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J Physiol (2003), 550.2, pp. 515-528
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.044149

Store-operated Ca²⁺ entry in first trimester and term human placenta

L. H. Clarson*†, V. H. J. Roberts*, B. Hamark‡, A. C. Elliott§ and T. Powell¶

	ABSTRACT

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We have examined whether store-operated Ca²⁺ entry, a common pathway for Ca²⁺ entry in non-excitable tissue, is apparent in the syncytiotrophoblast of both first trimester and term human placenta. Expression of transient receptor potential (TRPC) homologues, a family of channels thought to be involved in store-operated Ca²⁺ entry, was also studied at the mRNA and protein levels. [Ca²⁺]_i in syncytiotrophoblast of first trimester and term placental villous fragments was measured by microfluorimetry using the Ca²⁺-sensitive dye fura-2. Store-operated Ca²⁺ entry was stimulated using 1 µM thapsigargin in Ca²⁺-free Tyrode buffer (no added Ca²⁺ + 1 mM EGTA) followed by superfusion with control (Ca²⁺-containing) buffer. In term fragments, this protocol resulted in a rapid increase in [Ca²⁺]_i, which was inhibited in the presence of 150 µM GdCl₃, 200 µM NiCl₂, 200 µM CoCl₂ or 30 µM SKF96365 but was unaffected by addition of 10 µM nifedipine. It was not possible to stimulate such a rise in [Ca²⁺]_i in first trimester fragments. Messenger RNA encoding TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was identified in both first trimester and term placentas. From Western blotting, TRPC3 and TRPC6 proteins were detected in term, but not in first trimester, placentas, while TRPC1 protein was not detected. By immunocytochemistry, TRPC3 and TRPC4 were localised to cytotrophoblast cells in first trimester placentas and to the syncytiotrophoblast in term placentas. TRPC6 staining was present in the syncytiotrophoblast of both first trimester and term placenta, but the intensity was much greater in the latter. We propose that store-operated Ca²⁺ entry may be an important route for Ca²⁺ entry into the syncytiotrophoblast of term, but not first trimester placentas, and that in human placenta TRPC channels may underlie this entry mechanism.

(Resubmitted 31 March 2003; accepted after revision 23 April 2003; first published online 23 May 2003)
Corresponding author L. H. Clarson: Development, Growth and Function Division, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK. Email: l.clarson{at}rowett.ac.uk

	INTRODUCTION

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The human placenta is a dynamic organ which changes both morphologically and functionally over gestation. The transporting epithelium of the human placenta, the syncytiotrophoblast, is a multinucleate true syncytium, which is formed and replenished throughout gestation by differentiation and fusion of stem cytotrophoblast cells. It is the main site for maternofetal exchange of nutrients as well as production of hormones and autocrine/paracrine regulators of placental function. It has become increasingly apparent that many functions of the syncytiotrophoblast are regulated by [Ca²⁺]_i, including ion transport (K⁺ and Cl^-; Kibble et al. 1996; Clarson et al. 2002b), hormone secretion (human chorionic gonadotropin, hCG; Mathialagan & Rao 1989; Meuris et al. 1994; Polliotti et al. 1994), nitric oxide production (Myatt et al. 1993) and function of transport proteins (e.g. amino acid transporters; Karl et al. 1988; Krishna et al. 1995). However, the mechanisms by which [Ca²⁺]_i is controlled in the syncytiotrophoblast remain poorly understood. In previous studies it has been demonstrated that stimulation of cultured cytotrophoblast cells with agonists such as ATP, UTP and endothelin can raise [Ca²⁺]_i via release of Ca²⁺ from stores and by entry of extracellular Ca²⁺ (Petit & Belisle 1995; Karl et al. 1997; Cronier et al. 1999; Clarson et al. 2002b) but the specific route of entry is yet to be determined.

In other non-excitable tissue a major pathway for Ca²⁺ entry is via store-operated Ca²⁺ channels which are activated following depletion of Ca²⁺ from intracellular stores (also termed depletion-activated or capacitative Ca²⁺ entry; Putney, 1999, 2001). Stimulation by agonists such as ATP and UTP leads to activation of the phospholipase C (PLC) pathway, commonly though not exclusively via the G_q/11 G protein family. Stimulation of PLC results in production of 1,2-diacylglycerol (DAG), as well as inositol-1,4,5-trisphosphate (IP₃), which causes release of Ca²⁺ from IP₃-sensitive stores. Once the stores are depleted of Ca²⁺, Ca²⁺ channels on the plasma membrane are activated to allow entry of Ca²⁺ thereby replenishing stores and maintaining the [Ca²⁺]_i signal (Kiselyov & Muallem 1999; Putney 2001). Although the specific channel or channels underlying this mechanism for Ca²⁺ entry is yet to be identified, there are several candidates, primarily the transient receptor potential channels (TRPCs; Putney, 1999), which make up part of the TRP superfamily (Clapham et al. 2001; Montell et al. 2002). TRPCs are Ca²⁺ permeable and are the mammalian homologues of the trp and trpl channels first identified in Drosophila . Thus far, genes for seven channels have been cloned from human tissue: TRPC1, TRPC4 and TRPC5, which are thought to encode store-operated channels (Sossey-Alaoui et al. 1999; Liu et al. 2000; Philipp et al. 2000); TRPC6 and TRPC7, which appear to encode receptor-operated channels and can be directly activated by IP₃ or DAG (Zhang & Saffen 2001); TRPC3, which seems to act as either a store-operated or receptor-operated channel (Zhu et al. 1996, 1998; Kiselyov et al. 1998); and the pseudogene TRPC2 (Wes et al. 1995).

We have hypothesised that the syncytiotrophoblast of both first trimester and term human placentas exhibits a store-operated Ca²⁺ entry pathway. In order to test this hypothesis we have examined store-operated Ca²⁺ entry using the microfluorimetry technique in intact syncytiotrophoblast of villous fragments from first trimester and term placentas. We have also examined the expression of TRPC channel mRNA in placental tissue from all stages of gestation using RT-PCR, and expression of TRPC channel protein in first trimester and term placentas by both Western blotting and immunocyotchemistry.

Parts of this work have been presented in preliminary form to meetings of the European Placental Group (Clarson & Roberts 2001) and the International Federation of Placental Associations (Clarson et al. 2002a).

