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Institut National de la Santé et de la Recherche Médicale (INSERM) U427 (V.T., A.T., P.M., D.E.B.), Faculté des Sciences Pharmaceutiques et Biologiques de Paris, Université René Descartes, Paris V, 75006 Paris, France; and Unité INSERM U349 (J.M.G., N.S., A.J.), Centre Viggo Petersen, 75010 Paris, France
Abstract
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin gene. The objectives of this study were: 1) to determine the expression of CGRP and its receptor at the human implantation site, and 2) to examine the possible in vitro effect of this neuropeptide on two major partners of implantation, decidual cells and extravillous cytotrophoblasts. Immunohistological analysis of first-trimester placental chorionic villi showed CGRP in decidual cells and glandular cells, but not in extravillous trophoblast cells. CGRP expression was confirmed in cultured decidual cells by Southern blot analysis and immunocytochemistry and by RIA in culture medium. Transcripts of calcitonin receptor-like receptor were detected by Southern blot analysis of RT-PCR amplicons from both decidual and extravillous trophoblast cells, whereas transcripts for the receptor activity-modifying protein 1 were detected in decidual cells only. In vitro, CGRP stimulated cAMP production but not nitric oxide (NO) release by cultured decidual cells; in contrast CGRP increased NO release but not cAMP production in cultured extravillous trophoblasts. The presence of NO synthase (endothelial and inducible) was confirmed by immunodetection in extravillous trophoblasts, both in situ and in vitro. This study points to a paracrine and autocrine effect of CGRP on decidual and extravillous trophoblast cells, two major actors in implantation.
BY ITS MULTIPLE FUNCTIONS (exchanges, endocrine, and immunological functions), the placenta plays a key role in the development of pregnancy and in fetal growth. The main component of human placenta is the trophoblast, which may differentiate along two pathways: 1) villous trophoblasts, involved directly in materno-fetal exchanges and production of pregnancy-specific hormones, and 2) extravillous trophoblasts, involved in the anchorage of the placenta in the uterine wall.
The human placenta is characterized by extensive invasion of extravillous trophoblasts into the maternal uterus (1, 2). Trophoblast cells at the tip of some of the villi that juxtapose the uterine wall detach from the basement membrane and aggregate into multilayered columns of nonpolarized cells that rapidly invade the decidua and the first third of the myometrium and associated spiral arteries (3). The decidua that arises from hormonally transformed endometrium includes decidual cells and immunocompetent cells such as T lymphocytes and natural killer cells (4). Decidual cells secrete soluble factors such as IGF binding protein-1 (IGFBP-1; for review, see Ref. 5) and TGF-ß1 (for review, see Ref. 6) that are directly involved in the regulation of trophoblast invasion (for review, see Refs. 7, 8, 9, 10, 11).
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide produced by tissue-specific alternative splicing of the primary transcript (12, 13). CGRP acts at the cellular level by binding to a seven-transmembrane-domain G protein-coupled receptor (14, 15). This receptor requires the presence of modulating proteins with a single transmembrane domain known as receptor activity-modifying proteins (RAMPs; Ref. 16). CGRP has neurotropic and neurotrophic activity, regulates antigen presentation within the immune system, and is the most powerful endogenous vasodilator (17, 18).
The role of CGRP during pregnancy is still poorly known. CGRP is present in the maternal circulation, and plasma CGRP levels rise during gestation (19). Injection of CGRP to rats with an induced preeclamptic syndrome normalizes systemic pressure, suggesting a major vasodilatory role of this peptide during pregnancy (20). Recent immunohistochemical studies have shown the presence of CGRP in human decidua (21), which lacks innervation. The aim of this study was therefore to determine the expression of CGRP and its receptor at the human implantation site and to examine, in vitro, the possible paracrine or autocrine effect of this neuropeptide on two major partners of human implantation, namely decidual cells and extravillous cytotrophoblasts (EVCTs).
Materials and Methods
Tissues
Placental tissues were obtained during legal first-trimester (7–12 wk) abortions at the Obstetrics and Gynecology Departments of Broussais and Saint-Vincent de Paul Hospitals (Paris, France). The tissues were washed in Ca2+-, Mg2+-free Hank’s balanced salt solution supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin.
