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Expression of Calcium Channels along the Differentiation of Cultured Trophoblast Cells from Human Term Placenta¹

^a Laboratoire de Physiologie materno-foetale, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8

	ABSTRACT

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Placental transfer of maternal calcium (Ca²⁺) is carried out in vivo by the syncytiotrophoblast layer. Although this process is crucial for fetal development, it remains poorly understood. Cytotrophoblasts isolated from human term placenta undergo spontaneous syncytiotrophoblast-like morphological and biochemical differentiation in vitro and are thought to reflect in vivo syncytiotrophoblast. In the present study, we characterized the Ca²⁺ uptake potential and the expression of several Ca²⁺ channels by human trophoblasts during differentiation in vitro for up to 6 days. Secretion of hCG (specific differentiation marker) and uptake of Ca²⁺ by trophoblasts increased gradually as a function of days in culture. Both hCG secretion and Ca²⁺ uptake were maximal on Day 4 and declined on Days 5–6. Expression of the Ca²⁺ transporter proteins CaT1 and CaT2 was revealed by reverse transcription-polymerase chain reaction in cytotrophoblasts freshly isolated from human term placenta. In addition, messengers for two L-type Ca²⁺ channel isoforms (_1C and _1D) were also detected. Levels of CaT1, CaT2, and L-type Ca²⁺ channel mRNA increased gradually during culture, reaching a maximum between Days 2 and 3. In contrast to CaT1 and CaT2 expression that declined thereafter to levels observed on Day 1, L-type channel expression decreased by 50% but remained above the expression level of Day 1. Our results indicate that the pattern of CaT1 and CaT2 expression correlates with the Ca²⁺ uptake potential along the differentiation of cultured human trophoblasts isolated from term placenta. This correlation provides circumstantial evidence for a role of this family of channels in basal Ca²⁺ uptake by the syncytiotrophoblast.

calcium, placenta, trophoblast

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mammalian fetal nutrition during gestation is dependent on the transfer of maternal ions and nutrients by the placenta [1–4]. Throughout gestation, the fetus acquires all its Ca²⁺ from the maternal circulation for its development. This ion is actively transported across the placenta from the maternal to the fetal circulation; consequently, the total Ca²⁺ concentration in the fetal plasma over a wide range of mammalian species exceeds that in the mother, especially during the late part of gestation [5, 6]. This active Ca²⁺ transfer is carried out in vivo by the placental syncytiotrophoblast layer [7]. Cytotrophoblasts isolated from human term placenta undergo spontaneous syncytiotrophoblast-like morphological and biochemical differentiation in vitro and are thought to resemble in vivo syncytiotrophoblast [8]. Although the cell isolation procedure of Kliman et al. [8] has greatly improved the investigation of placental nutrient exchanges, the mechanisms of placental Ca²⁺ transfer remain to be established. Particularly, the membrane gates for the trophoblast Ca²⁺ entry have not been resolved. Primary culture of trophoblasts isolated from human term placenta represents a model of choice to investigate this aspect. Because these cells differentiate in vitro, investigation of the characteristics related to the Ca²⁺ uptake during the time in culture is mandatory.

Among candidates as membrane gates for Ca²⁺ entry in trophoblasts are L-type channels; Ca²⁺ channels with pharmacological characteristics of L-type channels have been functionally identified in trophoblasts in relation to the regulation of hormonal secretions [9–12]. These characteristics include voltage dependence of activation and sensitivity to dihydropyridine and phenylalkylamine organic Ca²⁺ channel modulators. Despite this pharmacological and functional evidence, characterization of trophoblast L-type Ca²⁺ channels at the molecular level remains to be performed. The L-type channel is a heteromeric protein complex composed of a pore-forming ₁ subunit and regulatory-associated ß and disulfide-linked ₂ subunits [13]. The properties of these Ca²⁺ channels are largely conferred by their pore-forming ₁ subunit [14]. Genes encoding the L-type channels include the _1C (cardiac), _1D (neuroendocrine), and _1S (skeletal) isoforms. Molecular cloning of the ₁ subunit has revealed that it is made up of four homologous domains (I–IV), each containing six putative transmembrane segments (S1–S6). Among other types of voltage-gated Ca²⁺ channels (N-, Q-, R-, and T-type), only expression of the T-type has been reported by Northern blot analysis in human placental tissue, though without any investigation of its functional role [15].