	METHODS

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Materials

Thapsigargin, GdCl₃, NiCl₂, CoCl₂ , 1-oleoyl-2-acetyl-sn-glycerol (OAG) and polylysine solution were purchased from Sigma-Aldrich (Poole, UK), pluronic F-127 and fura-2 AM from Molecular Probes Europe (Leiden, The Netherlands). All other reagents were analytical grade from standard suppliers.

Measurements of [Ca²⁺]_i

Tissue preparation and loading. The experimental protocol was based on that of Powell & Illsley (1996). All tissue collection was in accordance with procedures approved by the relevant local ethics committee. Term human placentas (38-41 weeks gestation), from normal uncomplicated pregnancies, were collected following vaginal delivery or Caesarian section. First trimester samples were collected following surgical termination of pregnancy (7-11 weeks gestation) for socio-economic reasons. In both cases, a 1 cm³ piece of villous tissue was removed, washed briefly in physiological saline (pH 7.4, 4 °C) and transferred to RPMI-1640 or Modified Earles' Medium (MEM) for transport.

Tissue was loaded with fura-2 by incubating small pieces of villous tissue ( 2-3 mm³) with control Tyrode buffer (mM: NaCl 135, KCl 5, CaCl₂ 1.8, MgCl₂ 1, glucose 5, Hepes 10; pH adjusted to 7.4 with NaOH) containing 10 µM fura-2 (which was diluted from a 5 mM stock in DMSO containing pluronic F-127 - the final concentration of DMSO and pluronic F-127 was 0.1 %) and 0.1 % BSA. Fragments were incubated for 30 min at 37 °C, rinsed briefly with control Tyrode buffer (37 °C) and washed twice for 15 min with excess control Tyrode buffer at 37 °C.

Microfluorimetry. After dye loading, villous tissue was finely minced with fine scissors in two drops of control buffer on a polylysine-coated glass coverslip. Excess buffer was removed allowing the fragments to immediately lightly attach to the polylysine coating. The coverslip was placed on the microscope stage and then superfused with buffer to remove any unattached pieces of villous tissue. The attached fragments were viewed under bright field to select a terminal villous sample with one cut end for term placenta or a branching mesenchymal villous/syncytial sprout in first trimester placenta. It was also important to ensure there was no movement of the selected fragment with flow of the superfusate. An area on the outer edge of the tissue, visually identified as syncytiotrophoblast, was selected and the field of study was limited using an adjustable diapraghm to include only syncytiotrophoblast. Experiments were performed on a Nikon Diaphot inverted microscope adapted for fluorescent measurement with the Photon Technology International (PTI, Lawrenceville, NJ, USA) M-series photometry system. Excitation light was provided by a 75 W xenon lamp, via two monochromators, at 340 and 380 nm. Emission light was collected by a photomultiplier tube, digitised and stored using PTI software. Fura-2 signals were not calibrated in terms of absolute values of [Ca²⁺]_i, since the accuracy of such estimates is debatable (Williams & Fay 1990) and since calibration was not necessary for the analysis of the data.

Protocol. In order to stimulate depletion-activated Ca²⁺ entry, villous fragments were superfused with Ca²⁺-free Tyrode buffer (composition as control buffer but with no added CaCl₂ and 1 mM EGTA) for 5 min, after which 1µM thapsigargin was applied for 2 min in Ca²⁺-free buffer to deplete stores and inhibit the sarco(endo)plasmic reticulum (SERCA) Ca²⁺-ATPase. The tissue was returned to Ca²⁺-free buffer alone for a further 2 min, before being superfused with control Ca²⁺-containing Tyrode buffer in the presence or absence of channel blockers (150 µM GdCl₃, 200 µM NiCl₂, 200 µM CoCl₂, 30 µM SKF96365 and 10 µM nifedipine). Channel blockers were applied for the last minute of superfusion in Ca²⁺-free buffer prior to Ca²⁺ repletion.

In separate experiments, we attempted to stimulate voltage-operated Ca²⁺ channels (VOCC) by depolarisation of the syncytiotrophoblast microvillous membrane. Fragments were exposed to 25 mM and 50 mM KCl after which they were returned to control buffer. Finally, term villous fragments were also exposed to 100 µM OAG in control Tyrode buffer. OAG is a cell-permeant analogue of DAG which can stimulate TRPC3 and TRPC6 channels (Hofman et al. 1999). The effect of addition of DMSO alone to control buffer was also examined to serve as a vehicle control for these experiments, since DMSO was the vehicle for OAG.

Expression of TRP channel mRNA

Total RNA was isolated from seven first trimester, five second trimester and eight term placentas using the method described by Chomczynski & Sacchi (1987). First trimester placentas were collected following surgical termination of pregnancy for socio-economic reasons; second trimester and term placentas were obtained within 30 min following vaginal delivery or Caesarian section. Villous tissue (approximately 1 g) was homogenised in 10 ml of 4 M guanidinium thiocyanate buffer within 15 min of collection and stored at -80 °C until required. The integrity of all RNA samples was confirmed by the presence of discrete 28S and 18S ribosomal RNA bands following electrophoresis through a 1.2 % agarose-6.3 % formaldehyde gel.

For PCR, human placental, human brain (BD Biosciences Clontech, Palo Alto, CA, USA) and human small intestine (Clontech) total RNA samples were reverse transcribed to cDNA. Total RNA samples (5 µg) were heat-denatured prior to the RT reaction. Each reaction was performed in the presence of 400 nM oligo dT₁₆ primer (Invitrogen), 10 mM dithiothreitol (DTT), 1 mM of each deoxyribonucleotide, 30 u 'RNA guard' ribonuclease inhibitor (Amersham Pharmacia Biotech UK Ltd), 1 reaction buffer (Invitrogen) and 200 u M-MLV reverse transcriptase (Invitrogen) in a total volume of 20 µl for 1 h at 37 °C. The reactions were terminated by incubating at 75 °C for 10 min.

