Evidence of solitary chemosensory cells in a large mammal: the diffuse chemosensory system in Bos taurus airways

Abstract

The diffuse chemosensory system (DCS) of the respiratory apparatus is composed of solitary chemosensory cells (SCCs) that resemble taste cells but are not organized in end organs. The discovery of the DCS may open up new approaches to respiratory diseases. However, available data on mammalian SCCs have so far been collected from rodents, the airways of which display some differences from those of large mammals. Here we investigated the presence of the DCS and of SCCs in cows and bulls (Bos taurus), in which the airway cytology is similar to that in humans, focusing our attention on detection in the airways of molecules involved in the transduction cascade of taste [i.e. α-gustducin and phospholipase C of the β2 subtype (PLCβ2)]. The aim of the research was to extend our understanding of airway chemoreceptors and to compare the organization of the DCS in a large mammal with that in rodents. Using immunocytochemistry for α-gustducin, the taste buds of the tongue and arytenoid were visualized. In the trachea and bronchi, α-gustducin-immunoreactive SCCs were frequently found. Using immunocytochemistry for PLCβ2, the staining pattern was generally similar to those seen for α-gustducin. Immunoblotting confirmed the expression of α-gustducin in the tongue and in all the airway regions tested. The study demonstrated the presence of SCCs in cows and bulls, suggesting that DCSs are present in many mammalian species. The description of areas with a high density of SCCs in bovine bronchi seems to indicate that the view of the DCS as made up of isolated cells totally devoid of ancillary elements is probably an oversimplification.

Introduction

A diffuse chemosensory system (DCS) has recently been described in the rat respiratory and digestive apparatuses. The DCS is composed of secondary receptor cells (i.e. solitary chemosensory cells, SCCs), which resemble taste cells but are not organized in end organs (Sbarbati & Osculati, 2003, 2005). The idea of the existence of a DCS on the epidermis and mucosal surfaces of vertebrates is not new – the same concept was expressed by Whitear in 1992 – but until 1998 the evidence was limited to fish. Bipolar cells in teleost epidermis were stained intra-vitam with methylene blue by Whitear (1952), who later identified and confirmed their innervation (Whitear, 1965, 1971). These studies demonstrated that the skin and the oropharyngeal surfaces of primary aquatic vertebrates are provided with a diffuse system of chemoreceptors, related to but distinct from the gustatory system. In recent years, several groups have contributed to a precise characterization of the func-tional role of SCCs in fish. SCC chemosense occurs in anamniote acquatic craniates and may be used for feeding or predator avoidance (Finger, 1997). The SCC system responds to mucus substances and may serve as a predator detector (Peters et al. 1991).

SCCs were later described in a mammal (Sbarbati et al. 1998) and the presence of the taste cell-related G protein α-gustducin confirmed a similarity with taste cells (Sbarbati et al. 1999). The presence of SCCs in mammals was then confirmed by several studies (Zancanaro et al. 1999; El-Sharaby et al. 2001a,b; for a review see also Sbarbati & Osculati, 2003). A relationship between SCCs and the gustducin-immunoreactive cells described in the digestive apparatus (Höfer et al. 1996) was also suggested (Sbarbati & Osculati, 2003, 2005).

Despite this morphological evidence, the first functional data were obtained only in 2003, when it was demonstrated that the SCCs localized in the nasal cavity operate in the detection of irritants (Finger et al. 2003). Recently, the same group also demonstrated that nasal SCCs proliferate and undergo rapid turnover (Gulbransen & Finger, 2005).

In the last 2 years, research into SCCs has opened exciting new possibilities. It has been demonstrated that SCCs are diffusely present in the airways, both in the larynx (Sbarbati et al. 2004a,b) and in the trachea/bronchi (Merigo et al. 2005). Differences between the innervation patterns of laryngeal SCCs and laryngeal taste buds have also been demonstrated (Finger et al. 2005).

The demonstration of a DCS in the airways raises questions about the role of chemoreceptors in control of complex functions (e.g. airway surface liquid secretion) and about the involvement of chemoreceptors in respiratory diseases. Preliminary data from our laboratory seem to suggest that this portion of the chemoreceptive apparatus provides information about the milieu covering the mucosa, which is rich in secretory and metabolic products of microorganisms.

In principle, these findings greatly extend the role of the DCS, but so far all the mammalian data have been obtained in rodents. This could be a major problem because small mammals have airways that are quite different from those of humans and large mammals; in particular, small mammalian airways are poor in mucous cells and rich in serous cells.