Other candidates for the trophoblast Ca²⁺ entry are members of a subfamily related to the transient receptor potential family of nonselective, Ca²⁺-permeable cation channels [16]. This subfamily is composed of novel, highly selective Ca²⁺ channels that recently have been cloned and shown to be expressed mainly in the epithelium involved in Ca²⁺ transfer. All these proteins represent a new family of Ca²⁺ transporter channels that is distinct from the well-characterized, voltage-dependent Ca²⁺ channels and that has structural homology to the capsaicin receptor, VR1 [17, 18]. Calcium transporter protein type 1 (CaT1/ECaC2 or CaT-like) has been identified in intestinal epithelium [19–23]. Calcium transporter protein type 2 (CaT2/ECaC) has been identified in rabbit [24], rat [25], and human [26] renal epithelium. The CaT channel activity is relatively insensitive to voltage-dependent Ca²⁺ channel blockers or activator [23–25] and shows calcium inactivation [27]. Recently, CaT1 was shown to share all the biophysical properties of the previously described Ca²⁺-released-activated Ca²⁺ channel [28]. The latter is a channel through which Ca²⁺ enters cells in response to depletion of internal Ca²⁺ stores, which is known as a capacitative Ca²⁺ entry or a store-operated Ca²⁺ entry. Recently, in our laboratory, Robidoux et al. [29] reported functional evidence for the presence of capacitative Ca²⁺ in the trophoblasts and its involvement in the regulation of corticotrophin-releasing factor secretion by the neuropeptide Y [29].

In the present study, we investigated the Ca²⁺ uptake potential and the expression of CaT1, CaT2, and 1C and 1D isoforms of L-type Ca²⁺ channels during the differentiation of cultured trophoblasts isolated from human term placenta.

	MATERIALS AND METHODS

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Materials

Dulbecco modified Eagle medium (DMEM), newborn calf serum, and TriZol were purchased from Invitrogen (Burlington, ON, Canada). Hanks balanced salt solution (HBSS), trypsin, DNase, Percoll, CaCl₂, and NaOH were from Sigma (Oakville, ON, Canada). The 24-well plates were obtained from Sarstedt (Montreal, QC, Canada). Fetal bovine serum (FBS) and ELISA for hCG were from Medicorp (Montreal, QC, Canada). Bovine serum albumin, EGTA, Hepes, and random hexamer primers were from Roche Molecular Biochemicals (Laval, QC, Canada). Radiolabeled Ca²⁺ (⁴⁵CaCl₂) was from ICN Biomedicals (Irvine, CA). The fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against cytokeratin 7 was from Accurate Chemical & Scientific Corp. (Westbury, NY). Bicinchoninic acid (BCA) reagent was purchased from Pierce (Brockville, ON, Canada). Omniscript RT, Oligotex Direct mRNA mini kit, and Taq polymerase chain reaction (PCR) core kits were from Qiagen (Mississauga, ON, Canada). Restriction enzymes (XhoI and SacI) were purchased from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). The thermal cycler GeneAmp PCR system 2400 was from Perkin Elmer (Markham, ON, Canada). All other products were from Sigma.

Human Placental Trophoblast Isolation and Purity Evaluation

Cytotrophoblasts were isolated from human term placentas according to the procedure of Kliman et al. [8]. The placentas were obtained from normal deliveries at the Pavilion St-Luc of the Centre Hospitalier Universitaire de Montréal (QC, Canada) and were immersed in DMEM for no more than 1 h after delivery. Briefly, fetal membranes and maternal decidua were removed, and villous tissue was cut into approximately 1-inch cubes and washed extensively with saline to remove blood. The tissue was then incubated three times in HBSS containing 1.5–1.6 mg/ml of trypsin and 0.2 mg/ml of DNase at 37°C for 30 min. After each incubation, the supernatant was removed and replaced by fresh digestion media. The supernatant was layered onto newborn calf serum and centrifuged at 1215 x g for 15 min. Pellets were resuspended in DMEM, deposited on top of a discontinuous 5–40% (v/v) Percoll gradient, and centrifuged at 507 x g for 20 min. Cytotrophoblast layers were removed and washed in DMEM. Cells were seeded at approximately 1.7 x 10⁶ cells/well in 24-well plates. The medium was refreshed daily with DMEM containing 10% FBS.

