Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells

The extracellular calcium (Ca²⁺)-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 (Ca²⁺, Mg²⁺, metals belonging to the lanthanides series such as La³⁺, 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 Ca²⁺ concentration [Ca²⁺]_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 [Ca²⁺]_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 intrapulmonary airways in all air-breathing vertebrate groups, makes them ideally placed to sense changes in the airway environment and transduce this information to the central nervous system (Adriaensen et al., 2003; Adriaensen et al., 2006; Brouns et al., 2009b; Brouns et al., 2012). The characteristics of the excitable NEB cells clearly allow the perception of environmental stimuli. When local stimuli activate the release of secretory products from NEB cells, sensory nerve fibers in synaptic contact are believed to depolarize, triggering action potentials that can reach the CNS. The best characterized mechanism so far is the [Ca²⁺]_i-mediated release of ATP from activated NEB cells (De Proost et al., 2009; Lembrechts et al., 2012) influencing the P2X_2/3 ATP receptors expressed on the terminals of a population of vagal afferents that contacts pulmonary NEBs in rodents (Brouns et al., 2009a).

In many species, the PNECs in NEBs are largely shielded from the airway lumen and the surrounding epithelial cells by a tight-junction-sealed specialized cell type, the Clara-like cells, which together with NEB cells and nerve terminals are referred to as the ‘NEB microenvironment’ (De Proost et al., 2008). In a previous report, we were able to show the delayed activation of Clara-like cells through P2Y₂ ATP receptors upon NEB stimulation and subsequent ATP release (De Proost et al., 2009). Because of the invariably observed close interaction between NEB cells, complex nerve terminals and Clara-like cells, in vitro models for functional studies should preferentially include all components of the NEB microenvironment. For this reason, we developed an ex vivo mouse lung vibratome slice model for confocal live-cell imaging of physiological reactions in the NEB microenvironment and surrounding epithelial cells (De Proost et al., 2008; Pintelon et al., 2005).

NEBs have been suggested to have several functions in the regulation of physiological processes in the lungs during fetal, perinatal, and postnatal life (Adriaensen et al., 2003; Cutz et al., 2008; Linnoila, 2006; Sorokin et al., 1997; Sorokin and Hoyt, 1989; Sorokin and Hoyt, 1990). Today, extensive evidence implicates both NEBs in several species, and NEB-related cell models in hypoxia sensing (Cutz et al., 2003; Cutz and Jackson, 1999; Kemp et al., 2003). The involvement of NEBs in postnatal mechanosensing has recently regained interest (Lembrechts et al., 2011; Lembrechts et al., 2012). Postnatally, the close association of NEB cells with afferent nerve terminals argues for a function as a sensory receptor (Adriaensen et al., 2006), although the exact stimuli and molecular transduction mechanisms that are involved in their activation remain a matter of debate.

Because of the multimodal sensing capacities of the CaSR, the present study aimed to investigate CaSR expression in the NEB microenvironment by laser microdissection (LMD), (quantitative) real-time RT-PCR (qRT-PCR) and immunohistochemical staining. To unravel the functionality and significance of the CaSR, confocal Ca²⁺ imaging of the NEB microenvironment in live lung slices was used. Together, our observations implicate the CaSR as an important mediator for sensing local changes in the microenvironment and/or for the physiology of NEB cells.

In GAD67–GFP Bl6 mice, mRNA encoding CaSR was amplified from samples of whole embryonic lung (ED14), as expected (Finney et al., 2008) (Fig. 1A, lane 4), whereas in whole postnatal (PD14) lung samples, CaSR expression could not be detected (Fig. 1A, lane 3). However, RT-PCR carried out on GAD67-positive NEBs, LMD from lung cryosections, indicated that CaSR mRNA was exclusively expressed in the NEB microenvironment (Fig. 1A, lane 1) and no such amplification of CaSR mRNA was possible from microdissected GAD67-negative intrapulmonary airway epithelium (Fig. 1A, lane 2). The reference gene, Eef2 (which encodes eEF-2) could be equally well detected in all samples (Fig. 1A), whereas the no-template and negative RT controls for both CaSR and eEF-2 showed no amplification (data not shown).