Isolation and purification of decidual cells
Decidual cells were isolated by using a technique based on that described by Braverman et al. (22). Small pieces of decidua (2 x 2 x 2 mm) obtained after first-trimester termination of pregnancies were treated in DMEM (Life Technologies, Inc., Grand Island, NY) with 0.25% collagenase (Sigma, Saint-Quentin Fallavier, France) and 6.25 U/mg DNase solution (DNase I type IV from bovine pancreas, Sigma) for 2 h in a shaking incubator at 37 C. Cell dispersion was facilitated by passing the suspension through a 22-gauge needle fitted to a sterile 5-ml syringe. The resulting suspension was filtered through a 50-µm membrane. The filtrate was then washed in DMEM, pelleted, resuspended in 20% isotonic Percoll, and layered on a 20–50% Percoll gradient (Pharmacia Biotech, Orsay, France). The tubes were centrifuged for 15 min at 30,000 x g. Cells with a density of about 1.048 g/ml were withdrawn with sterile pipettes and washed in DMEM.
Decidual cells were then suspended in DMEM supplemented with 10% calf serum (Biological Industries, Beth Hemeek, Israel), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml bovine-porcine insulin. They were plated in 100-mm Falcon culture dishes (Falcon Plastics, Los Angeles, CA) or on sterile glass slides. As recently published (23), extravillous trophoblasts do not survive on plastic dish, and villous trophoblasts fuse together on plastic dish to form multinucleated syncytiotrophoblasts that are easy to recognize. No contamination by trophoblast cells was observed.
Isolation and purification of trophoblasts differentiating into EVCTs
These cells were isolated using the method we recently established (23, 24). Briefly, chorionic villi were dissected, rinsed, and minced for cell isolation. Chorionic villi were incubated in Hank’s solution containing 0.125% trypsin (Difco Laboratories, Detroit, MI), 4.2 mM MgSO4, 25 mM HEPES, and 50 IU/ml DNase type IV (Sigma) for 35 min at 37 C without agitation.
After tissue sedimentation, the supernatant was removed and filtered (100-µm pore size). Hank’s solution was added to the tissue and sedimented twice. Trypsin digestion was stopped with 5% fetal calf serum (FCS). Cells were centrifuged at 300 x g for 10 min, diluted to a density of 5–6 x 105 cells/2 ml, and then plated on Matrigel-coated (5–6 mg/ml; Collaborative Biomedical Products, Le pont de Claix, France) 35-mm Falcon culture dishes.
Cells were maintained in DMEM (Life Technologies, Inc.), supplemented with 10% FCS (Biological Industries), 2 mM glutamine, 25 mM HEPES, 100 IU/ml penicillin, and 100 µg/ml streptomycin, and incubated in 5% CO2 at 37 C. After 3 h, cells were washed three times to eliminate debris and then incubated with complete medium. These purified primary EVCTs were characterized by using immunocytochemistry and real-time PCR as already described (23). EVCTs were shown to express in vitro the specific markers of human invasive trophoblasts described in situ.
Immunohistochemistry [CGRP/nitric oxide synthase (NOS)]
Decidual tissue was fixed in AFA solution (acetic acid 5%, formol 2%, absolute ethanol 75%, H2O 18%) for 1–12 h and then embedded in paraffin. Paraffin sections (4 µm) were mounted on aminopropyl- triethoxysilane (Tespa)-coated glass slides.
Sections were dewaxed in xylene and rehydrated. Immunostaining was performed using a universal streptavidin-alkaline phosphatase immunostaining kit (Immunotech, Margency, France). Nonspecific antibody binding was blocked by incubation for 15 min in a blocking reagent containing carrier protein. Then, 8 µg/ml 
-human CGRP (hCGRP) monoclonal antibody raised against the C-terminal part of hCGRP (a generous gift from Prof. D. Bellet, University Paris V) or 5 µg/ml endothelial NOS (eNOS) or inducible NOS (iNOS) polyclonal antibodies (Transduction Laboratories, Inc. Interchim, Montluçon, France) were incubated with the sections for 1 h and 3 h, respectively, at room temperature. Sections were washed in Tris-buffered saline (TBS)-0.1% Tween and incubated with a biotinylated secondary antibody for 1 h. Streptavidin-alkaline phosphatase was applied for 1 h. Staining was detected with the Fast Red chromogen after 5 min or 20 min. Nuclei were counterstained by incubation for 2 min with hematoxylin. Sections were mounted, examined, and photographed under an Olympus Corp. (Melville, NY) BX60 microscope. Controls were performed by omitting the primary antibody or by incubating the sections with nonspecific IgG at the same concentration as the primary antibody. The specificity of staining was also assessed by preincubation of the antibody with its peptide.
