Distribution of α7 and α4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela)

Tissue and Cell 36 (2004) 391–398

Distribution of ␣7 and ␣4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela) Salvatore Valiantea,∗ , Anna Capaldoa , Francesca Virgilioa , Rosaria Sciarrillob , Maria De Falcoa , Flaminia Gaya , Vincenza Laforgiaa , Lorenzo Varanoa a

Department of Evolutionary and Comparative Biology, University of Naples “Federico II”, 80134 Naples, Italy b Department of Biological and Environmental Sciences, University of Sannio, 82100 Benevento, Italy Received 18 March 2004; received in revised form 17 May 2004; accepted 30 June 2004

Abstract The distribution of neuronal and non-neuronal mRNAs for ␣7 and ␣4 nicotinic acetylcholine receptor subunits was investigated in Triturus carnifex tissues using the in situ hybridization approach. The findings reveal a composite pattern of expression only partially overlapping for the two subunits; subunit ␣7 seems to be expressed widely throughout nervous, gastrointestinal and skin tissues, while ␣4 is present in a restricted number of cells of nervous and gastrointestinal tissue. We also found a specific pattern for each subunit; ␣7 and ␣4 associated exclusively to the epidermal glands and hypophysis, respectively; this is probably due to alternative roles that nicotinic acetylcholine receptors play in regulating physiological functions of non-neuronal amphibian tissues, rather than as mere neurotransmitters in the nervous system. © 2004 Elsevier Ltd. All rights reserved. Keywords: Acetylcholine; Triturus carnifex; In situ hybridization; Nicotinic receptor; Amphibian

1. Introduction Nicotinic receptors belong to the ligand-gated ion channels superfamily, which includes receptors for glycine, ␥-aminobutyric acid (GABAA ) and serotonin 3 (5-HT3 ) (Albuquerque et al., 1997). They modulate rapid postsynaptic effects in the central and peripheral nervous system and in skeletal-muscle membranes (Sargent, 1993), being one of the components of the well-known cholinergic system involved in neurotransmission, although a complete cholinergic system, consisting of acetylcholine (ACh), its synthesizing enzyme choline acetyltransferase (ChAT), its inactivating enzyme acetylcholinesterase (AChE) and acetylcholine receptors (AChrs), has also been found in primitive organisms (Horiuchi et al., 2003) and in several non-excitable tissues suggesting an important role for ACh in non-neuronal tissues. Indeed, some of the basic cellular ∗

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functions such as proliferation, differentiation, apoptosis and secretion are under ACh control through the interaction with AChrs, as autocrine and/or paracrine modulation (Wessler et al., 1998; Grando et al., 2003). In particular, nicotinic acetylcholine receptors (nAChrs) seem to have a widespread distribution; they can be found on epithelial, mesothelial and endothelial cells in humans as in other animals (Wessler et al., 1998); they may play a role in angiogenesis through the nicotinic-induced enhancing of endothelial cell proliferation (Villablanca, 1998; Heeschen et al., 2001). Human keratinocytes synthesize ACh and express nicotinic subunits ␣3, ␣5 and ␣7 of AChrs due to their involvement in the keratinization of the epidermis (Nguyen et al., 2001; Grando et al., 1996). Moreover, lymphocytes isolated from several tissues express nAChrs (Kawashima and Fujii, 2000). nAChrs have been well characterized and classified in subfamilies sharing common features; subunits ␣7 and ␣4 belong to subfamilies I and III, respectively, the former has a binding site for ␣-bungarotoxin and can form homo-oligomers the latter, without a ␣-bungarotoxin binding site, is able to form

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only heteropentamer receptors, mainly with ␤-subunits. Subfamily I is probably evolved before separation between insects and vertebrates, while subfamily III appeared with chordates (Le Novere and Changeux, 1995). This evolutive distance and such pharmacological differences have been used for the choice of subunits in our experiments. We have previously shown the effects of ACh administration on the interrenal glands of the urodele Triturus carnifex, demonstrating the ACh role in the paracrine interactions between the two tissues forming these glands (chromaffin and steroidogenic) (Capaldo et al., 2002). Information on nonneuronal nAChrs in lower vertebrates is very scant although ACh is one of the oldest signalling molecules in nature, widely distributed in bacteria, animals and plants (Wessler et al., 1998). Against this background, we investigated the presence and distribution of nicotinic AChrs in the amphibian T. carnifex tissues, namely the brain, hypophysis, stomach, gut and skin, using the in situ hybridization approach.