CaSR mRNA expression levels in the LMD samples of NEB microenvironment, control airway epithelium and whole-lung tissue were quantified by qRT-PCR and normalized to the expression level of eEF-2. Fig. 1B shows that, whereas CaSR expression was very low in control airway epithelium samples and almost non-detectable in whole-lung tissue, a relatively high CaSR expression could be observed in the NEB microenvironment, suggestive of a preferential, selective expression of the receptor in this subset of cells of the postnatal lungs.

Immunostaining of cryosections of mouse lungs (PD14) revealed expression of CaSR in selective small cell groups in the airway epithelium (Fig. 2A,C,E). Co-staining with calcitonin gene-related peptide (CGRP) or synaptophysin (Syn), both markers for NEB cells, or single staining for the CaSR in GAD67–GFP mouse lung sections (PD14), resulted in a selective strong CaSR immunoreactivity in the plasma membrane of NEB cells, and in no other airway epithelial cell (Fig. 2A–F). Combined immunostaining for CaSR and P2X₃ or CB, which are each markers for a different subpopulation of intraepithelial vagal sensory nerve terminals that arborize between NEB cells (Brouns et al., 2009a), demonstrated the exclusive expression of the CaSR in NEB cells, whereas the P2X₃- and CB-immunoreactive nerve terminals in NEBs were negative (Fig. 2G–K).

Double staining for CaSR and Syn in adult mouse lungs confirmed the postnatal expression of the CaSR in pulmonary NEBs (Fig. 3A–B). Immunostaining of lung sections from mice at the ages of ED19 (just before birth) and PD0 (day of birth) showed absence of the CaSR in the intrapulmonary airway epithelium during the perinatal period (Fig. 3C,E). Co-immunostaining with CGRP or Syn also confirmed that NEB cells were negative in this time frame (Fig. 3D–F). The specificity of the CaSR antibody, tested on sections of mouse kidney tissue (PD14), showed strong expression of the CaSR in a subset of nephron tubules, but not in the glomeruli, as expected (data not shown) (Riccardi et al., 1998).

Acute exposure of 4-Di-2-ASP-stained and Fluo-4-loaded lung slices (PD14) to a physiological solution containing an elevated [Ca²⁺]_o (i.e. 5 mM) resulted in a transient rise in cytoplasmic Fluo-4 fluorescence selectively in NEB cells, representing a rise in [Ca²⁺]_i (Fig. 4A). The responses to 5 mM [Ca²⁺]_o started 10±3 seconds (n = 10) after the solution entered the perfusion bath. Exposure of the lung slice to a solution containing 2 mM Ca²⁺_o for 30 seconds evoked a much reduced increase in [Ca²⁺]_i. Fluo-4 fluorescence in NEB cells decreased towards baseline levels within 30 seconds of replacing this high [Ca²⁺]_o solution with a standard physiological solution containing 1.2 mM [Ca²⁺]_o. Subsequent exposure of the lung slice to the established positive control stimulus for NEB cells (50 mM K⁺, 5 seconds; Fig. 4A) invariably caused a prominent rise in [Ca²⁺]_i.

Acute, short-term perfusion of 4-Di-2-ASP-stained and Fluo-4-loaded lung slices (PD14) with standard physiological solution containing 1.2 mM [Ca²⁺]_o added with several known CaSR orthosteric agonists – spermine (1.5 mM), lanthanum (La³⁺; 50 µM), neomycin (300 µM) – or the positive allosteric modulator calcimimetic NPS-R568 (100 µM), resulted in strong transient rises in Fluo-4 fluorescence in NEB cells (Fig. 4B).

Fig. 5A shows that repeated applications of elevated [Ca²⁺]_o (5 mM), separated by 5 minute periods of 1.2 mM [Ca²⁺]_o, activated the NEB cells reversibly and reproducibly. A 3 minute pre-application and subsequent co-application of a CaSR negative allosteric modulator, Calhex-231 (20 µM) almost completely prevented NEB cell activation by the CaSR agonists [Ca²⁺]_o (5 mM) or spermine (1.5 mM) (Fig. 5B).