The purity of the cytotrophoblast preparations was evaluated by flow cytometry using FITC-conjugated monoclonal antibody against cytokeratin 7 following the procedure described by Campbell et al. [30]. Approximately 500 000 cells from the preparations were fixed in methanol at -20°C for 20 min, washed in PBS containing 0.9 mM CaCl₂, 2.7 mM KCl, 1.5 mM KH₂PO₄, 0.5 mM MgCl₂, 136.9 mM NaCl, and 0.8 mM Na₂HPO₄ and then incubated in PBS containing serum (1:50 ;obv/v;cb dilution) for 30 min to eliminate nonspecific binding. Thereafter, cells were washed with PBS and incubated with mouse monoclonal anti-human cytokeratin 7 in PBS containing 0.2% BSA for 45 min at room temperature in the dark. Controls were performed by omitting the antibody. Cells were then washed in PBS and analyzed by flow cytometry using FACScan system (Becton Dickinson, San Jose, CA) with WinMDI software (San Diego, CA). It has been reported that anticytokeratin 7 antibody is specific for trophoblast cells [30, 31].

Secretion of hCG by Trophoblasts

Secretion of hCG by primary culture of trophoblast was evaluated by ELISA following the manufacturer's instructions as described previously [12]. For daily analysis of hCG secretion, incubation media were collected and centrifuged, and the supernatants were frozen at -20°C until measurement. Briefly, samples were applied in 96-well plates coated with an antibody against hCG. A second antibody against hCG conjugated to horseradish peroxidase (HRP) was added to each well, and the antigen-antibody complexes were revealed with a chromogenic substrate for HPR (3,3',5,5'-tetramethylbenzidine).

Calcium Uptake Studies

Calcium uptake studies were performed on trophoblasts after 1–6 days of culture. Briefly, cells were washed twice with the Ca²⁺ uptake buffer (HBSS containing 1.26 mM CaCl₂, 10 mM Hepes, and 0.1% BSA) and allowed to equilibrate in the same buffer (250 µl) for 10 min. Thereafter, cells were incubated at 37°C for different intervals of time following the addition of 250 µl of uptake buffer containing ⁴⁵CaCl₂ (2–4 µCi/well). The incubation was stopped by aspiration of the uptake buffer. The cells were washed three times with 1 ml of ice-cold PBS containing 4 mM EGTA (to eliminate the nonspecific component of the uptake according to a procedure reported elsewhere [32]) and then solubilized in 0.1 M NaOH. The cell-associated radioactivity was measured by a ß-scintillation 1400 Wallac counter (Turky, Finland). The cellular protein content of each well was evaluated by spectrophotometric quantification using the BCA reagent with BSA as standard. In the present study, Ca²⁺ uptake is expressed as nmole of Ca²⁺ (from specific activity) per mg of cellular proteins.

