Purification, characterization, and in vitro differentiation of cytotro-phoblasts from human term placentae. Endocrinology 118:1567–1582.

Kofuji P, Hadley RW, Kieval RS, Lederer WJ, Schulze DH. 1992.Expression of the Na-Ca exchanger in diverse tissues: A study usingthe cloned human cardiac Na-Ca exchager. Am J Physiol 263:C1241–C1249.

Kraev A, Chumakov I, Carafoli E. 1996. The organization of the humangene NCX1 encoding the sodium-calcium exchanger. Genomics37:105–112.

Lafond J, Leclerc M, Brunette MG. 1991. Characterization of calciumtransport by basal plasma membranes from human placentalsyncytiotrophoblast. J Cell Physiol 148:17–23.

Li Z, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP,Lifton RP, Philipson KD. 1994. Cloning of the NCX2 isoforms of theisoform of the plasma membrane Naþ-Ca2þ exchanger. J Biol Chem269:17434–17439.

Munro H, Pilistine S, Fant M. 1983. The placenta in nutrition. Ann RevNutr 3:97–124.

Nicoll DA, Longoni S, Philipson KD. 1990. Molecular cloning andfunctional expression of the cardiac sarcolemmal Naþ-Ca2þ exchan-ger. Science 250:562–565.

Nicoll DA, Quednau BD, Qui Z, Xia YR, Lusis AJ, Philipson KD. 1996.Cloning of a third mammalian Naþ-Ca2þ exchanger, NCX3. J BiolChem 271:24914–24921.

Philipson KD, Nicoll DA. 1993. Molecular and kinetic aspects ofsodium-calcium exchange. Int Rev Cytol 137C:199–227.

Pitkin RM. 1985. Calcium metabolism in pregnancy and the perinatalperiod. Am J Obstet Gynaecol 151:99–109.

Santiago-Garcia J, Mas-Oliva J, Saavedra D, Zarain-Herzberg A. 1996.Analysis of mRNA expression and cloning of a novel plasmamembrane Ca2þ-ATPase splice variant in human heart. Mol CellBiochem 155:173–182.

Schauberger CW, Pitkin RM. 1979. Maternal-perinatal Ca2þ relation-ship. Obstet Gynecol 53:74–76.

Schulze DH, Kofuji P, Valdivia C, He S, Luo S, Ruknudin A. 1996.Alternative splicing of the Naþ-Ca2þ exchanger gene,NCX1. Ann NYAcad Sci 779:46–57.

Shennan D, Boyd C. 1987. Ion transport by the placenta: A review ofmembrane transport system. Biochem Biophys Acra 906:437–457.

Shieh BH, Xia Y, Sparkes RS, Klisak I, Lusis AJ, Nicoll DA, PhilipsonKD. 1992. Mapping of the gene for the cardiac sarcolemmal Naþ-Ca2þ

exchanger to human chromosome 2p21-p23. Genomics 12:616–617.

Stauffer TP, Hilfiker H, Carafoli E, Strehler EE. 1993. Quantitativeanalysis of alternative splicing options of human plasma membranecalcium pump genes. J Biol Chem 268:25993–26003.

Strehler EE, Zacharias DA. 2001. Role of alternative splicing ingenerating isoform diversity among plasma membrane calciumpumps. Physiol Rev 81:21–50.

Strid H, Powell TL. 2000. ATP-dependent Ca2þ transport is up-regulated during third trimester in human syncytiotrophoblast basalmembranes. Pediatr Res 48:58–63.

Tuan RS, Bigioni N. 1990. Ca2þ-activated ATPase of the mousechorioallantoic placenta: Developmental expression, characteriza-tion, and cytohistochemical localization. Development 110:505–513.

Tuan RS, Kushner T. 1987. Calcium-activated ATPase of the humanplacenta: Identification, characterization, and functional involve-ment in calcium transport. Placenta 8:53–64.

Van Eylen F, Bollen A, Herchuelz A. 2001. NCX1 Na/Ca exchangersplice variants in pancreatic islet cells. J Endocrinol 168:517–526.

Williams JM, Abramovich DR, Dacke CG, Mayhew TM, Page KR. 1991.Inhibitor action on placental calcium transport. Calcif Tissue Int48:7–12.

288 R. MOREAU ET AL.