In this study we therefore investigated the presence of the DCS and of SCCs in a large mammal species, i.e. cows and bulls (Bos taurus), in which the airway cytology is similar to that of humans, focusing our attention on detection in the airways of molecules involved in the transduction cascade of taste, i.e. α-gustducin and phospholipase C of the β2 subtype (PLCβ2).

Our aim was to extend understanding of airway chemoreceptors and to compare the organization of the DCS in a large mammal with that previously found in rodents.

Materials and methods

Animals

The study was conducted on adult cows and bulls (weight 400–450 kg; Carni Martinelli S.R.L., Montecchia di Crosara, Verona, Italy). The animals were anaesthetized and killed with a compressed air gun and handled in accordance with the guidelines for animal experimentation laid down by Italian law.

Immunoblotting

Various bovine tissues, i.e. circumvallate papillae, epiglottis, arytenoids, trachea, kidney and bronchi (from a pool of three animals) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (6–10%), loading 20 µg of protein into each lane. Proteins were subsequently transferred to nitrocellulose, blocked with 3% bovine serum albumin in Tris-buffered saline and 0.1% Tween 20 (pH 7.4), and incubated for 24 h at 4 °C with the primary antibody diluted with Tris-buffered saline and 0.1% Tween 20 : rabbit polyclonal antibody anti-α-gustducin 1 : 2000 (Santa Cruz Biotechnology, Heidelberg, Germany). As a secondary antibody, horseradish peroxidase-labelled goat anti-rabbit immunoglobulin G was used (Vector Laboratories, Burlingame, CA, USA) at a working concentration of 0.5–5.0 µg mL⁻¹. Bound immunoglobulins were visualized by the chemiluminescence technique using enhanced chemiluminescent reagents (Amersham, Freiburg, Germany).

Immunocytochemistry

Bovine circumvallate papillae, fungiform papillae, larynges, trachea and bronchi were removed and fixed by immersion in 4% paraformaldehyde in 0.1 m phosphate buffer (PB), pH 7.4, for 4 h at 4 °C. After fixation, specimens were placed in 30% sucrose overnight and cut at a thickness of 10–30 µm on a freezing microtome (Reichert-Jung, Vienna, Austria). Sections were collected on polylysine-coated slides, dried overnight at 37 °C and processed for immunocytochemistry. Briefly, endogenous peroxidase was quenched by immersion in a solution of 0.3% hydrogen peroxide in methanol for 30 min. Sections were blocked for 1 h in blocking solution consisting of 0.3% Tween 20, 1% bovine serum albumin (BSA), 1% normal swine serum in 0.1 m phosphate-buffered saline (PBS). Subsequently, sections were incubated overnight at room temperature with primary antibodies raised in rabbits and directed against rat α-gustducin (1 : 400; Santa Cruz Biotechnology) and rat PLCβ2 (1 : 1500; Santa Cruz Biotechnology), diluted with blocking solution. After three washes, sections were then reacted with biotinylated swine anti-rabbit immunoglobulins (DAKO, Milan, Italy) diluted 1 : 400 for 1 h. The immunoreaction was detected using the avidin–biotin complex (ABC) technique with a Vectastain Elite ABC kit (Vector Laboratories) and visualized with 3,3′-diaminobenzidine tetrahydrocloride (DAKO) for 5–10 min. Finally, sections were dehydrated through a descending ethanol series and twice with xylene and mounted with Entellan. Control sections were produced omitting the primary antibodies to confirm the specificity of the antibody. No controls exhibited immunolabelling. Sections were observed by means of an Olympus BX51 photomicroscope equipped with a KY-F58 CCD camera (JVC). Electronic images were analysed and stored using Image-ProPlus software (Media Cybernetics, Silver Spring, MD, USA).

Results

Using immunocytochemistry for α-gustducin, the bovine taste bud cells of both circumvallate (Fig. 1a–c) and fungiform (Fig. 1d–f) papillae of the tongue were stained. Numerous immunoreactive taste buds were also visible on the arytenoid epithelium (Fig. 1g–i). In the bovine trachea (Fig. 1l–n) and bronchi (Fig. 1o–q), α-gustducin-immunoreactive SCCs were frequently found. In the trachea, these cells were elongated bipolar elements with an apical process reaching the free surface. In the bronchi, flask-shaped elements devoid of a basal process were also found.

Fig 1.