Cell Expression of Calcium Channels

The presence of transcripts for CaT1 (accession no. AF304463), CaT2 (accession no. AF304464), and _1C (accession no. XM_052409), _1D (accession no. NM_000720), and _1S (accession no. NM_000069) subunits of L-type Ca²⁺ channels was evaluated in trophoblasts by PCR using specific primers for CaT1 (sense primer, 5'-CTCTGCCTATGGAGCAAGTTCTGC-3'; antisense primer, 5'-GAGAGTCGAGGTCAGTGGTCC-3') and CaT2 (sense primer, 5'-GGCCTATGAGACACGTGAAGATATC-3'; antisense primer, 5'-ATAGAATTGCCCCAGACTGGT-3'); with degenerate primers for _1C, _1D, and _1S (sense primer, 5'-TTCCAGAAGCTSCGGGAGAAG-3'; antisense primer, 5'-GGATGCCACYARGTTGCTCA-3'); and with specific primers for _1S (sense primer, 5'-GGAGTGCAGGGGCTACTACTACG-3'; antisense primer, 5'-GGTTCATCTGCTCCGACTGG-3'). Each RNA preparation was subjected to PCR amplification of an 945-base pair (bp) fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; accession no. XM_006959) using specific primers (sense primer, 5'-GAGTCAACGGATTTGGTCGTATTG-3'; antisense prime, 5'-GCTGTAGCCAAATTCGTTGTC-3') to verify the quality of the RNA sample. Total RNA was isolated from cultured cells using TriZol and mRNA isolated using Oligotex Direct mRNA mini kit according to the manufacturers' instructions. Isolated RNA was reversed transcribed using random hexamer primers and Omniscript RT kit. The PCR reactions were performed using Taq PCR core kit in a thermal cycler GeneAmp PCR system 2400. The specificity of the amplicon was evaluated with restriction enzymes.

Statistical Analysis

Statistical analyses were performed using ANOVA followed by the Tukey test for Figures 1 and 2, whereas the Student t-test was using for Table 1. Differences were considered to be significant at P < 0.05.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Secretion of hCG in trophoblast primary culture from human term placenta as a function of the time of culture. Data are expressed as relative secretion of hCG ± SEM compared to secretion at 4 days of culture of three to seven cell preparations from placentas at 37–40 wk of pregnancy (P < 0.0001, ANOVA). *P < 0.05 and **P < 0.001 compared to Day 1 of culture (Tukey test)

View larger version (27K): [in this window] [in a new window]   FIG. 2. Kinetics of Ca2+ uptake by trophoblast cells as a function of the time of culture. Cells were incubated at 37°C in HBSS containing 1.26 mM CaCl2 and a trace amount of 45CaCl2 for different intervals of time. The transport was stopped by removing the incubation medium, and the cells were washed with PBS containing 4 mM EGTA. The results represent the mean ± SEM of three to four cell preparations (ANOVA, P < 0.0002; Tukey test, P < 0.001 and P < 0.01 for Day 4 compared to Days 1 and 2, respectively)

	RESULTS

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Hormonal Status of the Primary Cultures of Human Trophoblasts

Following the isolation of cytotrophoblasts from human term placenta, the purity of the cell preparations was ascertained by flow cytometry using mouse monoclonal antibody against human cytokeratin 7 (trophoblast specific). For all cell preparations used in the present study, more than 96% of cells were positive for cytokeratin 7 (data not shown), and this percentage of purity has been considered to be acceptable by other investigators [8, 30, 31].

To follow the differentiation of isolated cytotrophoblast cells into functional syncytiotrophoblast, we measured for a period of 7 days the secretion of hCG, a well-known marker of this process [8]. Figure 1 shows a profile of the relative secretion of hCG by isolated trophoblasts from placentas at 37–40 wk of pregnancy. The hCG secretion was modest during the first 24 h of culture. The secretion then increased, reaching a peak at 4 days of culture and declining thereafter. The absolute values of hCG released in the medium were variable in relation to weeks of pregnancy, being highest at 4 days of culture for a 37-wk placenta (5495.8 mU/well per 24 h) and lowest for a 40-wk placenta (449.6 mU/well per 24 h). Maximum hCG secretion was always observed on Day 4 of culture; therefore, we expressed our data as relative hCG secretion compared to the maximum obtained at 4 days of culture.

Ca²⁺ Uptake Studies

The Ca²⁺ uptake experiments on trophoblasts were performed at each day of culture. The kinetics of Ca²⁺ uptake were rapid for the first 2 min of incubation and were followed by establishment of a plateau (Fig. 2). The Ca²⁺ uptake potential of the cultured trophoblasts estimated at the plateau increased gradually on Day 3 (1.60-fold compared to Day 1) and reached a maximum at 4 days of culture (1.84-fold compared to Day 1). We observed a subsequent decline (compared to Day 4) of the Ca²⁺ uptake at the plateau for Days 5 and 6 (Table 1). The initial velocity (Vi) of Ca²⁺ uptake along the time of culture correlated with the relation observed between days of culture and the plateau. The Vi on Day 4 was maximal (5.34-fold compared to Day 1) and decreased on Days 5 and 6 (Table 1). On Day 6 of culture, the plateau was not different than on Day 1, but the Vi remained higher (3.34-fold).