Immunocytochemistry
Antibodies.
Antibodies used for immunocytochemistry and immunohistochemistry are listed in Table 1
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). Cells were washed in TBS-0.1% Tween, then incubated with a secondary antibody, a rhodamine-conjugated goat antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and with a fluorescein-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:250 in the dark for 1 h. Slides were mounted in mounting medium (Vector Laboratories, Inc., Burlingame, CA) and examined and photographed on an Olympus BX60 epifluorescence microscope. Controls were performed by omitting the primary antibody or by using nonspecific mouse or rabbit IgG.
Immunoassays
For CGRP, 48-h conditioned medium was collected and stored at -20 C; the peptide was measured by means of a specific and sensitive RIA (25) after purification of the culture supernatants on an AMPREP column (Amersham, les Ulis, France). Eluted CGRP (Bachem, Bâle, Switzerland) was lyophilized and dissolved in 500 µl RIA phosphate buffer. Serial dilutions (10–100 µl) were incubated for 4 d with sheep antiserum against hCGRP (26) at a final dilution of 1:250,000 in 0.05 M phosphate buffer containing 0.3% albumin and 10 mM EDTA in a final volume of 400 µl. Then, 100 µl [125I]-CGRP (Amersham) was added, and incubation was continued for 3 d. Free and bound hormones were separated by charcoal adsorption in RIA buffer in the presence of 0.25% gelatin.
Prolactin (PRL) was measured in the conditioned medium by enzyme-linked fluorescence assay (Vidas System, BioMerieux, Marcy l’Etoile, France).
RT-PCR
Total RNA was extracted from decidual cells, EVCTs, and TT cells using the QIAGEN (Courtabeuf, France) procedure. TT cells are an established cell line derived from a thyroid carcinoma, and they produce CGRP in large amounts. cDNA was synthesized from 1 µg total RNA heated for 10 min at 70 C. The reaction mixture had a final volume of 20 µl and contained 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 10 mM dithiothreitol, 10 U RNAsine, 200 U Superscript reverse transcriptase (Life Technologies, Inc.), 1 mM each dNTP, and 50 pmol of a 3' oligo-dT primer. Primer extension was performed for 50 min at 42 C. The reaction mixture was half-diluted, and 18 µl were made up to 50 µl with Taq polymerase buffer containing 0.5 U of Taq polymerase (Life Technologies, Inc.) in the presence of 1.5 mM MgCl2 for calcitonin receptor-like receptor (CRLR), CGRP, and actin, 1 mM for RAMP1, 20 pmol of each specific primer (Table 2). For the actin control, 2 µl of the diluted RT reaction mixture was treated in the same way. Actin and RAMP1 amplifications were run for 25 cycles and 27 cycles, respectively, of 30 sec at 95 C (denaturation), 30 sec at 55 C (annealing), and 30 sec at 72 C (extension). CGRP and CRLR amplifications were run for 30 cycles, with 1 min for each cycle segment. The reaction was stopped after 5 min of elongation.
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CGRP amplification product (15 µl) and actin amplification product (10 µl) were analyzed by electrophoresis on 2% agarose gel, visualized with ethidium bromide, and transferred to membranes (GeneScreen, NEN Life Science Products) after denaturation in 0.4 N NaOH. The amplified cDNAs were hybridized with specific cDNA (CGRP, actin) or oligonucleotide (CRLR, RAMP1) probe labeled with 32P by using, respectively, a random priming method (specific activities > 108 cpm/µg DNA) or a 3' labeling assay with deoxynucleotidyl terminal transferase, respectively. After prehybridization for 1 h at 42 C in 50% formamide, 1% SDS, 2x SSC, 10% dextran sulfate, the membranes were hybridized at 42 C in the same buffer containing the specific probe, and then subjected to the following washing steps: 2x SSC for 10 min at room temperature, twice in 2x SSC 1% SDS for 20 min, twice in 0.2x SSC 1% SDS for 20 min at the hybridization temperature, and for 2 min in 0.2x SSC at room temperature for cDNA hybridization. Washing steps for oligonucleotide hybridization consisted of 2x SSC 0.1% SDS at room temperature followed by 30 min at 47 C, then 2x SSC 0.1% SDS for 20 min and 5 min in 2x SSC at room temperature. Membranes were dried, and autoradiography was performed with intensifying screens for 1 h.