A 10 minute pre-application and subsequent co-application of SKF96365 (25 µM), a selective TRPC channel antagonist, consistently attenuated the increase in [Ca²⁺]_i in NEB cells evoked by the CaSR agonists, [Ca²⁺]_o (5 mM) or spermine (1.5 mM) (Fig. 5C).

Lung slices from 2-week-old mice were maintained in a solution containing 0.8 mM [Ca²⁺]_o, which is below the CaSR threshold of activation. Indeed, at the physiological [Ca²⁺]_o of 1.2 mM, the CaSR is already half-maximally active (Brown et al., 1993). Under these conditions, 2 mM [Ca²⁺]_o (30 seconds) evoked a clear rise in [Ca²⁺]_i in NEB cells (Fig. 6A) that was greater than that observed when baseline [Ca²⁺]_o was 1.2 mM (compare with Fig. 4A). Remarkably, Fluo-4 fluorescence did not return to the initial baseline level in NEB cells but stabilized at a higher fluorescence value, representing a higher [Ca²⁺]_i. Similar to control slices (compare with Fig. 4A), subsequent short stimulation with 5 mM [Ca²⁺]_o resulted in a further increase in Fluo-4 fluorescence in NEB cells, but also in an additional increase in baseline [Ca²⁺]_i (Fig. 6A).

When lung slices were kept in the solution with a [Ca²⁺]_o of 0.8 mM, supplemented with the calcimimetic NPS-R568 (1 µM), no baseline [Ca²⁺]_i changes were seen after exposure of NEB cells to 2 mM and 5 mM [Ca²⁺]_o (Fig. 6B).

When lung slices were kept in standard physiological solution (1.2 mM [Ca²⁺]_o), activation of NEB cells with elevated [Ca²⁺]_o or with selective agonists of the CaSR invariably resulted in a [Ca²⁺]_i rise in NEB cells. Frequently, this was followed by a delayed (5–8 second) rise of [Ca²⁺]_i in Clara-like cells of the NEB microenvironment (Fig. 7A), as has previously been shown when NEBs are activated by depolarization (De Proost et al., 2008).

Short-term stimulation of Fluo-4-loaded lung slices with ATP (50 µM, 10 s) evoked a rise in [Ca²⁺]_i in all Clara cells, including the Clara-like cells surrounding NEBs. In agreement with our previous work (De Proost et al., 2009), no rise in [Ca²⁺]_i was observed in NEB cells (Fig. 7B).

Application of ATP (50 µM, 10 seconds) to lung slices in the presence of the calcimimetic NPS-R568 (10 µM) resulted in a similar [Ca²⁺]_i-mediated activation of Clara-like cells, which could be followed by a delayed [Ca²⁺]_i rise in a neighboring NEB cell (Fig. 7C), suggesting CaSR-dependent paracrine regulation of NEB cells by Clara-like cells.

The present study demonstrated a functional expression of the CaSR, selectively in NEB cells in the intrapulmonary airway epithelium of postnatal mouse lungs (PD14/adult), by (q)RT-PCR, immunostaining, and Ca²⁺ imaging in an in vivo lung slice model.

In fetal mice, CaSR has been detected in airway epithelium, with a high level of expression between ED11.5 and ED16.5 where it regulates branching morphogenesis. Receptor expression rapidly declines after this time-frame (Finney et al., 2008). RT-PCR analysis, performed in the present study, confirmed the expression of CaSR in embryonic mouse lung samples. By contrast, randomly microdissected lung tissue of 2-week-old mice appeared to lack CaSR expression, which is consistent with previously published data concerning whole postnatal mouse lungs (Finney et al., 2008).

The NEB microenvironment accounts for only a minor part of the airway epithelium and, inherently, of the lung in general (<1%). Consequently, it is not surprising that analysis on whole-lung tissue or complete airway epithelium can mask the presence of the very small amounts of mRNA that are specifically expressed in rare cell types. In the present study, LMD allowed for the generation of an enriched pool of cells from the NEB microenvironment, which were shown to express CaSR mRNA by RT-PCR analysis. Moreover, relative quantification of the CaSR, normalized to the expression level of eEF-2, showed that the expression of CaSR was relatively high in the NEB microenvironment, and virtually absent in airway epithelium and whole lung tissue. These experiments highlight the need for tissue microdissection for gene expression analysis of the NEB microenvironment.