Sections taken from circumvallate papillae (a–c), fungiform papillae (d–f), arytenoids (g–i) and various regions of the respiratory tract (trachea, l–n; bronchi, o–q) of adult cows and bulls, showing α-gustducin-immunoreactive taste bud cells and SCCs. Scale bars = 150 µm (a), 100 µm (d, e, g, h), 50 µm (b, c, f, i), 5 µm (l–q).

Using immunocytochemistry for PLCβ2 (Fig. 2A), the staining pattern was found to be basically similar to those seen with α-gustducin. Both bovine taste buds and SCCs were PLCβ2 immunoreactive (Fig. 2Aa–i). Figure 2(Ah) and 2(Ai) show an area with a high density of SCCs. These SCCs appear to be arranged in parallel lines, with their apical processes running toward the free surface.

Fig 2.

Sections taken from circumvallate papillae (a–c), fungiform papillae (d–f) and various regions of the respiratory tract (trachea, g; bronchi, h, i) of adult cows and bulls showing PLCβ2-immunoreactive taste bud cells and SCCs. Scale bars = 300 µm (d), 100 µm (a, b, e), 50 µm (c, f), 5 µm (g–i).

An immunoblotting experiment (Fig. 3) confirmed the expression of α-gustducin in the bovine airway. The result was positive in the circumvallate papillae of the tongue and in all the airway regions tested (epiglottis, arytenoids, trachea and bronchi). The result was negative in the control tissue (kidney).

Fig 3.

Immunoblotting of bovine tissue reacted with the polyclonal anti-α-gustducin antibody. 1, circumvallate papillae; 2, epiglottis; 3, arytenoids; 4, trachea; 5, kidney; 6, bronchi. With the anti-α-gustducin antibody in cows and bulls we obtained a band at 40 kDa. Markers (kDa): 45 000 and 30 000.

There was no sex difference in the distribution of taste buds and SCCs.

Discussion

The gustatory system in bulls and cows

Although bovine taste cells are used for molecular biology experiments (Ming et al. 1998) previous studies on the bovine gustatory system are rare (Scala et al. 1995). In these studies the presence of laryngeal taste buds has been described as related to rumination. This was confirmed in our study, which found a pluristratified epithelium in the arytenoids and in the tongue, and also a sufficient number of laryngeal taste buds to confirm the probable functional role of these laryngeal receptors as protectors of the deep airways in ruminants, preventing food particles from entering the larynx in the regurgitation phase.

Using immunocytochemistry it has been demonstrated that bovine taste buds contain α-gustducin (Tabata et al. 2003). Our experiments confirm and extend previous results, showing how another chemoreceptorial cascade molecule (i.e. PLCβ2) is also expressed.

Taken together, therefore, our data demonstrate that taste markers previously described in rodents and in humans seem to mark chemoreceptor cascade molecules in bulls and cows, both in the tongue and in the airways.

The diffuse chemosensory system

This was the first study to demonstrate the presence of SCCs in a large mammal. The general organization of the system described in rodents (Sbarbati & Osculati, 2005) appears to be confirmed in cows and bulls in the present study. Nevertheless, some differences must be noted. In B. taurus, SCCs appear to be longer than in rodents. Whereas in rodents, SCCs generally have a flask profile, in bulls and cows they tend to have an accentuated bipolar profile. These differences are probably related to the greater thickness of the epithelium in bulls and cows.

Moreover, SCCs in B. taurus present a singular pattern of organization, particularly in the bronchi. They are arranged in parallel intercalated with epithelial elements, very different from what is observed in rodent distal airways. This organization strongly resembles the arrangement of SCCs in fish, particularly in the areas where SCCs are most common (e.g. in vibratile rays) (Whitear, 1992).

This arrangement seems to suggest that in large animals, along with a greater extent of the respiratory tree, there are distal areas with unusual sensitivity. The possible relation between sensorial corpuscles and neuroepithelial bodies, which are thought to be respiratory gas receptors, should be studied in detail.

Concluding remarks

Although they were only recently discovered, research on SCCs in mammalian airways is rapidly expanding and the data obtained by various research groups are very consistent. The presence of SCCs has been verified in all mammalian species studied so far, which suggests that they are widely present in mammalian species. The analogy between SCCs and various elements which in the past were named in different ways (for a review see Sbarbati & Osculati, 2005), represents a strong confirmation of this hypothesis. The finding of areas with a high density of SCCs in bovine bronchi (which had already been noted in the rodent larynx) would seem to indicate that the view of the DCS as comprising simple separate elements, totally devoid of ancillary elements, is probably an oversimplification.