PCR was performed on 2 µl cDNA from placental samples or from cDNA obtained commercially (positive control samples). A negative control was included in each PCR reaction where cDNA was replaced with ultrapure water. Each reaction was performed in the presence of 500 nM 5' and 3' gene-specific primers (see Table 1), 200 µM of each deoxyribonucleotide, the appropriate Mg²⁺ concentration as determined for each primer pair (see Table 1), 1 reaction buffer (Promega, Southampton, UK) and 0.5 u Taq DNA polymerase (Promega, Southampton, UK) in a total volume of 20 µl. The reaction mixtures were denatured for 2.5 min at 95 °C and then amplified in 35 sequential cycles, consisting of the following steps: 95 °C for 1 min, 1 min at the optimum annealing temperature for each primer pair (see Table 1) and 72 °C for 2 min. After the last cycle, the samples were incubated at 72 °C for 5 min to ensure cDNA extension. Amplified products were electrophoresed through a 1.2 % agarose gel containing 0.5 µg ml^-1 ethidium bromide in 1 TAE (40 mM Tris, 5.7 % acetic acid, 1 mM EDTA) to determine product size.

The identity of the RT-PCR products was confirmed by restriction enzyme digest. PCR products were eluted from the agarose gel using the Qiagen Qiaex kit and incubated with restriction enzymes chosen to cut at one site within the product length (PstI: TRP1, 3; PvuII: TRP4; HindIII: TRP5, 6), for 1 h at 37 °C. Cut products were electrophoresed through a 1.2 % agarose gel to confirm product sizes. Finally, PCR products were also confirmed by sequencing. This was carried out by the Sequencing Service Department at the Manchester University Medical School. The nucleotide sequence was determined and product specificity was checked by entering each sequence into the BLAST facility of the NCBI site.

Expression of TRP channel protein

Western blotting. A membrane-enriched homogenate was prepared from human term and first trimester placental tissue (collected as described above). Villous tissue was washed briefly in phosphate-buffered saline (PBS; 4 °C) and homogenised on ice in 10 ml buffer A (12 mM Hepes, 300 mM mannitol pH 7.6 with saturated Tris) containing 0.1 mM of the serine protease inhibitor 4-[2-aminoethyl]-benzenesulfonylflouride hydrochloride (AEBSF - 100 mM stock in dH₂O; ICN Biomedicals, Basingstoke, UK). Homogenised tissue was centrifuged at 2500 g for 15 min at 4 °C, and the supernatant was removed and recentrifuged at 100 000 g for 45 min at 4 °C. The final pellet was resuspended in buffer A containing AEBSF, passed through a 21 gauge needle to shear the membranes, and stored at -80 °C until required. Protein content was evaluated using a Bio-Rad protein microassay (Bio-Rad Laboratories, Hemel Hempstead, UK).

For Western blotting, protein was heat-denatured at 95 °C for 5 min with 2-mercaptoethanol and separated out on an SDS-PAGE mini-gel electrophoresis system at 120 V for 70 min using a 3 % stacking gel and 6.5 % (TRP1) or 7.5 % (TRP3, TRP6 and -actin) resolving gel. Protein was transferred to nitrocellulose membranes by semi-dry electrophoretic transfer (trans-blot SD semi-dry transfer cell; Bio-Rad Laboratories). Following transfer, membranes were blocked overnight with PBS containing 5 % powdered milk and 0.025 % sodium azide.

Nitrocellulose membranes were incubated with affinity-purified polyclonal antibodies to TRPC1, TRPC3 and TRPC6 (1:200; Alomone labs, Jerusalem, Israel) for 2 h at room temperature in PBS containing 5 % milk protein and 0.025 % sodium azide. The anti-TRPC1 antibody was raised against the human peptide sequence, while anti-TRPC3 and TRPC6 antisera were raised against the mouse peptide sequences, which are highly homologous to human (13/14 and 15/16 residues identical, respectively). Membranes were also incubated with a mouse monoclonal antibody to -actin (1:2500; Sigma, Poole, UK) for 1 h at room temperature in Tris-buffered saline (TBS)-0.1 % TWEEN (polyoxyethylene sorbiton monolaurate; Sigma), containing 5 % milk protein. In order to confirm specificity, membranes were also incubated in the absence of primary antibody (negative control) or following antibody preabsorption (preabsorption control) with the specific immunising peptides (Alomone Labs). For preabsorption, TRPC1, TRPC3 and TRPC6 antibodies were incubated with 1 µg peptide per 1 µg antibody for 1 h at room temperature prior to incubation with the membrane as described above. Membranes were then incubated with either HRP-conjugated goat anti-rabbit antibody (TRPC1, TRPC3, TRPC6 or negative control - 1:8000 in PBS containing 5 % non-fat milk protein (Marvel; Sigma) or HRP-conjugated sheep anti-mouse antibody (-actin - 1:1000 in TBS/0.1 % TWEEN containing 5 % non-fat milk protein) for 1 h at room temperature. Signals were visualised using an enhanced chemiluminescence detection system (ECL; Amersham Bioscience Ltd) and detected on photo-sensitive film (hyperfilm-ECL; Amersham Bioscience Ltd).

Immunocytochemistry. First trimester and term placental tissue, samples were rinsed in ice cold physiological saline and fixed in zinc buffer for 10 h (zinc buffer: 2.8 mM calcium acetate, 23 mM zinc acetate, 37 mM ZnCl₂ in 0.1 M Tris buffer pH 6.8). Fixed tissue was rinsed in PBS and dehydrated through graded ethanol to xylene, embedded in paraffin, sectioned to 4 µm thickness and mounted on positively charged slides (Superfrost plus, Menzel-Gläser, Germany). Tissue was deparaffinised by heating the slide to 60 °C for 20 min and then passing though xylene, graded ethanol and then deionised water to rehydrate the tissue. Slides were boiled in 10 mM citrate buffer pH 6.0 for 10 min and then allowed to cool to room temperature for 30 min. After washing the slides in PBS, tissue was blocked in blotto (4 % non-fat milk protein, 4 % horse serum in PBS) for 60 min at room temperature. Primary antibodies (TRPC3, TRPC4 and TRPC6) were diluted in 1:50 blotto and incubated with tissue sections in a moisture chamber for 60 min at room temperature. Primary antibodies were detected using a Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA). Essentially, slides were washed in PBS then incubated with the biotinylated secondary antibody (anti-rabbit IgG) for 45 min at room temperature. The secondary antibody was rinsed off with PBS and the ABC reagent was added and incubated for 45 min at room temperature. Finally slides were treated with 3,5-diaminobenzidine and incubated at room temperature until a black product appeared. Slides were then washed in PBS, dehydrated through graded ethanol, cleared in xylene and mounted.