2. Materials and methods Animals were captured in the region of Campania (southern Italy), fed ad libitum and exposed to natural photoperiod and temperature. The experiments were admitted by institutional committees (Italian Ministry of Health) and organized to minimize the number of animals used (number = 30). Throughout the experiments solutions were made exclusively with double distilled water treated with 0.1% of diethylpyrocarbonate (Sigma) and autoclaved. Immediately after anaesthesia and killing, tissues were excised and fixed 24 h in Bouin’s fluid (71% formaldehyde, 5% acetic acid and 24% picric acid) for both Mallory’s staining and in situ hybridization or 2 h in paraformaldehyde 4%/PBS for in situ experiments. Samples were then embedded in paraffin wax and sectioned at 5–7 ␮m on a rotary microtome, sections were lifted onto baked superfrost glass slides (Menzel-Glaser). A Clustal X (version 1.81) (Thompson et al., 1997) comparison of known amino acidic sequences for ␣7 and ␣4 nAChrs subunits was carried out to determine the degree of conservation through tetrapods; the percentage of conservation of sequences (identities plus single amino acidic substitution) was about 70% and 83% for ␣7 and ␣4, respectively. These high values of conservation and the high stringency of washings and temperature in the experimental procedure (see below) allowed us to use a heterologous probe source without affecting the significance of results. Four riboprobes, two

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Table 1 Degree of expression of ␣7 and ␣4 in T. carnifex tissues Tissue

␣7

␣4

Brain Anterior parvocellular nuclei Medulla oblongata

+++ +

+ +−

Stomach Longitudinal muscular fibres Tubular glands Epithelial cells

+++ − +++

+− − +

Intestine Goblet cells Muscular fibres

+++ ++

++ −

Integumentary system Mucous glands Granular glands Epidermic cells Connective cells

++ − − −

− − − −

Hypophysis Adenohypophysis Neurohypophysis

− −

++ −

(−) undetectable, (+/−) very low expression level (>0% up to 25% of positive cells), (+) low/medium expression level (>25% up to 50% of positive cells), (++) medium/high expression level (>50 up to 75% of positive cells), (+++) high expression level (>75% up to 100% of positive cells).

sense and two antisense of 800 bp each, were synthesized using as template cDNAs for ␣7 and ␣4 chicken nicotinic AChrs, coding for 3 region and for cytoplasmic loop 3 -end of subunits ␣7 and ␣4, respectively, kindly provided by Prof. Ballivet and Dr. Barabino (Geneva), inserted in PBluescript plasmid (Stratagene). After plasmid purification using an Ultraclean miniplasmid kit (MoBio Laboratories) and cleavage with the appropriate restriction enzymes, RNA probes were synthesized and labeled with digoxigenin using the DIG RNA labeling kit (Boeringher-Mannheim) or T3 RNA polymerase (Fermentas) following the manufacturer’s instructions. They were checked for labeling efficiency by spotting them on positively charged nylon membranes (Boeringher-Mannheim) with the NBT/BCIP (Sigma) colorimetric method. After removing paraffin wax with xylene and rehydrated sections in alcohol, slides were dipped into PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ) and 4% paraformaldehyde, then in Tris–HCl 1 M pH 7.0, EDTA 0.5 M pH 7.2 containing proteinase K 10 ␮g/ml. A second paraformaldehyde step was carried out, after which slides were immersed in prehybridization buffer, 50% deionized

Fig. 1. In situ hybridization performed with ␣7 antisense probe on several tissues of T. carnifex. 20 mm scale bar corresponds to: (a–a1–c–c1–f) 50 ␮m; (b–e) 16.5 ␮m; (d) 6.5 ␮m. (a) Longitudinal section of T. carnifex stomach. Longitudinal muscular fibres marked for ␣7 antisense probe (∗ ). Strong signal arises from epithelial cells ( ). Cells of the tonaca sottomucosae (TS) appear only weakly labeled. (a1) Sense control for ␣7 riboprobe on T. carnifex stomach, showing no signal. (b) Mallory trichromic stain of intestinal section showing the histology of gut. Connective tissue (CT), goblet cells (→). (c) Consecutive intestinal section showing labeling for subunit ␣7. Connective tissue (CT) is clear, while goblet cells (→) express strong signal. (c1) T. carnifex gut treated with sense ␣7 riboprobe. The absence of hybridization signal is evident in the whole section. (d) Higher magnification of the gut section, where ␣7 labeling occurs in goblet cells (GC) cytoplasm. (e) Epidermal gland in T. carnifex skin strongly labeled with ␣7 antisense probe. (f) Mallory’s trichromic stain of the skin section. The histology of amphibian skin can be noted. Dermis (D), epidermis (E), melanophore (→), epidemal glands (EG).