Immunostaining revealed that CaSR expression in the NEB microenvironment is restricted to the plasma membrane of NEB cells in 2-week-old and adult mouse lungs, but absent in the airways in general and in NEBs in particular in the perinatal period, implicating an exclusive role for the CaSR in NEB cell physiology after birth.

In perinatal and neonatal airways, NEB function is believed to be related to hypoxia-chemosensing and important for respiratory adaptation during transition to air-breathing (Cutz and Jackson, 1999), a time window in which carotid body function – a well-accepted arterial hypoxia sensor in control of breathing – is still immature (Bollé et al., 2000; Cutz and Jackson, 1999). Because of the lack of CaSR expression in perinatal NEBs, their suggested role as chemo or hypoxia sensors appears to be independent of CaSR signaling.

Both prenatal and postnatal pulmonary NEBs can respond to extracellular environmental changes with a rise in [Ca²⁺]_i and subsequent exocytosis of ATP (De Proost et al., 2008; De Proost et al., 2009; Lembrechts et al., 2012; Schnorbusch et al., 2012). In this study on postnatal mouse lung slices, application of several known CaSR activators –high [Ca²⁺]_o, trivalent cations such as La³⁺, the polyamine spermine and the calcimimetic NPS-R568 – induced a rise in Fluo-4 fluorescence and consequently a [Ca²⁺]_i-mediated activation of NEB cells. Two observations point to an involvement of the CaSR in this process. First, the membrane-impermeant La³⁺, spermine and R568 mimic the effects of [Ca²⁺]_o in their ability to increase [Ca²⁺]_i, and second, their ability to activate NEB cells was consistently inhibited by the selective CaSR-negative allosteric modulator Calhex-231. Together, these observations indicate that the cation-dependent increases in [Ca²⁺]_i in NEB cells are mediated by CaSR.

An important advantage of our lung-slice imaging model is the simultaneous visualization of physiological activity in different epithelial cell types, including the Clara-like cells of the NEB microenvironment (De Proost et al., 2008). In this way, we were able to demonstrate that ATP released from activated NEB cells typically gives rise to a delayed activation of Clara-like cells (De Proost et al., 2009; Lembrechts et al., 2012). In the present study, activation of CaSR expressed on NEB cells resulted in a delayed [Ca²⁺]_i rise in Clara-like cells, indicating that CaSR activation in NEB cells leads to [Ca²⁺]_i mediated exocytosis. These observations are in line with experiments performed on cholecystokinin (CCK)-producing diffuse neuroendocrine cells in the small intestine, that were reported to release CCK after CaSR stimulation (Wang et al., 2011). Recently, we were able to demonstrate that pulmonary NEBs are airway epithelial mechanotransducers, in which the osmosensitive and mechanosensitive channel TRPC5, which is selectively expressed in the apical membrane of NEB cells, was responsible for osmosensing and mechanosensing by NEB cells (Lembrechts et al., 2012). In various other cell types (e.g. smooth muscle cells, breast cancer cells, ventricular cardiomyocytes) CaSR activation has been reported to mediate Ca²⁺ entry through TRPC-encoded receptor-operated and store-operated channels (Chow et al., 2011; El Hiani et al., 2009; Sun et al., 2010). In the present study, application of SKF96365, a general blocker of TRPC channels, inhibited the [Ca²⁺]_i rise in NEB cells evoked by CaSR-dependent stimuli, thereby suggesting a role for TRPC channels in mediating the CaSR-dependent [Ca²⁺]_i influx.