Data analysis and statistics

The change in fura-2 fluorescence ratio (R) was calculated as the percentage difference in ratio in Ca²⁺-free buffer to the peak response in Ca²⁺-containing control buffer ± blocker. All data were expressed as median ± range, with n given as number of villous fragments examined (the number of different placentas used is also given). Data were analysed statistically using either the non-parametric Kruskal-Wallis test followed by a Dunn's multiple comparisons test or by a Mann-Whitney U test.

	RESULTS

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Measurement of [Ca²⁺]_i

Term placenta. When term fragments were superfused with Ca²⁺-free Tyrode buffer there was a rapid decrease in the 340:380 fluorescence ratio. Application of 1 µM thapsigargin caused a variable change in signal ratio, ranging from a small peak increase (for example Figs 1A, 2A, 3B and 3C), to no apparent change (for example Fig. 7). This variablility might be due to variable store depletion following a 5 min exposure to Ca²⁺-free medium. Following thapsigargin application, re-addition of extracellular Ca²⁺ caused a rapid increase in fluorescence ratio in 25 out of 28 fragments (see examples Figs 1, 2 and 3A) indicative of depletion-activated Ca²⁺ entry. Such a response was not seen in the absence of thapsigargin suggesting Ca²⁺ entry required store depletion (Fig. 1B). Overall, Ca²⁺ repletion after thapsigargin treatment increased the fluorescence ratio by 8.4 % (0.94-20 %) (median and range, n = 10, Fig. 2C), 14.8 % (4.18-44 %) (median and range n = 12, Fig. 3D) and 7.8 % (2.44-13.81 %) (median and range n = 6, Fig. 4D) in three different series of experiments. In the first series of experiments, 150 µM Gd³⁺, a blocker of Ca²⁺-permeable channels, significantly reduced the Ca²⁺ repletion response (P = 0.0147 Mann-Witney U test; see Fig. 2). Two other inorganic Ca²⁺ channel blockers, NiCl₂ and CoCl₂, had similar effects, as 200 µM NiCl₂ or CoCl₂ significantly reduced the Ca²⁺ repletion response (P < 0.001 and P < 0.01, respectively, Kruskal-Wallis with Dunn's multiple comparisons test; Fig. 3). Addition of 30 µM SKF96365 also reduced the store-mediated Ca²⁺ entry, although the effect was less pronounced than with the inorganic blockers (P = 0.052 Mann-Whitney U test), but addition of 10 µM nifedipine was without effect (Fig. 4).

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Figure 1. Store-mediated Ca2+ entry in Fura-2-loaded term fragments A, store-mediated Ca2+ entry was stimulated by exposure of the tissues to Ca2+-free buffer in the presence or absence of 1 µM thapsigargin (tg) and then returning tissue to control buffer. R, change in 340:380 fluorescence ratio. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. B, averaged data on the percentage change in 340:380 ratio. All data are given as median ± range; + thapsigargin, n = 16, 15 placentas; - thapsigargin, n = 4, 4 placentas. ** P < 0.01 Mann-Whitney U test.

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Figure 2. Effect of 150 µM GdCl3 on changes in [Ca2+]i evoked by store-depletion in fura-2-loaded term fragments Store-operated Ca2+ entry was stimulated by exposure of the tissues to Ca2+-free buffer in the presence of 1 µM thapsigargin. The preparation was then returned to A, control buffer or B, control buffer in the presence of 150 µM GdCl3. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. C, averaged data on the percentage change in 340:380 ratio. All data are given as median ± range; control, n = 10, 10 placentas; control + GdCl3, n = 10, 10 placentas. * P < 0.05 Mann-Whitney U test.

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Figure 3. Effect of 200 µM CoCl2 and NiCl2 on changes in [Ca2+]i in fura-2-loaded term fragments Store-operated Ca2+ entry was stimulated by exposure to Ca2+-free buffer in the presence of 1 µM thapsigargin then returned to A, control buffer, B, control buffer in the presence of 200 µM CoCl2 or C, control buffer in the presence of 200 µM NiCl2. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. D, combined data for percentage change in 340:380 ratio. All data are give as median ± range; control, n = 12, 9 placentas; CoCl2, n = 9, 4 placentas; NiCl2, n = 7, 5 placentas. *** P < 0.001 vs. control, ** P < 0.01 vs. control, Kruskal-Wallis with Dunn's multiple comparisons test.

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Figure 4. Effect of 10 µM nifedipine and 30 µM SKF96365 on changes in [Ca2+]i in fura-2-loaded term fragments Store-operated Ca2+ entry was stimulated by exposure to Ca2+-free buffer in the presence of 1 µM thapsigargin then returned to A, control buffer, B, control buffer in the presence of 10 µM nifedipine or C, control buffer in the presence of 30 µM SKF96365. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. D, combined data for percentage change in 340:380 ratio. All data are give as median ± range, control, n = 6, 5 placentas; nifedipine, n = 7, 6 placentas; SKF96365, n = 5, 5 placentas. P = 0.052 vs. control, Mann-Whitney U test.

Depolarisation of the syncytiotrophoblast plasma membranes in term fragments. The plasma membrane of term syncytiotrophoblast was depolarised by addition of 25 mM and then 50 mM KCl to control buffer, in order to directly stimulate any voltage-gated Ca²⁺ channels present. Addition of neither 25 mM nor 50 mM KCl caused any increase in [Ca²⁺]_i (Fig. 5). On returning the fragment to control buffer, however, a spike increase in [Ca²⁺]_i was evident in six out of seven fragments.

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Figure 5. Effect of 25 and 50 mM KCl on fura-2-loaded term fragments A, tissue was stimulated by addition of 25 mM and then 50 mM KCl to control Tyrode buffer; the response after returning the fragment to control buffer is also shown. B, averaged data for percentage change in 340:380 ratio. All data are given as median ± range; 25 mM or 50 mM KCl, n = 7, 5 placentas; control buffer after KCl, n = 6, 5 placentas.

Stimulation of term fragments with OAG. Term villous fragments were stimulated with 100 µM OAG, a non-metabolisable analogue of DAG, which has previously been shown to directly stimulate TRPC3 and TRPC6 channels (Hofmann et al. 1999). Following addition of 100 µM OAG, there was an increase in fluorescence ratio of 16.1 % (-0.6 to 67.7 %) (median and range, n = 12; see Fig. 6). As OAG was dissolved in DMSO, we also tested the effect of the vehicle alone, which had no stimulatory effect (-2.9 %, -7.4 to 0.6 %, median and range, n = 6).

