Journal of Cell Science Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells Robrecht Lembrechts 1 , Inge Brouns 1 , Kathy Schnorbusch 1 , Isabel Pintelon 1 , Paul J. Kemp 2 , Jean-Pierre Timmermans 1 , Daniela Riccardi 2 and Dirk Adriaensen 1, * 1 Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, BE-2020 Antwerp, Belgium 2 Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK *Author for correspondence (dirk.adriaensen@ua.ac.be) Accepted 30 June 2013 Journal of Cell Science 126, 4490–4501 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.131656 Summary The Ca 2+ -sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca 2+ ] o . In addition to sensing [Ca 2+ ] o , CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca 2+ ] o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67–GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca 2+ ] o with a rise in intracellular Ca 2+ ([Ca 2+ ] i ); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex- 231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca 2+ ] i , suggesting that Ca 2+ influx through TRPC channels contributes to the total [Ca 2+ ] i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca 2+ ] i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche. Key words: Airway stem cell niche, Immunohistochemistry, Live-cell imaging, Pulmonary neuroepithelial body microenvironment, qRT-PCR, Extracellular calcium-sensing receptor Introduction The extracellular calcium (Ca 2+ )-sensing receptor (CaSR) is a cell- surface protein with multimodal sensing capabilities that has been shown to play an important role in mineral ion metabolism. The CaSR belongs to group C of the G-protein-coupled receptor superfamily (GPCRs) and has been demonstrated to be sensitive to a variety of metabolic signals including di- and trivalent cations (Ca 2+ , Mg 2+ , metals belonging to the lanthanides series such as La 3+ , etc.), pH, ionic strength, aromatic amino acids (L-Phe, L- Trp) and polyamines such as spermine (for reviews, see Chang and Shoback, 2004; Riccardi et al., 2009; Riccardi and Kemp, 2012). Genetic studies in humans have shown that the best documented role of the CaSR is in systemic homeostasis of extracellular free ionized Ca 2+ concentration [Ca 2+ ] o through the regulation of parathyroid hormone secretion by the parathyroid glands (Brown and MacLeod, 2001; Conigrave et al., 2000). However, expression of the CaSR has been reported in many other specialized cells and tissues that are not primarily involved in [Ca 2+ ] o homeostasis, such as in the brain, skin and lungs, suggesting that the receptor might be involved in cellular functions other than divalent cation homeostasis (Hebert et al., 2004; Riccardi et al., 2009; Yano et al., 2004). The CaSR has been reported to be expressed in cells of the diffuse neuroendocrine system, such as endocrine pancreatic cells (Gray et al., 2006), intestinal cholecystokinin secreting cells (Wang et al., 2011), and in a subset of innervated sensory epithelial cells in taste buds (San Gabriel et al., 2009). In taste cells, the CaSR was shown to act as a multimodal sensor, able to evoke responses such as secretion of specific substances upon activation, and therefore appears to fulfil an important role in taste perception (Bystrova et al., 2010). In developing mouse lungs, CaSR mRNA and protein is present in airway epithelium within a narrow developmental window, between embryonic day (ED) 11.5 and ED16.5, regulating lung branching morphogenesis. After ED16.5, CaSR expression progressively decreases and has so far not been detected in postnatal lung tissue (Finney et al., 2008; Riccardi et al., 2009). However, mRNA expression of CaSR in postnatal lungs was studied in whole-lung tissue blocks only, without the ability of precise localization of potential mRNA expression at the level of less-abundant cell types. Pulmonary neuroepithelial bodies (NEBs) (Lauweryns et al., 1972) are organized as highly specialized clusters of pulmonary neuroendocrine cells (PNECs), closely associated with a large number of mainly vagal sensory nerve terminals. Their location as an integrated component of the epithelial lining of 4490 Research Article