In Fluo-4-loaded live-mouse lung slices, NEB cell clusters reveal a high baseline fluorescence, compared with the surrounding much ‘darker’ Clara-like cells (De Proost et al., 2008), suggesting a relatively higher basal [Ca²⁺]_i concentration. Because NEB cells are neuroendocrine secretory cells, maintaining the basal [Ca²⁺]_i at a relatively strictly controlled high concentration might be crucial for normal NEB physiology. Until now, however, no specific mechanism has been described to support this hypothesis. We therefore explored whether the CaSR is involved in maintaining a stable and relatively high basal [Ca²⁺]_i in NEB cells. The IC₅₀ for CaSR activation has been reported to be reached at around 1.2 mM [Ca²⁺]_o (Brown and MacLeod, 2001). When lung slices were kept in a physiological solution containing 0.8 mM [Ca²⁺]_o, and then subjected to acute 2 or 5 mM [Ca²⁺]_o pulses, [Ca²⁺]_i did not return to the initial baseline. When the sensitivity of the CaSR was shifted leftwards by adding the calcimimetic NPS-R568 to the 0.8 mM [Ca²⁺]_o solution, [Ca²⁺]_i always went back to baseline after the acute [Ca²⁺]_o pulses, suggesting an involvement of the CaSR in setting basal [Ca²⁺]_i in pulmonary NEBs.

The NEB microenvironment is a sensory airway receptor complex in which NEB cells, Clara-like cells and nerve fibers are closely associated (Brouns et al., 2009a; Brouns et al., 2012; De Proost et al., 2009), and well-organized intercellular communication could be essential for normal physiology. Apart from its role in interactions between synaptic NEB cells and nerves (Sorokin and Hoyt, 1989), paracrine signaling between ATP-releasing NEB cells and P2Y₂ ATP-receptor-expressing Clara-like cells has been described after depolarization (De Proost et al., 2009) and specific stimulation of NEB cells (Lembrechts et al., 2012). Until now, Ca²⁺ imaging using agonists for Clara cells and Clara-like cells, such as ATP, was never reported to result in a direct or delayed [Ca²⁺]_i rise in NEB cells (De Proost et al., 2009). In many excitable cell types, agonist-evoked [Ca²⁺]_i signaling events are associated with subsequent active extrusion of Ca²⁺ across the plasma membrane, which would thereby lead to a local [Ca²⁺]_o increase close to the surface of the activated cell (Hofer et al., 2000). In this way, [Ca²⁺]_i mobilization in one cell could produce an extracellular signal that can be detected in closely associated cells, sharing the same microenvironment and expressing the CaSR. The CaSR might therefore mediate a universal form of intercellular communication that allows cells to be informed of the [Ca²⁺]_i signaling status of their neighbors (Hofer et al., 2000). To demonstrate the existence of such a paracrine CaSR-dependent signaling in the NEB microenvironment, we applied the calcimimetic NPS-R568 to lower the threshold for CaSR activation by rendering the CaSR more sensitive to changes in [Ca²⁺]_o. In lung slices subjected to ATP under these conditions, Clara-like cells in the NEB microenvironment showed a typical rise in [Ca²⁺]_i that was occasionally followed by a delayed rise in [Ca²⁺]_i in NEB cells. Because this secondary Ca²⁺-mediated activation of NEB cells, which was never seen under control conditions, is likely to be due to the increased sensitivity of the CaSR, this potential [Ca²⁺]_i signaling is reminiscent of the mechanism proposed first in cell models (Hofer et al., 2000) and subsequently in the intact gastric mucosa (Caroppo et al., 2004).

Apart from its potential role in the physiology of NEB cells, it might also be worthwhile to consider a role for the CaSR as a more general [Ca²⁺]_o sensor in the NEB microenvironment, which is now believed to be a potential stem cell niche in the epithelium of intrapulmonary airways (Bishop, 2004; Giangreco et al., 2007; Guha et al., 2012; Liu et al., 2006; Rawlins and Hogan, 2006; Snyder et al., 2009). Several signaling pathways involved in cell growth and differentiation, adult stem cell proliferation and specification, have been reported to be highly influenced by changes in [Ca²⁺]_o through the CaSR (Riccardi and Kemp, 2012). Activation of CaSR has been linked to a variety of intracellular signaling events that are associated with cell proliferation and/or differentiation (Mamillapalli and Wysolmerski, 2010), depending on the cell type and developmental time-frame (for reviews, see Manning et al., 2006), but also to carcinogenesis (Manning et al., 2006; Saidak et al., 2009). Indeed, aberrant CaSR signaling is found during malignant transformation and is linked to excessive secretion of parathyroid-hormone-related peptide (PTHrP). Furthermore, a CaSR polymorphism found in a hypercalcemia-inducing lung squamous cell carcinoma enhances the CaSR affinity for [Ca²⁺]_o, leading to PTHrP secretion (Lorch et al., 2011). Thus, expression of the CaSR on the plasma membrane of NEB cells, the endocrine cell compartment of the NEB microenvironment, might be an important component in control of proliferation or differentiation in this stem cell niche, for maintenance of the healthy airway epithelium, repair after injury, and for pathological cell proliferation and malignant progression.