Expression of mRNA for Ca²⁺ Channels in Cytotrophoblasts Freshly Isolated from Human> Term Placenta

Freshly isolated cells from human term placenta are phenotypically identified as cytotrophoblasts. We evaluated the expression of CaT1, CaT2, and L-type channels in these cells. Figure 3A shows representative results of PCR reactions performed on cell preparations from two placentas. The PCR amplifications led to the observation of amplicons of the expected size for CaT1 (795 bp, lane 2), CaT2 (448 bp, lane 3), and _1C (671 bp) and _1D (727 bp) of L-type channels (lane 4). The degenerate primers designed for the three isotypes of L-type channels did not permit the observation of an amplicon for the _1S (expected 606-bp amplicon with degenerate primers). Therefore, we performed PCR using specific primers for the _1S subunit. An amplicon of the expected size (544 bp) was obtained with total RNA of BeWo cells used as control (Fig. 3B, lane 3). However, no PCR product was obtained with total RNA from cytotrophoblasts (Fig. 3B, lane 2) or with mRNA from cytotrophoblasts. Specificity of the amplicon corresponding to CaT1 was demonstrated following digestion with SacI, with two digestion fragments of 490 and 305 bp (Fig. 3C, lane 3). Specificity of the amplicon corresponding to CaT2 was demonstrated following digestion with XhoI, with two digestion fragments of 282 and 166 bp (Fig. 3D, lane 3).

View larger version (76K): [in this window] [in a new window]   FIG. 3. Representative results of the expression of Ca2+ channels in freshly isolated cytotrophoblast cells from human term placenta. A) PCR was performed on total RNA with specific primers for CaT1 (lane 2) and CaT2 (lane 3) and with degenerate primers for L-type channel 1 subunits (lane 4). Lane 1: 100-bp ladder. B) PCR was performed on total RNA of cytotrophoblasts (lane 2) or of BeWo cells (lane 3) with specific primers for 1S subunit. Lane 1: 50-bp ladder. C) Amplicon of CaT1 (lane 2) and digested with SacI (lane 3). Lane 1: 100-bp ladder. D) Amplicon of CaT2 (lane 2) and digested with XhoI (lane 3). Lane 1: 50-bp ladder. Independent experiments were performed on cells isolated from two placentas

Expression of Ca²⁺ Channels along the Differentiation of Trophoblasts in Culture

The expression of CaT1, CaT2, and L-type channel _1C and _1D subunits was evaluated by reverse transcription-PCR using mRNA from trophoblasts cultured for 1–6 days. Expression of the _1S subunit was not further evaluated, because PCR performed with mRNA from freshly isolated trophoblasts was negative. Figure 4 shows representative results of PCR amplifications performed on cells isolated from two placentas. The expression of CaT1 mRNA increased on Days 2, 3, and 4 of culture (3.84-, 3.42-, and 3.21-fold, respectively) compared to Day 1 following normalization with the GAPDH expression level. Expression of CaT1 declined thereafter on Day 5 (0.75-fold) and Day 6 (0.06-fold) compared to Day 1. The normalized expression of CaT2 mRNA increased on Days 2 and 3 of culture (1.68- and 5.24-fold, respectively) compared to Day 1 and declined on Day 4 (1.63-fold), Day 5 (0.48-fold), and Day 6 (0.14-fold) of culture (Fig. 5). The expression of both CaT1 and CaT2 declined dramatically on Days 5 and 6, and mRNAs were barely detectable on Day 6. Figure 6 shows that normalized expression of L-type _1C and _1D subunits increased by 3.56-fold on Day 2 and reached a maximum on Day 3 of culture (20-fold) compared to expression on Day 1. The relative expression level of _1C compared to _1D was similar at all days of culture. As for CaT1 and CaT2, the normalized expression of _1C and _1D decreased on Days 4, 5, and 6 compared to the maximum on Day 3 but remained higher than the expression on Day 1 (9.80-, 7.78-, and 8.79-fold, respectively).