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Figure 6. Effect of 100 µM OAG on fura-2-loaded term fragments A, tissue was stimulated by addition of 100 µM OAG to control Tyrode buffer; an example of the term response seen following addition of the DMSO vehicle alone is also shown. The open bar indicates application of 100 µM OAG or DMSO vehicle. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. B, averaged data for percentage change in 340:380 ratio. All data are given as median ± range; 100 µM OAG, n = 12, 6 placentas; DMSO, n = 6, 4 placentas. ** P < 0.01, Mann-Whitney U test

Term vs. first trimester placenta. Superfusion with Ca²⁺-free Tyrode solution rapidly reduced the 340:380 fluorescence ratio in first trimester placental villous fragments, similar to results with term tissue. Addition of 1 µM thapsigargin to first trimester tissue produced a variable response, as also seen in term tissue. However, in contrast to term tissue, in first trimester placenta Ca²⁺ repletion following application of thapsigargin caused only a small increase in fluorescence ratio (3.1 %, -3.0 to 9.34 %, median and range, n = 23, Fig. 7). The response was significantly lower than that for term fragments (P < 0.0001 Mann-Whitney U test, Fig. 3C). Indeed, the response to control Tyrode solution in first trimester fragments was not statistically different from that seen in term fragments in the absence of thapsigargin or following application of Ni²⁺ or Co²⁺ (1.86 %, 0.14-2.93 % n = 4, 2.03 %, -1.0 to 3.15 % n = 7 and 3.84 %, -0.92 to 5.68 %, n = 9, respectively, median and range), suggesting that first trimester tissue has little or no depletion-activated Ca²⁺ entry.

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Figure 7. Comparison of store-operated Ca2+ entry in fura-2-loaded term and first trimester fragments Store-operated Ca2+ entry was stimulated by Ca2+ depletion-repletion exactly as in Figs 1 and 2 in A, term fragments or B, first trimester fragments. Arrows indicate the point of measurement for assessment of the percentage change in 340:380 ratio. C, combined data for percentage change in 340:380 ratio. All data are given as median ± range; term tissue, n = 12, 9 placentas; first trimester tissue, n = 23, 6 placentas. *** P < 0.001, Mann-Whitney U test.

Expression of TRP channel mRNA and protein

Expression of mRNA. Expression of mRNA for TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was examined in first trimester, second trimester and term placenta by RT-PCR. PCR products of the expected size were obtained for each primer pair (Fig. 8), and confirmed as TRPC fragments by restriction digests and sequencing of PCR products (data not shown). A strong signal was obtained for TRPC1, TRPC3 and TRPC6 in all three placental groups, as shown in Fig. 8A, B and E. The signal for the TRPC5 product in all placental samples was very weak, although a relatively strong signal for the positive control tissue (human brain) was evident, suggesting the PCR amplification had been successful (Fig. 8D). The signal with TRPC4 gene-specific primers was more variable, with only a faint product present in the first trimester sample. Once again, a strong signal was evident in the positive control sample (human brain), second trimester and term placenta (Fig. 8C).

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Figure 8. RT-PCR detection of TRPC mRNA RT-PCR was performed using gene-specific primers for A, TRPC1, B, TRPC3, C, TRPC4, D, TRPC5, E, TRPC6 and F, -actin. PCR product sizes are indicated. L, 100 bp ladder; -, negative control (H2O); SI, small intestine (Clontech); HB, human brain (Clontech); I, first trimester placenta (n = 7 pooled); II, second trimester placentas (n = 5 pooled); III, term placentas (n = 8 pooled).

Expression of protein. We went on to assess expression of TRPC1, TRPC3 and TRPC6 protein by Western blotting, using rat brain as a positive control for each antibody. Western blotting with -actin was also carried out to confirm that the protein samples were intact and to confirm equal loading of samples. All three antibodies gave bands of the expected size with the rat brain samples (218 kDa, TRPC1; 130 kDa, TRPC3; 100 kDa + 45 kDa, TRPC6). We also attempted to assess TRPC4 protein levels by Western blotting, but could not obtain reproducible results with the commercial anti-TRPC4 polyclonal antibody. Neither first trimester nor term placental homogenate samples expressed detectable TRPC1 protein, although TRPC1 was clearly detected in the rat brain positive control tissue (Fig. 9A and B). Incubation of the placental homogenate with antibodies to both TRPC3 and TRPC6 revealed bands of 43 and 60 kDa, respectively, for human term samples. Whilst these molecular weights were different from those seen in rat brain, the bands disappeared following preabsorption with peptide-specific antigens (Fig. 9C and E). No bands were evident on the negative control where the primary antibody was absent (data not shown). Tissue from four first trimester and term placental samples was examined to assess whether expression was altered with gestation. Both TRPC3 and TRPC6 were expressed in term tissue but could not be detected in first trimester tissue (see Fig. 9D and E).

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Figure 9. Western blotting for TRPC1 (A, B), TRPC3 (C, D) and TRPC6 (E, F) Protein sizes are given. Preabsorption of each primary antibody with antigen (Ag) shows removal of specific bands (A, C, E). ECL detection was for 30 min for preabsorption studies and 1 h for comparison of first trimester and term tissue. RB, rat brain (positive control tissue); T, term placenta; F, first trimester placenta. In B, D, F, and G each lane corresponds to samples prepared from four different first trimester and term placentas.

Immunocytochemistry

We localised TRPC3, TRPC4 and TRPC6 protein in both first trimester and term placenta by immunocytochemistry. Both TRPC3 and TRPC4 were mainly located in the cytotrophoblast cells underlying the syncytiotrophoblast in first trimester tissue, with faint staining of the syncytiotrophoblast cytosol. There was no apparent staining of the microvillous membrane of the first trimester syncytiotrophoblast for either TRPC3 or TRPC4 (Fig. 10A and C). By contrast, in term tissue, staining with TRPC3 and TRPC4 was localised to the syncytiotrophoblast, with patches of staining identifiable in both the microvillous and basal plasma membranes (Fig. 10B and D). TRPC6 staining was evident in the syncytiotrophoblast of both first trimester and term tissue. However, the intensity was much greater in the latter and, in first trimester syncytiotrophoblast, TRPC6 staining was confined to the cytosol (Fig. 10E and F). In all cases, there was no staining in the negative control (no addition of primary antibody; Fig. 10G and H).