Mouse NEBs express CGRP (Brouns et al., 2009a; Uddman et al., 1985), a neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing (Rosenfeld et al., 1983); calcitonin was also reported to be expressed in mouse NEBs (Luts et al., 1991). As a secretory product of thyroid C-cells, calcitonin is well known as an important mediator in thyroid and parathyroid control of [Ca²⁺]_o homeostasis (Freichel et al., 1996; Garrett et al., 1995), in which the CaSR is the key [Ca²⁺]_o sensor (Chang and Shoback, 2004; Riccardi and Kemp, 2012). The literature indicates that the effects of thyroid and parathyroid gland removal on [Ca²⁺]_o homeostasis and plasma calcitonin levels are limited, that the lungs represent an important secondary source of calcitonin, and that the number of pulmonary endocrine cells appears to be upregulated (Becker et al., 1980; Becker and Silva, 1981; Kasacka et al., 2001). It is therefore not unlikely that NEBs, which express both [Ca²⁺]_o-regulating and -sensing proteins, might serve as a back-up system for [Ca²⁺]_o homeostasis in case of thyroid or parathyroid dysfunction.

The CaSR is generally accepted as a polymodal sensor for a broad range of physiologically relevant extracellular molecules. The functional expression of CaSR in the plasma membrane of a complex sensory system such as the NEB, suggests that this receptor senses, integrates and transduces local changes in multiple physiological and pathological stimuli in the airway NEB microenvironment.

In conclusion, the present study revealed a novel role for the CaSR outside the [Ca²⁺]_o homeostatic system, as a key regulator of the postnatal NEB microenvironment. Here, the receptor integrates multiple physiopathological signals and coordinates the dynamic changes in [Ca²⁺]_i and [Ca²⁺]_o, which are essential for functional integrity of the NEB microenvironment. Given the importance of the NEB microenvironment as a sensory receptor and an intrapulmonary airway stem cell niche, this information opens new perspectives for unraveling the postnatal function(s) of pulmonary NEBs.

Lung tissue was obtained from ED14 (n = 3), ED19 (n = 3), newborn (n = 3), postnatal day (PD) 14 (n = 18), and adult wild-type (WT; n = 3) C57-Bl6 mice (Janvier; Bio Services, Uden, The Netherlands), and from GAD67–GFP C57-Bl6 mice (PD14; n = 5; The Jackson Laboratory, Charles River, l'Arbresle, France). The latter is a C57-Bl6 based GAD67–GFP knock-in mouse strain that displays GFP fluorescent pulmonary NEBs, and is referred to here as ‘GAD67–GFP mouse’(Schnorbusch et al., 2013). Kidney tissue was obtained from PD14 WT C57-Bl6 mice. All animals were housed with their mothers in acrylic cages in an acclimatized room (12/12 hour light-dark cycle; 22±3°C) and were provided with water and food ad libitum. National and international principles of laboratory animal care were followed and experiments were approved by the local animal ethics committee of the University of Antwerp. All animals were killed by intraperitoneal injection of an overdose of sodium pentobarbital (Nembutal 200 mg/kg, CEVA Santé Animale, Brussels, Belgium).

Lungs of GAD67–GFP mice (PD14, n = 3) were dissected, immediately snap-frozen in liquid nitrogen and preserved at −80°C. 25-µm-thick cryosections were thaw-mounted on poly ethylene terephthalate (PET) Frameslides (Leica, Wetzlar, Germany) and immediately refrozen and kept at −80°C until further use. NEB microenvironments, control airway epithelium and random whole-lung tissue were excised from the slides by LMD (Leica LMD7000 system) and collected in the cap of a 0.2 ml Eppendorf tube filled with RLT Plus lysis buffer (Qiagen, Hilden, Germany). An embryonic (ED14) mouse lung sample was snap-frozen and processed together with the LMD samples as a positive control.