View larger version (48K): [in this window] [in a new window]   FIG. 4. Representative results of CaT1 channel expression along the time of culture of trophoblast cells. A) PCR was performed with specific primers for CaT1 or GAPDH on mRNA isolated after 1–6 days of culture (lanes 1–6, respectively). Lane 7: 100-bp ladder. B) Densitometric data of CaT1 expression after normalization with GAPDH expression level. Independent experiments were performed on cells isolated from two placentas

View larger version (42K): [in this window] [in a new window]   FIG. 5. Representative results of CaT2 channel expression along the time of culture of trophoblast cells. A) PCR was performed with specific primers for CaT2 or GAPDH on mRNA isolated after 1–6 days of culture (lanes 1–6, respectively). Lane 7: 100-bp ladder. See Figure 4 for other details

View larger version (41K): [in this window] [in a new window]   FIG. 6. Representative results of L-type channel expression along the time of culture of trophoblast cells. A) PCR was performed with degenerate primers for L-type channel or GAPDH on mRNA isolated after 1–6 days of culture (lanes 1–6, respectively). Lane 7: 100-bp ladder. See Figure 4 for other details

	DISCUSSION

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In the present study, we report, to our knowledge for the first time, the expression of CaT1 and CaT2 in cytotrophoblasts isolated from human term placenta. The specificity of the amplicons for both channels was confirmed by restriction enzyme-generated digestion fragments. These cells differentiated in culture into syncytiotrophoblast during 4 days of culture as indicated by the increase in hCG secretion, confirming previous findings [8]. The hCG secretion was modest during the first 24 h of culture, suggesting that our cytotrophoblast preparations were highly purified, without syncytial fragments. We observed that the expression of both CaT1 and CaT2 increased with days of culture, reaching a maximum between Days 2 and 3. Interestingly, the trophoblast Ca²⁺ uptake potential also increased during differentiation, reaching a maximum on Day 4 of culture. The Ca²⁺ uptake potential of the trophoblasts declined thereafter on Days 5 and 6. Our results indicate that the pattern of CaT1 and CaT2 expression correlates with the Ca²⁺ uptake potential along the differentiation of cultured human trophoblasts isolated from term placenta. This correlation provides circumstantial evidence for a role of this family of channels in basal Ca²⁺ uptake by the syncytiotrophoblast.

Because the primary culture of trophoblasts represents a dynamic cell model that undergoes spontaneous differentiation, investigation of the Ca²⁺ uptake potential and Ca²⁺ channel expression along the differentiation were mandatory to understand the global trophoblast Ca²⁺ regulation. The kinetics of specific Ca²⁺ uptakes by the trophoblasts (without nonspecific Ca²⁺ adsorption [32]) were rapid for the first 2 min of incubation and were followed by establishment of a plateau at all days studied. The initial Ca²⁺ uptake (Vi, up to 2 min) represents the Ca²⁺ uptake potential at the cell membrane level. The establishment of a gradual plateau around 5 min of incubation has been generally viewed as reflecting an equilibrium of the radiolabel. Indeed, the Ca²⁺ uptake studies using whole cells are a reflection of the dynamic homeostasis of cellular Ca²⁺, in which mechanisms of Ca²⁺ entry and exit coexist and result in the maintenance of an adequate intracellular Ca²⁺ level. The level of ⁴⁵Ca²⁺ observed at the plateau reflects the extent of intracellular Ca²⁺ retention, which depends on factors such as binding of Ca²⁺ to intracellular proteins or its sequestration in intracellular membrane compartments. Considering these two aspects (Vi and plateau), we observed that the Ca²⁺ uptake by trophoblasts cultured for 1 and 2 days was similar and relatively low. The trophoblast Ca²⁺ uptake potential increased on Day 3, reaching a maximum on Day 4 of culture. After 4 days of culture, Vi and plateau had increased by 5.34- and 1.84-fold, respectively. Interestingly, the Ca²⁺ uptake potential of the trophoblasts declined on Days 5 and 6. It should be noted that the Ca²⁺ uptake potential of trophoblast primary culture as a function of days of culture correlates with the hCG secretion pattern. The decrease in hCG secretion and Ca²⁺ uptake potential on Days 5 and 6 have not been investigated further. However, apoptotic phenotype progression may be suspected, because differentiation and fusion of trophoblast cells involved a preapoptotic phenotype [33].