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Figure 10. Immunocytochemistry showing localisation for TRPC3, TRPC4 and TRPC6 in first trimester (A, C and E) and term placenta (B, D and F) Negative controls for first trimester (G) and term placenta (H) are also shown. Samples were magnified 1000; a scale bar is given in H. S, syncytiotrophoblast; C, cytotrophoblast; VC, villous core; M, microvillous membrane; B, basal membrane.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Depletion-activated entry

In electrically non-excitable cells, store-operated Ca²⁺ entry is one of the main routes for entry of extracellular Ca²⁺ (Luckhoff & Clapham 1994; Putney 1997, 2001; Ng & Gurney 2001). Although this mechanism has not previously been measured directly in human placenta, several studies have suggested it could be present. In primary culture of cytotrophoblast cells, isolated from human term placenta and used as a model of the syncytiotrophoblast, stimulation with extracellular ATP, UTP and endothelin produces a biphasic increase in [Ca²⁺]_i (Petit & Belisle 1995; Karl et al. 1997; Cronier et al. 1999; Clarson et al. 2002b), which is dependent on both release of Ca²⁺ from intracellular stores and entry of extracellular Ca²⁺. Whilst the pathway for Ca²⁺ entry was not determined in any of these studies, it was suggested to be a voltage-independent, store-operated channel. Robidoux et al. (2000) also suggested, on the basis of pharmacological data, that store-operated Ca²⁺ channels were present in cultured cytotrophoblast cells. The data in the present study demonstrate directly for the first time that store-operated Ca²⁺ entry does indeed occur in human term placenta, and suggest that it may be a key pathway for Ca²⁺ entry into the syncytiotrophoblast and one which is developmentally regulated.

Previous investigations have suggested that VOCCs could underlie Ca²⁺ entry into the syncytiotrophoblast of term placenta (Meuris et al. 1994; Cemerikic et al. 1998; Moreau et al. 2001, 2002). However, this is controversial, since other studies, which have examined the presence of these channels in human placenta by Ca²⁺ measurements or whole-cell recording, could not identify a functional role for VOCCs in the syncytiotrophoblast (Bax et al. 1994; Cronier et al. 1999; Robidoux et al. 2000). We were unable to find any evidence for the presence of VOCCs, since depolarisation did not raise [Ca²⁺]_i, and nifedipine did not reduce depletion-activated Ca²⁺ entry. This supports the view that VOCCs do not have a functional role in the syncytiotrophoblast of human placenta. A spike increase in [Ca²⁺]_i was evident on returning the fragment to control buffer, but this was probably due to osmotic changes or hyperpolarisation of the membrane when high external K⁺ was removed. Other studies have proposed a role for receptor-operated Ca²⁺ channels (Bax et al. 1994). However, the rise in [Ca²⁺]_i seen in this study after depleting stores directly using thapsigargin cannot be attributed to receptor-operated channels. Indeed in the absence of thapsigargin we were unable to stimulate any marked increase in [Ca²⁺]_i. Nontheless, it remains possible that the same Ca²⁺ entry channel or channels might underlie both receptor-operated and store-operated entry.

In first trimester human placenta, we were unable to demonstrate store-mediated Ca²⁺ entry into the syncytiotrophoblast. Interestingly, our previous preliminary studies also revealed that the [Ca²⁺]_i response to extracellular ATP was significantly smaller in the syncytiotrophoblast of first trimester placentas than term placentas (Clarson et al. 2002a). These data together suggest that [Ca²⁺]_i homeostasis shows clear differences in first trimester compared to term placentas. Notwithstanding the present study, first trimester placentas are likely to be able to elevate [Ca²⁺]_i, since Ca²⁺-dependent secretion of hormones, such as hCG, is evident at this stage (Mathialagan & Rao 1989; Sharma & Rao 1992, 1993). It has been suggested that this increase in [Ca²⁺]_i is from entry of Ca²⁺ through voltage-operated Ca²⁺ channels (Meuris et al. 1994; Sharma & Rao, 1997; Cemerikic et al. 1998). However, we were unable to identify any evidence for voltage-operated Ca²⁺ entry into the syncytiotrophoblast of term placenta in this study, and it seems unlikely that this would be a route for Ca²⁺ entry into first trimester tissue. This leaves receptor-operated Ca²⁺ entry as an untested possibility.

Expression of TRPC channels

Whilst store-operated Ca²⁺ entry is now well established as one of the main routes for Ca²⁺ entry in non-excitable tissue, the channel or channels underlying this pathway have not been conclusively identified, although several molecular candidates have been proposed. We therefore examined the expression, at both the mRNA and protein levels, of TRPC channels, a family of proteins which are thought to be involved in store-operated Ca²⁺ entry (Putney 1999; Clapham et al. 2001; Montell et al. 2002). We found that both first trimester and term human placenta expressed mRNA for TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6. Expression of TRPC5 was extremely low relative to the others, perhaps unsurprisingly as TRPC5 expression is generally associated with brain and neural tissue (Sossey-Alaoui et al. 1999) and placental tissue is not innervated. We did not investigate expression of TRPC2 mRNA since this is a pseudogene in humans and therefore cannot encode a functional channel (Wes et al. 1995).

At the protein level we examined expression of TRPC1, TRPC3, TRPC4 and TRPC6 and found that term placenta expressed TRPC3, TRPC4 and TRPC6 but not TRPC1. This is the first study demonstrating the expression of any TRPC channel protein in human placenta. From Western blotting, the band sizes identified for TRPC3 and TRPC6 protein in human term placenta were different from those for rat brain positive control tissue. However, both the bands in rat brain and human placental samples were absent following preabsorption with peptide-specific antigens suggesting specificity of the signal. The difference in size may be species specific; for instance TRPC1 expression in human tissue produces a protein of ~92 kDa (Wang et al. 1999) compared to that given for rat brain ~300 kDa (Alomone Labs). There is also variablility in protein size between tissue types (Brereton et al. 2000; Zhang & Saffen 2001), which may be due to splice variants (Sakura & Ashcroft 1997; Ohki et al. 2000; Zhang & Saffen, 2001). It is possible, therefore, that in human placenta we have identified a splice variant or truncated form of TRPC3 and TRPC6. The lack of TRPC1 protein expression is perhaps not surprising as previous studies using Northern multiblots demonstrated only very weak bands for TRPC1 in human placenta (Wes et al. 1995; Zhu et al. 1995). Interestingly we obtained a strong TRPC1 band following RT-PCR using gene-specific primers. There are known to be at least four spliced variants for TRPC1 (Sakura & Ashcroft, 1997), so it is possible that we identified a splice variant using RT-PCR, which cannot be functionally expressed at the protein level.

