Expression of the parathyroid Ca 2+ -sensing receptor in cytotrophoblasts from human term placenta R A Bradbury, K L Sunn, M Crossley, M Bai 1 , E M Brown 1 , L Delbridge 2 and A D Conigrave Departments of Biochemistry and 2 Surgery, University of Sydney, New South Wales 2006, Australia and 1 Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA (Requests for offprints should be addressed to A D Conigrave, Department of Biochemistry (G08), University of Sydney, New South Wales 2006, Australia) Abstract Fura-2-loaded human cytotrophoblasts responded to elevated extracellular Ca 2+ concentration ([Ca 2+ ] o ) with monophasic or, in the case of large (>20 μm) extravillous cells, biphasic elevations in intracellular free Ca 2+ ion concentration ([Ca 2+ ] i ) that returned to baseline levels after restoration of control [Ca 2+ ] o . Large extravillous cytotrophoblasts also responded to elevated [Mg 2+ ] o with transient elevations in [Ca 2+ ] i , consistent with the behav- iour of the parathyroid Ca 2+ -sensing receptor. Expression of the parathyroid Ca 2+ -sensing receptor in placental cells was confirmed using Northern blot and reverse transcrip- tion (RT)-PCR analysis. However, the major transcript in human placental cells (6·2 kb) differed from that expressed by human parathyroid cells (5·6 kb). RT-PCR analysis and DNA sequencing of key PCR products also revealed the presence of a splice variant in placental and parathyroid cells that lacks exon 3. Journal of Endocrinology (1998) 156, 425–430 Introduction Recent studies have shown that placental cytotrophoblasts are sensitive to changes in extracellular Ca 2+ concentration (Juhlin et al. 1990, Hellman et al. 1992) and that an elevation in extracellular Ca 2+ concentration ([Ca 2+ ] o ) suppresses the release of parathyroid hormone-related protein (PTHrP) (Hellman et al. 1992). In this respect cytotrophoblasts resemble Ca 2+ -sensitive chief cells of the parathyroid that are responsible for modulating [Ca 2+ ] o via the controlled release of parathyroid hormone. A Ca 2+ - sensing receptor from human parathyroid cells has recently been cloned, sequenced and expressed in cultured cell lines (Garrett et al. 1995a, Bai et al. 1996). This protein is a G-protein-coupled receptor (Brown et al. 1993) that responds to a variety of divalent and polyvalent cations with elevations in intracellular Ca 2+ concentration ([Ca 2+ ] i ) via the release of inositol 1,4,5-trisphosphate and is transcribed as a seven exon primary transcript in which the first exon encodes a 5 untranslated region and the last six exons encode the full-length amino acid sequence (Pearce et al. 1995). The distribution of this receptor also includes thyroid parafollicular cells, where it appears to regulate calcitonin release (Garrett et al. 1995b), renal tubular cells, where it regulates the excretion of Ca 2+ ions and water (Riccardi et al. 1995), the brain (Ruat et al. 1995, Ye et al. 1996) and antral gastrin cells (Ray et al. 1997). However, it is not known whether the parathyroid Ca 2+ -sensor protein is responsible for Ca 2+ sensing in placental cells. In this study we have used a combination of fura-2 microfluorimetry, Northern blot analysis, reverse transcription (RT)-PCR and DNA sequencing to dem- onstrate that the parathyroid Ca 2+ -sensor protein is expressed in human placental cells. A splice variant of the receptor that lacks exon 3 of the full length mRNA was identified in both placental and adenomatous parathyroid cells during this study. Materials and Methods Cell preparation Human term placentas were obtained immediately after spontaneous vaginal delivery from either the Royal Hospital for Women, Paddington NSW or King George V Hospital, Camperdown, NSW, Australia. Placental tissue was released with the approval of the hospitals’ ethics committees. Approximately 10 g placental tissue was minced and then digested for 30 min at 37 C in pre-oxygenated physiological saline solution (PSS) that contained collagenase (Worthington type II, 1 mg/ml, Scimar, Templestowe, Victoria, Australia) and DNase I (Sigma Type IV, 0·4 mg/ml, Sigma Chemical Co., St Louis, MO, USA). The composition of PSS was as follows (in mM): NaCl 145, KCl 5·0, H-HEPES 10, CaCl 2 1·0, MgCl 2 1·2, D-glucose 10, Na 2 HPO 4 1·0 (pH 7·4 adjusted with NaOH). The cell suspension was filtered, washed by centrifugation and layered on top of a pre-formed Percoll 425 Journal of Endocrinology (1998) 156, 425–430  1998 Journal of Endocrinology Ltd Printed in Great Britain 0022–0795/98/0156–0425 $08.00/0 Downloaded from Bioscientifica.com at 08/18/2019 07:30:51AM via free access