Total RNA of the LMD samples and the embryonic lung sample (ED14) was isolated using the RNeasy Plus Micro kit (Qiagen). Concentration and integrity of the RNA samples was examined by an Experion automated electrophoresis system (Bio-Rad, Hercules, CA), using the Experion HighSens analysis kit. cDNA was prepared using 2 ng (for the LMD samples) or 1 µg (for the embryonic lung sample) of total RNA, and random hexamers of the SuperScriptIII First-Strand Synthesis SuperMix (Invitrogen, Life Technologies, Gent, Belgium) in a reaction volume of 20 µl, on an MJ Mini Cycler (Bio-Rad).

Taqman gene expression analysis was used for (q)RT-PCR analysis in a multiwell-plate-based system (LightCycler480; LC480; Roche Applied Science, Penzberg, Germany). The primer and probe design was based on the general guidelines for real-time PCR primer design using the Lasergene (DNASTAR) software. A BLAST analysis was performed to confirm the specificity of the primers and the probes. All primers were designed to be intron-spanning to prevent the replication of residual contaminating DNA, and to obtain an amplicon length of 60–120 bp (supplementary material Table S1). Real-time PCR was performed on 5 µl cDNA using the LC480 Probes Master (Roche Applied Science) in a final reaction volume of 20 µl in LC480 white 96 Multiwell Plates (Roche Applied Science). All samples were run in triplicate and both no-template (blanco) and negative RT controls were included in all runs to exclude possible DNA contamination. Reactions were carried out as follows: after an initial denaturation-activation step at 95°C for 10 minutes, amplifications consisted of 50 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 1 second, and were ended with a cooling step at 40°C for 10 seconds. The eukaryotic translation elongation factor-2 (Eef2 which encodes eEf-2) was used as a reference gene (Kouadjo et al., 2007). Relative gene expression differences (qRT-PCR) between LMD NEBs, control airway epithelium, and whole lung tissue of three 2-week-old mice, were calculated using the ΔΔCT-method in the LC480 Software (Roche Applied Science), with Eef2 as a reference gene for normalization and with the NEB sample as calibrator group. The relative gene expression is reported as the mean value ± s.d. Amplification products were separated on a 2% agarose gel and visualized under UV illumination.

For cryosectioning, lungs of WT C57-Bl6 and GAD67–GFP mice were transcardially perfused with standard physiological solution and subsequently filled with 4% paraformaldehyde through the trachea. Lungs, trachea, esophagus and heart were dissected en bloc, degassed and immersion-fixed in the same fixative for 30 minutes. After rinsing in phosphate-buffered saline (PBS; 0.01 M; pH 7.4), tissues were stored overnight in 20% sucrose (in PBS; 4°C), and mounted in Tissue Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands). Cryostat sections (25 µm thick) of the whole tissue blocks were thaw-mounted on poly-L-lysine-coated microscope slides, dried at 37°C (2 hours) and processed for immunolabeling. Immunocytochemical incubations were performed in a closed humidified container (22°C). All primary and secondary antisera were diluted in PBS containing 10% normal horse serum, 0.1% BSA, 0.05% thimerosal and 0.01% NaN₃ (PBS*). Before incubation with the primary antisera, cryostat sections were incubated for 1 hour with PBS* containing 1% Triton X-100. To block non-specific endogenous mouse IgG, sections were preincubated with PBS* containing mouse-on-mouse blocker (M.O.M; Vector labs, Burlingame, CA). Sections were then incubated overnight with one of the primary antibodies listed in supplementary material Table S2. For visualization of the immunostaining, sections were further incubated for 4 hours with secondary antibodies (supplementary material Table S3). After a final wash in PBS, the sections were mounted in Citifluor (19470; Ted Pella, Redding, CA). Negative staining controls for all immunocytochemical procedures were performed by substituting the primary antisera with non-immune sera. To check for possible cross-reactivity after consecutive multiple staining when using two rabbit primary antisera, the results of single immunostaining for the different antisera were evaluated and compared with those from multiple labeling experiments. Kidney tissue cryosections were used as a positive control for the CaSR antibody (Riccardi et al., 1998).