TRPC3 and TRPC6 protein could not be identified in the plasma membranes of first trimester placenta using Western blotting. When this was investigated further by immunocytochemistry, it was evident that TRPC3 and TRPC6 could be detected in the syncytiotrophoblast of first trimester placenta. However, the staining was weak and localised to the cytosol, rather than being evident on the microvillous or basal membrane as in term placenta. Immunocytochemistry showed TRPC4 expression clearly localised to the cytotrophoblast cells in first trimester placenta, but expression then switched to the syncytiotrophoblast at term. A similar pattern was evident with TRPC3, but to a lesser extent. These data suggest that first trimester placenta has the ability to encode these channel proteins but possibly requires a post-translational switch in order to express them in a functional form in the plasma membrane of the syncytiotrophoblast.

In other cells and tissues it has been suggested that TRPC3 and TRPC6 are associated with receptor-operated, rather than store-operated, Ca²⁺ entry (Boulay et al. 1997; Zitt et al. 1997; Hofmann et al. 1999; Liu et al. 2000; McKay et al. 2000), although there is also evidence that TRPC3 may be able to function as both a store-operated and receptor-operated channel (Kiselyov et al. 1998; Thyagarajan et al. 2001; Wu et al. 2000; Zhu et al. 1996; Zhu et al. 1998). Both TRPC3 and TRPC6 are stimulated directly by DAG, which is produced on stimulation of PLC-coupled receptors, and its analogues (Hofmann et al. 1999). In this study we were able to directly stimulate an increase in [Ca²⁺]_i with 100µM OAG, a membrane-permeant analogue of DAG, which is consistent with functional expression of TRPC6 and TRPC3. However, the stimulation of Ca²⁺ entry by thapsigargin treatment clearly implies the existence of a store-operated Ca²⁺ entry pathway. This could conceivably involve TRPC3. However, we also detected TRPC4, which along with TRPC1 and TRPC5, is thought to encode a store-operated Ca²⁺ channel (McKay et al. 2000; Philipp et al. 2000). In this study TRPC4 was clearly evident on the plasma membranes of the syncytiotrophoblast in term but not first trimester tissue. Therefore, this channel protein could potentially be responsible for the store-mediated Ca²⁺ entry mechanism apparent in term tissue but absent in first trimester tissue.