cAMP determination
At 48 h of extravillous trophoblast and decidual cell culture, the medium was removed, and the cells were washed in serum-free medium and then incubated for 1 h in serum-free medium with 0.1 mM isobutylmethylxanthine.
Cells were treated with -hCGRP (Novabiochem, Meudon, France) at the indicated concentrations for 90 min. Conditioned medium was centrifuged and frozen until use. The cells were counted. The cAMP concentration in the culture media was determined by RIA, as recommended by the kit manufacturer (Amersham, Saclay, France). The results are expressed as femtomoles of cAMP per 106 cells. Each experiment was repeated at least twice, and intra-assay variations (triplicate determinations) were ±10%.
Nitrate and nitrite determination
Nitric oxide (NO) release by extravillous and decidual cells was determined spectrophotometrically by measuring the accumulation of nitrate and nitrite in culture medium. Twenty hours after plating, cells were cultured with or without -h CGRP (Novabiochem) at 10-10, 10-9, and 10-8 M in DMEM without phenol red and supplemented with 10% FCS, 2 mM glutamine, 25 mM HEPES, 4.5 g/liter glucose, 100 IU/ml penicillin, and 100 µg/ml streptomycin and incubated in humidified 5% CO2-95% air at 37 C. -CGRP was added each day at the different concentrations. On the third day, the culture media were centrifuged at 1200 x g for 10 min to remove particles. The cells were counted.
Nitrate was reduced to nitrite by incubation of 150-µl samples for 30 min at 37 C with 0.1 U/ml nitrate reductase (Roche Molecular Biochemicals), 50 µM NADPH (Sigma), and 5 µM flavin adenine dinucleotide (Sigma) in a final volume of 160 µl. The samples were then incubated with 10 U/ml lactate dehydrogenase (Sigma) and 10 mM sodium pyruvate for 15 min at 37 C in a final volume of 170 µl.
Total nitrite was determined by the Griess reaction. Samples were placed on ice, and 1 mM sulfanilamide and 0.1 M HCl were added to a final volume of 200 µl. The samples were centrifuged at 10,000 x g for 15 min at 4 C. Then, 150 µl were transferred to well microtiter plates, and 10 µl (1 mM) of naphthylethylene-diamine were added; the plates were incubated for 30 min in the dark before reading at 550 nm.
Statistical analysis
Results are shown as means ± SD of triplicate determinations. Comparisons between the different groups were made with the Kruskall-Wallis test, and significant differences were further analyzed by pairwise comparison with the Mann-Whitney U test. P values less than 0.05 were considered statistically significant.
Results
The implantation site
At the tip of the anchoring villi (Fig. 1), cytotrophoblasts proliferate and differentiate into extravillous trophoblasts with invasive properties. These EVCTs migrate through the decidua and invade the spiral arterial walls to replace endothelial cells. They also form bi- or trinucleated giant cells inside the decidua (27). The decidua is a complex tissue composed of decidual cells, immunocompetent cells (T cells, B cells, macrophages, granulocytes, and natural killer cells) and glandular cells (8). The two pictures (Fig. 1, A and B) illustrate the morphological aspects of isolated extravillous trophoblasts cells and decidual cells in culture.
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As shown in Fig. 2, specific CGRP immunostaining was detected at the materno-fetal interface in large polygonal cells with an open vesicular nucleus (8) separated by an abundant matrix (27), corresponding to decidual cells. No immunostaining was observed in either EVCTs (large round cells with a hyperchromatic nucleus) or immune cells (small round cells). As shown in Fig. 2E, CGRP was also strongly detected in the glandular cells of the decidua.