A standard physiological solution was used, containing (in mM): 130 NaCl, 5 KCl, 1.2 CaCl₂.H₂O, 1 MgSO₄.7H₂O, 11 D-glucose, 20 HEPES, adjusted to pH 7.42 with NaOH. Unless stated otherwise, all drugs and chemicals were purchased from Sigma-Aldrich (Bornem, Belgium). Fluo-4 AM (F14201) and 4-Di-2-ASP (D-289) were provided by Molecular Probes (Invitrogen, Life Technologies, Merelbeke, Belgium), Calhex-231 by Santa Cruz Biotechnology (Santa Cruz, CA), NPS-R568 and SKF96365 hydrochloride by Tocris Bioscience (Bristol, UK). All experimental solutions were applied to lung slices that were submerged in physiological solution in a tissue bath (2 ml) mounted on the microscope stage, perfused by a gravity-fed system (flow rate >5 ml/minute) with triggered valves that allowed the fast and reproducible exchange of solutions. For the physiological live-cell imaging experiments, lung slices were restrained in the perfusion chamber with a golden ring spanned with a sheet of nylon mesh.

Tissue slices were prepared from individual mouse lung lobes (WT C57-Bl6; PD14; n = 15) and stained for NEBs using 4-Di-2-ASP as previously published (De Proost et al., 2008; Pintelon et al., 2005). In short, lung tissue was stabilized by instillation of a 2% agarose solution (low-melt agarose A4018). Lung slices were cut using a vibratome (HM650 V; Microm International, Walldorf, Germany) and subsequently incubated for 4 minutes with 4 µM 4-Di-2-ASP in Dulbecco's modified Eagle's medium/F-12 (DMEM-F-12; Invitrogen) at 37°C, rinsed, and kept in DMEM-F-12 in an incubator (37°C; 5% CO₂/95% air) for further use within 12 hours after killing the animal.

Lung slices stained with 4-Di-2-ASP were incubated in physiological solution with 10 µM of the fluorescent [Ca²⁺]_i indicator Fluo-4 AM, 100 µM sulfobromophtalein, 0.1% DMSO and 0.02% Pluronic F-127 for 1 hour at 22°C. The slices were subsequently washed (10 minutes; 22°C) in physiological solution to allow for complete deesterification of intracellular Fluo-4 AM.

An epifluorescence microscope (Zeiss Axiophot, Carl Zeiss, Jena, Germany) equipped with filters for the visualization of FITC/GFP (Zeiss 17; BP475-495/FT510/BP515-565) and Cy3 (Zeiss 14; LP510-KP560/FT580/LP590) was used to screen quickly the immunostaining results. All high-resolution images and live-cell imaging data were obtained using a microlens-enhanced dual spinning disk confocal microscope (UltraVIEW VoX; PerkinElmer, Zaventem, Belgium) equipped with 488 nm and 561 nm diode lasers for excitation of FITC/GFP and Cy3. Images were processed and time-lapse recordings analyzed using Volocity 6.1.1 software (PerkinElmer). For analysis, individual images were studied as grey value datasets. Regions of interest (ROIs) were drawn manually around identified cells of interest. For every ROI, the Fluo-4 fluorescence intensity changes (ΔFluo-4), expressed as arbitrary units (A.U.), were plotted against time. To facilitate interpretation of the results, the presented grey values are relative, and the initial baseline fluorescence intensity was set to zero.

The traces shown for NEBs represent one example of the average changes in Fluo-4 fluorescence intensity of all cells in one NEB (mostly 10–15 cells). Each condition has been tested on multiple NEBs in different slices of several mice.

We thank S. Thys, I. Micalessi and Dr. G. Boulet for their essential help with gene expression studies, F. Terloo for technical assistance, D. De Rijck for help with imaging and illustrations, D. Vindevogel for aid with the manuscript and S. Kockelberg for administrative help.