J Physiol 561(Pt 2): 449-458

Calcium channels activated by endothelin-1 in human trophoblast

^{CNRS UMR 6187, Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, 40, Avenue du Recteur Pineau, 86022 Poitiers Cedex, France}

^{INSERM U427, Développement, humain et différenciation, Faculté de pharmacie, 75270 Paris cedex 6, France}

Corresponding author L. Cronier: CNRS UMR 6187, Institut de Physiologie et Biologie Cellulaires, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France. Email: rf.sreitiop-vinu@reinorc.tnerual

Revised 2004 Jul 30; Accepted 2004 Sep 1.

Abstract

Ca^{transfer across the syncytiotrophoblast (ST) of the human placenta is essential for normal fetal development. However, the nature of Ca}^{conductance in the ST and the mechanisms by which it is regulated are poorly understood. With the major signal transduction pathway of endothelin-1 (ET1) acting via phospholipase C (PLC) and Ca}^{, we used ET1 to analyse the nature of Ca}^{channels on cultured trophoblastic cells by means of cytofluorimetric analysis using the ratiometric Ca}^{indicator Indo-1. Results indicate that ET1 (10}m) stimulates a biphasic (transient and sustained) increase in [Ca^]_i in trophoblastic cells. This response is mediated by the endothelin receptor B (ETB) coupled to PLC, since treatment with BQ788 (10m) or U73122 (2 μm) totally abolished the response. Persistence of the rapid transient rise in [Ca^]_i in Ca^{-free extracellular medium confirms the release of Ca}^{from intracellular stores in response to ET1 stimulation. Furthermore, abolition of the sustained increase in [Ca}^]_i in Ca^{-free extracellular medium argues in favour of the entry of Ca}^{during the plateau phase. Abolition of this plateau phase by Ni}^{(1 m}m) in the presence of extracellular Ca^{confirmed the existence of an ET1-induced Ca}^{entry. No evidence for the presence of voltage-operated channels was demonstrated during ET1 action since nifedipine (10}m) did not reduce the Ca^{response and depolarization with a hyper-potassium solution had no effect. Pharmacological studies using the imidazole derivatives SK&F96365 (30 μ}m) and LOE 908 (10 μm) partially inhibited the ET1-evoked Ca^{response, thus providing evidence for the presence of both store-operated Ca}^{channels and non-selective cationic channels in the human ST.}

Abstract

In the human placenta, the chorionic villi, which are in direct contact with the maternal blood, consist of trophoblasts surrounding a core of connective tissue that includes the fetal vessels, fibroblasts and Hofbauer cells. The villous trophoblast differentiates from the fusion of cytotrophoblastic (CT) cells into a multinucleated true syncytium, the syncytiotrophoblast (ST). The ST is a transporting epithelium engaged in numerous placental functions required for fetal growth and development (different forms of exchange, metabolism and production of biologically active substances).

Ca^{ion transfer across the ST is essential for normal fetal development. Its transport takes place in an active manner against a concentration gradient, as the Ca}^{concentration in the fetal circulation is considerably higher than that of the mother. This active transfer is carried out by the ST, for which the first step is that of membrane-gated Ca}^{entry. It has been suggested that the influx of Ca}^{across the ST microvillous membrane is performed by a facilitated diffusion process (}Kamath et al. 1992). Recently, the presence of Ca^{transporter types 1 and 2 was demonstrated in cultured trophoblastic cells (}Moreau et al. 2002a). Furthermore, Ca^{is required in multiple cellular functions that include secretion, ionic conductance, cell-cycle regulation and programmed cell death (}Berridge et al. 2000). However, the nature of Ca^{conductance in the ST, and the mechanisms by which it is regulated, are poorly understood. Previous studies have demonstrated an increase in intracellular Ca}^{in the ST following exposure to various biologically active substances acting in both autocrine and paracrine fashions: GnRH (}Currie et al. 1993), ATP and UTP (Petit & Belisle, 1995), ATP and angiotensin II (Karl et al. 1997), endothelin (Cronier et al. 1999), ATP (Clarson et al. 2002). However, the nature of Ca^{channel activation in the ST remains controversial, with voltage-operated Ca}^{channels (VOCC;}Meuris et al. 1994; Petit & Belisle, 1995; Robidoux et al. 2000), non-selective cationic channels (NSCC; Grosman and Reisin, 2000; Llanos et al. 2002; Long & Clarson, 2002), store-operated Ca^{channels (SOCCs;}Clarson et al. 2003) and receptor-operated Ca^{channels (ROCCs;}Bax et al. 1994) all having been described.

We previously demonstrated the presence of endothelin (ET) receptors A and B on the human trophoblastic membrane (Malassiné et al. 1993b). With the major signal transduction pathway of endothelin-1 (ET1) acting via phospholipase C (PLC) and the mobilization of intracellular Ca^{, we have thus used cytofluorimetry and pharmacological analysis to determine the nature of ET1-mediated Ca}^{entry into ST cells in primary culture.}

Acknowledgments

We are grateful to the Clinique du Fief de Grimoire for providing us with placental tissue. We would like to thank Boehringer Ingelheim for the generous gift of LOE 908 and Dr F. Gaillard (UMR 6187 CNRS) for his expertise in the statistical analysis. This research was supported in part by grants from the Ligue nationale contre le cancer to C. Niger.

Acknowledgments