In summary, we have shown that the syncytiotrophoblast of human term placenta possesses store-operated Ca²⁺ entry, and expresses TRPC3, TRPC4 and TRPC6 protein. We also showed that store-operated Ca²⁺ entry was not evident in the syncytiotrophoblast of first trimester placentas. Interestingly, we were unable to identify TRPC3, TRPC4 or TRPC6 protein in the plasma membranes of first trimester syncytiotrophoblast. We therefore propose that there may be a gestationally regulated switch for expression of functional TRPC channels in human placenta and, that these channels could be involved in store-operated Ca²⁺ entry in term human placental syncytiotrophoblast.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Bax C, Bax B, Bain M & Zaidi M (1994). Ca2+ channels in human term trophoblast cells in vitro. A study using the Ca2+-sensitive dye FURA 2. Trophoblast Res 8, 573-580
Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E & Birnbaumer L (1997). Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 272, 29672-29680	[Abstract/Full Text]
Brereton HM, Harland ML, Auld AM & Barritt GJ (2000). Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes. Mol Cell Biochem 214, 63-74	[Medline]
Cemerikic B, Zamah R & Ahmed MS (1998). Identification of L-type calcium channels associated with kappa opioid receptors in human placenta. J Mol Neurosci 10, 261-272	[Medline]
Chomczynski P , & Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159	[Medline]
Clapham DE, Runnels LW & Strubing C (2001). The TRP ion channel family. Nat Rev Neurosci 2, 387-396	[Medline]
Clarson LH, Hamark B & Powell T (2002a). Intracellular Ca2+ homeostasis in the syncytiotrophoblast of villous fragments from first trimester and term placentas. Placenta 23, A17
Clarson LH , & Roberts VHJ (2001). Expression of Ca2+-permeable transient receptor potential (TRP) channel proteins in human placenta and trophoblast cells. Placenta 22, A43
Clarson LH, Roberts VH, Greenwood SL & Elliott AC (2002b). ATP-stimulated Ca(2+)-activated K(+) efflux pathway and differentiation of human placental cytotrophoblast cells. Am J Physiol Regul Integr Comp Physiol 282, R1077-1085	[Abstract/Full Text]
Cronier L, Dubut A, Guibourdenche J & Malassine A (1999). Effects of endothelin on villous trophoblast differentiation and free intracellular calcium. Trophoblast Res 13, 69-86
Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T & Schultz G (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259-263	[Medline]
Karl PI, Chang B & Fisher SE (1988). Calcium-sensitive uptake of amino acids by human placental slices. Pediatr Res 23, 9-13	[Abstract]
Karl PI, Chusid J, Tagoe C & Fisher SE (1997). Ca2+ flux in human placental trophoblasts. Am J Physiol 272, C1776-1780	[Medline]
Kibble JD, Greenwood SL, Clarson LH & Sibley CP (1996). A Ca2+-activated whole-cell Cl- conductance in human placental cytotrophoblast cells activated via a G protein. J Membr Biol 151, 131-138	[Medline]
Kiselyov K , & Muallem S (1999). Fatty acids, diacylglycerol, Ins(1,4,5)P3 receptors and Ca2+ influx. Trends Neurosci 22, 334-337	[Medline]
Kiselyov K, Xu X, Mozhayeva G, Kuo T, Pessah I, Mignery G, Zhu X, Birnbaumer L & Muallem S (1998). Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478-482	[Medline]
Krishna BR, Gude NM, King RG & Brennecke SP (1995). Calcium dependence of alpha-aminoisobutyric acid transfer in human bilaterally perfused placental lobules. J Perinat Med 23, 343-351	[Medline]
Liu X, Wang W, Singh BB, Lockwich T, Jadlowiec J, O'Connell B, Wellner R, Zhu MX & Ambudkar IS (2000). Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells. J Biol Chem 275, 3403-3411	[Abstract/Full Text]
Luckhoff A , & Clapham DE (1994). Calcium channels activated by depletion of internal calcium stores in A431 cells. Biophys J 67, 177-182	[Abstract]
Mathialagan N , & Rao AJ (1989). A role for calcium in gonadotrophin-releasing hormone (GnRH) stimulated secretion of chorionic gonadotrophin by first trimester human placental minces in vitro. Placenta 10, 61-70	[Medline]
McKay RR, Szymeczek-Seay CL, Lievremont JP, Bird GS, Zitt C, Jungling E, Luckhoff A & Putney JW JR (2000). Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3. Biochem J 351, 735-746	[Medline]
Meuris S, Polliotti B, Robyn C & Lebrun P (1994). Ca2+ entry through L-type voltage-sensitive Ca2+ channels stimulates the release of human chorionic gonadotrophin and placental lactogen by placental explants. Biochim Biophys Acta 1220, 101-106	[Medline]
Montell C, Birnbaumer L & Flockerzi V (2002). The TRP channels, a remarkably functional family. Cell 108, 595-598	[Medline]
Moreau R, Daoud G, Bernatchez R, Simoneau L, Masse A & Lafond J (2002). Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta. Biochim Biophys Acta 1564, 325-332	[Medline]
Moreau R, Simoneau L & Lafond J (2001). Characteristics of calcium uptake by BeWo cells, a human trophoblast cell line. Placenta 22, 768-775	[Medline]
Myatt L, Brockman DE, Langdon G & Pollock JS (1993). Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree. Placenta 14, 373-383	[Medline]
Ng LC , & Gurney AM (2001). Store-operated channels mediate Ca(2+) influx and contraction in rat pulmonary artery. Circ Res 89, 923-929	[Abstract/Full Text]
Ohki G, Miyoshi T, Murata M, Ishibashi K, Imai M & Suzuki M (2000). A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart. J Biol Chem 275, 39055-39060	[Abstract/Full Text]
Petit A , & Belisle S (1995). Stimulation of intracellular calcium concentration by adenosine triphosphate and uridine 5'-triphosphate in human term placental cells: evidence for purinergic receptors. J Clin Endocrinol Metab 80, 1809-1815	[Abstract]
Philipp S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalie A, Hoth M & Flockerzi V (2000). TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 275, 23965-23972	[Abstract/Full Text]
Polliotti B, Lebrun P, Robyn C & Meuris S (1994). The release of human chorionic gonadotrophin and placental lactogen by placental explants can be stimulated by Ca2+ entry through a Na+-Ca2+ exchange process. Placenta 15, 477-485	[Medline]
Powell TL , & Illsley NP (1996). A novel technique for studying cellular function in human placenta: gestational changes in intracellular pH regulation. Placenta 17, 661-668	[Medline]
Putney JW Jr, (1997). Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry. Cell Calcium 21, 257-261	[Medline]
Putney JW Jr, (1999). TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry. Proc Natl Acad Sci U S A 96, 14669-14671	[Full Text]
Putney JW Jr, (2001). Channelling calcium. Nature 410, 648-649	[Medline]
Robidoux J, Simoneau L, St-Pierre S, Masse A & Lafond J (2000). Characterization of neuropeptide Y-mediated corticotropin-releasing factor synthesis and release from human placental trophoblasts. Endocrinology 141, 2795-2804	[Abstract/Full Text]
Sakura H , & Ashcroft FM (1997). Identification of four trp1 gene variants in murine pancreatic beta-cells. Diabetologia 40, 528-532	[Medline]
Sharma SC , & Rao AJ (1992). Role of calcium in secretion of chorionic gonadotropin by first trimester human placenta. Indian J Exp Biol 30, 1105-1110	[Medline]
Sharma SC , & Rao AJ (1993). Effect of calcium depletion on the secretion of newly synthesised human chorionic gonadotropin by first trimester human placenta. Cell Calcium 14, 601-607	[Medline]
Sharma SC , & Rao AJ (1997). Effect of calcium ion channel antagonists on chorionic gonadotropin secretion. Biochem Mol Biol Int 43, 1101-1106	[Medline]
Sossey-Alaoui K, Lyon JA, Jones L, Abidi FE, Hartung AJ, Hane B, Schwartz CE, Stevenson RE & Srivastava AK (1999). Molecular cloning and characterization of TRPC5 (HTRP5), the human homologue of a mouse brain receptor-activated capacitative Ca2+ entry channel. Genomics 60, 330-340	[Medline]
Thyagarajan B, Poteser M, Romanin C, Kahr H, Zhu MX & Groschner K (2001). Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels. J Biol Chem 276, 48149-48158	[Abstract/Full Text]
Wang W, O'Connell B, Dykeman R, Sakai T, Delporte C, Swaim W, Zhu X, Birnbaumer L & Ambudkar IS (1999). Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein. Am J Physiol 276, C969-979	[Medline]
Wes PD, Chevesich J, Jeromin A, Rosenberg C, Stetten G & Montell C (1995). TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci U S A 92, 9652-9656	[Abstract]
Williams DA , & Fay FS (1990). Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium 11, 75-83	[Medline]
Wu X, Babnigg G & Villereal ML (2000). Functional significance of human trp1 and trp3 in store-operated Ca(2+) entry in HEK-293 cells. Am J Physiol Cell Physiol 278, C526-536	[Abstract/Full Text]
Zhang L , & Saffen D (2001). Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization. J Biol Chem 276, 13331-13339	[Abstract/Full Text]
Zhu X, Chu PB, Peyton M & Birnbaumer L (1995). Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett 373, 193-198	[Medline]
Zhu X, Jiang M & Birnbaumer L (1998). Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J Biol Chem 273, 133-142	[Abstract/Full Text]
Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E & Birnbaumer L (1996). trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85, 661-671	[Medline]
Zitt C, Obukhov AG, Strubing C, Zobel A, Kalkbrenner F, Luckhoff A & Schultz G (1997). Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J Cell Biol 138, 1333-1341	[Abstract/Full Text]

Acknowledgements

We thank the staff of the Central Delivery Unit at St Mary's Hospital, Manchester, UK and the Maternity Unit at East Hospital, Gothenburg, Sweden for their assistance in collection of the tissue. This work was funded by the Medical Research Council, UK, the Swedish Research Council (14555) and Jubileums Fond, Sweden.

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