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As shown in Fig. 3, CGRP expression was detected in cultured isolated decidual cells. Colocalization of PRL, a specific marker of decidual cells (Fig. 3D) and also CGRP (Fig. 3, A and C), was observed by immunochemistry. Decidual cells also were stained with a monoclonal antivimentin antibody (a marker of mesenchymal cells, Fig. 3B). In contrast, no CGRP staining was observed in isolated purified EVCTs in culture. These results were confirmed by RIA detection of CGRP release in the culture medium of decidual cells (169 pg/ml ± 85/106 cells after 48 h; data not shown). No CGRP was detected in the culture medium of EVCT cells (data not shown). Decidual cells in culture also produced PRL, as shown by a specific enzyme-liked immunoassay (8.5 ng/ml ± 6.1/106 cells after 48 h, data not shown). Moreover, CGRP mRNA was detected in decidual cells by Southern-blot hybridization of RT-PCR amplicons. As expected, a specific 435-bp product was amplified from RNA extracted from decidual cells and in positive control TT cells (Fig. 4).
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The expression of CGRP receptors was confirmed by Southern blot hybridization of CRLR and RAMP1 RT-PCR products in decidual cells (Fig. 4). As expected, a 362-bp amplicon (CRLR) and a 219-bp amplicon (RAMP1) were generated from RNA extracted from decidual cells and from TT cells (positive controls). No RAMP1 transcripts were detected in cultured extravillous trophoblast cells (Fig. 4), but CRLR mRNA was expressed in these cells. Thus, a functional CGRP receptor appears to be present in decidual cells but not in extravillous trophoblast cells.
CGRP stimulates cAMP and NO production at the implantation site
As shown in Fig. 5A, cAMP release into culture medium was increased 10-fold 90 min after addition of 1 nM CGRP to isolated decidual cells, but the same concentration had no effect on EVCT cells (Fig. 5C). The release of cAMP was maximal with 1 nM CGRP (Fig. 5A).
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) was significantly increased by 0.1 nM CGRP in trophoblast cells. No change in nitrate release was found in decidual cells after 72 h of stimulation by CGRP (Fig. 5B). NOS expression by extravillous trophoblast cells
Expression of iNOS and eNOS was demonstrated in situ by immunodetection at the implantation site in villous and extravillous trophoblasts (Fig. 6). Expression of iNOS and eNOS was also found by immunocytochemistry in cultured EVCTs (data not shown). eNOS and iNOS transcript expression was confirmed by real-time quantitative RT-PCR (data not shown).
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Human trophoblastic invasion is precisely regulated (3). Expression of integrins (28, 29), cell adhesion molecules (30, 31), metalloproteinases (32), and cell cycle proteins (33) throughout this process has been documented by immunohistochemistry and in situ hybridization. Interestingly, although extravillous trophoblast cells migrate within the lumen of arterioles, they express endothelium-specific markers such as specific cell-cell adhesion molecules (30) and eNOS (34). In this study, we confirmed by immunohistochemistry and immunocytochemistry on isolated purified cells that extravillous trophoblast cells express both eNOS and iNOS. These data are in keeping with previous reports (35). In addition, using the cell model that we recently established (24), we showed that cultured extravillous trophoblast cells produce NO and that this production is increased by CGRP in a concentration-dependent manner.
Interestingly, the cells that expressed this neuropeptide are neighboring decidual cells. Decidual cells are large polyhedral cells that arise from differentiation of endometrial stromal cells induced by estrogen and progesterone. These cells express specific markers such as PRL (36, 37, 38) and IGFBP-1 (39). Although the role of PRL is unknown, it is established that IGFBP-1 modulates trophoblast migration (40, 41). In addition, decidual cells secrete TGF-ß, which is directly involved in inhibiting trophoblast invasion (42). In this study, we found that decidual cells express CGRP both in situ and in culture, and that CGRP stimulates cAMP release by these cells.