Primary cultures of trophoblasts isolated from human term placenta are generally used for experiments after 4 days of culture [8, 9, 11, 12, 29]. In regard to Ca²⁺ uptake, our results indicate that trophoblasts have reached their maximal potential by Day 4 of culture.

Peng et al. [34] recently reported that the relative expression of CaT1 was much higher than that of CaT2 in whole placenta. This quantification was performed using the real-time PCR technique. Considering PCR conditions, intensity of bands, and volume ratio of the same reverse transcription preparation versus total volume of PCR used for CaT1 (4%) and CaT2 (10%), we also observed in the present study a higher expression of CaT1 compared to CaT2. However, further quantitative investigation is warranted to establish the exact relative levels of CaT1 and CaT2 expression in cultured trophoblast. Furthermore, we observed that the expression of both CaT1 and CaT2 increased with days of culture, reaching a maximum between Days 2 and 3. Unfortunately, no antibody is currently available to evaluate the level of both protein expressions. Considering a lapse of time between the maximum expression of mRNA and the translation and membrane localization processes, a delay of a few to several hours must be considered in view of the functional maximum related to the Ca²⁺ uptake. Interestingly, the Ca²⁺ uptake potential of trophoblasts increased by Day 3 of culture, and a maximum was reached on Day 4. Therefore, we observed a correlation of CaT1 and CaT2 expression with the increase in trophoblast Ca²⁺ uptake.

Pharmacological and functional evidence has been previously obtained for the presence of L-type channels in human trophoblasts, mainly in relation to regulation of hormonal secretion [9–12]. However, to our knowledge, characterization of trophoblast L-type Ca²⁺ channels at the molecular level remained to be performed. Here, we report that L-type channel _1C and _1D subunits are expressed by freshly isolated cytotrophoblasts. Expression of the _1S subunit was not revealed with degenerate primers. A lower level of _1S subunit expression compared to that of the other two may have resulted in primer sequestration. Therefore, we performed PCR amplifications with specific primers for the L-type channel _1S subunit. No amplicon was obtained with mRNA of cytotrophoblasts and 3-day cultured trophoblasts. However, these primers revealed expression of the _1S subunit in the trophoblast cell line BeWo. Accordingly, we recently demonstrated by Western blot analysis the presence of L-type channels in BeWo cells [32]. When the expression of L-type channel _1C and _1D subunits was evaluated as a function of days of culture, we observed an increase in the expression of both subunits for all days of culture compared to Day 1. Expression of the L-type channel _1C and _1D subunits was maximal on Day 3 and declined thereafter. Considering also an interval of time between the maximum mRNA expression and the presence of functional Ca²⁺ channels at the cell membrane, a delay of a few to several hours must be considered in view of the maximum Ca²⁺ uptake. The increase of L-type subunit expression correlates with the Ca²⁺ uptake augmentation as a function of days of culture.

Interestingly, the Ca²⁺ uptake plateau on Days 5 and 6 almost declined to the value of Day 1, suggesting that the intracellular Ca²⁺ retention characteristics of the trophoblasts were lost. However, Vi on Days 5 and 6 remained higher than on Day 1, suggesting that the Ca²⁺ uptake potential at the membrane level was decreased compared to Day 4 but remained higher compared to Day 1. Expression of CaT1 and CaT2 had declined after 4 days of culture, and their expression on Days 5 and 6 was similar or lower than their expression on Day 1. Therefore, decreased expression of CaT1 and CaT2 on Days 5 and 6 correlates with concomitant decline of trophoblast Ca²⁺ uptake potential. The L-type channel expression on Days 4–6 remained higher than that on Day 1. However, considering the voltage dependence of activation [13], their involvement in trophoblast basal Ca²⁺ uptake is unlikely. To best define the decline of mRNA expression and Ca²⁺ uptake potential in trophoblasts, the half-life of the respective mRNA and proteins must be investigated in the trophoblast.