CGRP has a number of effects in a variety of systems, implying the existence of several intracellular signaling pathways. cAMP production and/or NO release is/are increased after CGRP stimulation. In smooth muscle cells (SMC) of guinea pig ileum, CGRP-induced relaxation involves cAMP production in both circular and longitudinal intestinal SMC, but also NO release in circular SMC (43). In human colon SMC, CGRP induces relaxation via both the cGMP and cAMP pathways (44). An increase in cAMP production has been reported after addition of 10 nM CGRP to rabbit aortic SMC (45). In mongrel dogs, cardiac inotropic signaling by nitroxyl anion (NO-), acting independently from cGMP, was mediated by CGRP (46). In this study, we found that CGRP stimulated NO release in extravillous trophoblast cells despite the absence of RAMP1. Even if recent progress suggests the existence of additional receptor subtypes (47), CGRP receptors are always classified into CGRP subtype 1 and CGRP subtype 2. This classification is based on the affinity of diverse antagonists, like CGRP (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) or the new nonpeptidic CGRP antagonist BIBN4096RS (48). The molecular feature of CGRP 2 receptor is as yet unknown, but the CGRP 1 subtype has been identified to be the CRLR (15). The present work suggests that cAMP production by decidual cells was coupled to the presence of CGRP 1 receptor (CRLR + RAMP1) but that signaling for NO production by extravillous trophoblast cells does not require the presence of RAMP1 because the latter was not expressed in these cells. Alternatively, our data do not exclude the expression of a CGRP 2 receptor subtype coupled to NOSs in extravillous trophoblast cells.
In this study, we found that CGRPs secreted by decidual cells appear to activate cAMP secretion by decidual cells through an autocrine process and NO production by extravillous trophoblast cells through a paracrine effect. The physiological role of this peptide at the materno-fetal interface remains to be elucidated. We previously showed, in the model of embryonal teratocarcinoma cells, that CGRP is a potent mitogenic and chemotactic autocrine agent (49), suggesting a possible role of CGRP in cell migration. NO production during CGRP stimulation might be involved in the migration, and therefore the motility, of extravillous trophoblasts. Indeed, numerous growth factors and cytokines stimulate cytoskeleton motility via NO (for review, see Ref. 50). However, in the invasion assay that we recently described (24), we observed no effect of CGRP or of exogenous NO or cGMP on trophoblast cell motility (Tarrade, A., unpublished data) as previously described with a trophoblast cell line (51). This suggests that exogenous NO or activation of cyclic GMP-dependent pathways alone are not sufficient to stimulate trophoblast motility.
CGRP produced by decidual cells might be involved in other important events occurring at the implantation site, such as the complex immunomodulation that abrogates rejection of trophoblast cells by decidual cells and immunocompetent cells present in the decidua. Indeed, the immunomodulatory role of CGRP has already been shown in other systems such as human skin (52, 53). In addition, the CGRP produced by decidual cells might be involved in dilation of the vasculature of the chorionic plate (54). Increased CGRP levels are observed in preeclampsia (55), which involves complex abnormal placenta vascularization. CGRP given to rats with an induced preeclamptic syndrome is capable of normalizing systemic pressure and preventing neonatal death (20). Finally, in this study we observed strong immunodetection of CGRP in the uterine glands. Recent studies have shown that secretions by these uterine glands might be involved in feeding the fetus during the first trimester of human pregnancy (56).
In conclusion, despite the absence of innervation, various neuropeptides appear to be expressed at the human materno-fetal interface (21). Here, we show that CGRP secreted by decidual cells activates different signal transduction pathways in two major cell types involved in implantation, namely decidual cells and trophoblast cells.
Acknowledgments
We thank Prof. D. Bellet and J. L. Janneau (Institut Gustave Roussy, Villejuif, France) for providing anti-CGRP antibodies and technical assistance, respectively.
Footnotes
Address all correspondence and requests for reprints to: Danièle Evain-Brion, Institut National de la Santé et de la Recherche Médicale U 427, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France. E-mail: .
This work was supported by a grant from the Fondation pour la Recherche Médicale.
V.T. and A.T. should both be considered first authors.
Abbreviations: CGRP, Calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; eNOS, endothelial NOS; EVCT, extravillous cytotrophoblast; FCS, fetal calf serum; hCGRP, human CGRP; IGFBP-1, IGF binding protein-1; iNOS, inducible NOS; NO, nitric oxide; NOS, nitric oxide synthase; PRL, prolactin; RAMP, receptor activity-modifying proteins; SMC, smooth muscle cells; TBS, Tris-buffered saline.
Received January 31, 2002.
Accepted May 23, 2002.
References
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