The trophoblast differentiation into syncytiotrophoblast implies biochemical differentiation and cell fusion to form a multinucleate, nonmitotic syncytium. The expression of several genes has been reported to be altered during trophoblast differentiation [35, 36]. Our results indicate that the expression of CaT1, CaT2, and L-type _1C and _1D subunits is also modified during trophoblast differentiation. In regard to the regulation of CaT channel expression, limited information is presently available. Expression of CaT1 channel has been reported to correlate with the malignancy of prostate cancer [21, 22]; CaT1 is highly expressed in prostatic carcinoma cells and in metastatic and androgen-insensitive prostatic lesions in comparison to benign prostatic hyperplasia and healthy prostate tissue. Furthermore, the CaT1 message level decreased in the presence of androgen and increased in the presence of a specific androgen-receptor antagonist (Casodex), suggesting hormonal regulation of expression [21]. In the intestinal cell line Caco-2, 1,25-dihydroxyvitamin D₃ increases the expression of CaT1 [37]. In view of the increased expression during differentiation, regulation of CaT channel expression in trophoblast needs further investigation. In regard to L-type channels, alteration of expression has been reported with cell differentiation. Expression of L-type channels in rat aortic vascular smooth muscle cells depends on the state of cell differentiation [38]. Retinoic acid-induced differentiation of the human teratocarcinoma cell line NT2 into NT2N neurons is associated with an increase in L-type channel expression [39]. Interestingly, we previously observed that the trophoblast cell line BeWo did not express L-type channel _1C and _1D subunits but did express the _1S subunit. Accordingly, expression of the _1S isoform was also found in the osteoblast-like transformed cell lines but not in normal osteoblasts [40].

Expression of CaT1/ECaC2 and CaT2/ECaC is mainly restricted to epithelium involved in Ca²⁺ transfer, with the exception of pancreas and prostate [19–26]. The L-type channels are rather more largely expressed by exciting cells and also by nonexciting cells [13]. It is therefore generally believed that the former channels mediate cellular Ca²⁺ uptake as part of the transcellular pathway of Ca²⁺ transport in kidney, intestine, and placenta. The correlation observed in the present study between the trophoblast Ca²⁺ uptake potential and the CaTs expression pattern during differentiation may implicate this family of channels in basal Ca²⁺ uptake by the syncytiotrophoblast and ion transfer to the fetus. Studies using the patch-clamp technique [41] have indicated activity of CaT1 and CaT2 at a membrane potential that could easily correspond to the resting membrane potential of human trophoblast cell culture [42, 43]. In comparison, the opening probability (P_o) of L-type channels would be relatively low at the trophoblast resting potential [13]. Recently, we reported that basal Ca²⁺ uptake by BeWo cells is mainly independent of voltage and is not influenced by L-type Ca²⁺ channel modulators [32], suggesting that L-type Ca²⁺ channel activity appears to be a minor contribution to BeWo cell basal Ca²⁺ uptake. Similar characteristics were observed for the basal Ca²⁺ uptake by 4-day cultured trophoblasts isolated from human term placenta (unpublished observations). We hypothesize that the role of L-type Ca²⁺ channels in the trophoblasts more likely may involve cell signaling and regulation of protein secretion (especially for hCG and placental lactogen). The expression pattern of CaT channels by the trophoblasts during differentiation may characterize the Ca²⁺ transfer potential of the syncytiotrophoblast.

	FOOTNOTES

 
¹ Supported by a grant from the March of Dimes, Saving Babies Together.

² Correspondence: Julie Lafond, Département des Sciences Biologiques, Laboratoire de Physiologie materno-ftale, Université du Québec à Montréal, C.P. 8888, Succursale "Centre-Ville", Montréal, QC, Canada H3C 3P8. FAX: 514 987 4647; lafond.julie{at}uqam.ca

Received: 11 March 2002.

First decision: 12 April 2002.

Accepted: 12 June 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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