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Fig. 2. In situ hybridization performed with ␣7 antisense or sense probe on brain and hypophysis of T. carnifex. 20 mm scale bar corresponds to: (a–b) 50 ␮m; (c–d) 16.5 ␮m. (a) Section of T. carnifex brain treated with ␣7 antisense probe. Hybridization signal occurs widely in the anterior preoptic parvocellular nuclei (PPN) surrounding the preoptic recess (PR). (b) Consecutive slide treated with ␣7 sense probe. No labeling in any brain structure is evident; ventral portion of thalamus (VT). (c) Hypophysis of T. carnifex stained with Mallory’s staining. Adenohypophysis (AH), neurohypophysis (NH). (d) Consecutive hypophysis slide treated with ␣7 antisense riboprobe showing no signal.

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formamide (Applichem), SSC 5× (Applichem), Denhardt solution 1× (Sigma), denatured salmon sperm DNA 100 ␮g/ml (Applichem), tRNA 100 ␮g/ml (Boeringher-Mannheim), 20% dextran sulphate (Sigma), for 1 h at 70 ◦ C. The hybridization step was performed overnight at 70 ◦ C in the same buffer with 1 ng/␮l of ␣7 and ␣4 sense or antisense riboprobes. Post-hybridization washings were carried out consecutively in SSC 0.5× and 20% formamide (Sigma) prewarmed to 60 ◦ C, then in NTE (NaCl 0.5 M Tris 10 mM pH 7.2, EDTA 5 mM), containing RNAse A 10 ␮g/ml (Applichem) at 37 ◦ C for 30 min. Sections were then treated with blocking solution, maleic acid 100 mM with 2% blocking reagent (BoeringherMannheim) and 10% normal sheep serum (Sigma). A 1:5000 dilution of anti-DIG alkaline phosphatase conjugated antibody (Boeringher-Mannheim) was used and after several TBS (100 mM NaCl, 10 mM Tris–HCl pH 8.0) washings, slides were covered with a solution of 0.1% Tween 20 (Sigma), 0.5 mg/ml levamisole (Sigma) and BM Purple (Boeringher-Mannheim) to ensure colour development reaction. Finally sections were mounted with Acquovitrex (Carlo Erba), hybridization signal analyzed with Axioskope System (Zeiss) and images acquired by KS 300 software (Zeiss). Three observers separately evaluated, using KS 300 software (Zeiss), the hybridization signal of each probe on each tissue obtaining the percentage of positive cells. The level of concordance, expressed as the percentage of agreement between the observers was 93%; our results are summarized in Table 1. All control slides were treated with sense probe for ␣7 or ␣4 nAChr subunits. All experiments were performed in triplicate giving the same labeling pattern.

3. Results 3.1. ␣7 riboprobe In the digestive tract labeling occurs in the stomach, where positivity is evident in the longitudinal muscular fibres (Fig. 1a), while tubular glands of stomach seem to be only weakly labeled for ␣7 (Fig. 1a). Further labeling occurs in the epithelial cells of stomach lumen (Fig. 1a). The expression pattern of ␣7 in the small intestine is associated with goblet cells (Fig. 1c) which are strongly labeled. At higher magnification, it is evident that labeling occurs near the cell membrane of goblet cells close to enterocytes (Fig. 1d). Controls show no labeling for ␣7 sense probe (Fig. 1a1 and c1).

The integumentary system of this amphibian is rich in mucous glands and granular glands (Fig. 1f) that are involved in many environmental interactions, mainly through the secretion of mucous and poison and/or peptides, respectively. In the integumentary system labeling for ␣7 is very selective, being present only within epidermal glands embedded in the dermis, while the other histological structures of the dermis and epidermis, like several layers of epidermic cells, are not labeled (Fig. 1e). ␣7 mRNA has a widespread distribution in the T. carnifex brain as assessed by in situ hybridization experiments (Fig. 2a). Cellular bodies are strongly labeled in the anterior parvocellular preoptical nuclei, indicating a high degree of expression for this messenger (Fig. 2a). All tissues treated with sense riboprobe do not show any kind of signal (Fig. 2b). The hypophysis of T. carnifex is divided into an anterior portion which contains secretory cells (Fig. 2c) and a posterior portion formed by nerve endings that secrete neurohormones (Fig. 2c). Neither adeno- or neurohypophysis are labeled for ␣7 antisense probe (Fig. 2d). 3.2. ␣4 riboprobe Skin does not show labeling for ␣4 mRNA both in epidermis and dermis, in particular neither mucous glands nor epidermic cells are labeled (Fig. 3a). In situ hybridization performed with ␣4 anti-sense riboprobe in the brain shows the expression of ␣4 mRNA restricted to cellular bodies of neurons (Fig. 3b) and weak labeling occurs in the same regions of ␣7 expression. The main result can be evidenced in the hypophysis of T. carnifex where an exclusive pattern for ␣4 mRNA is found: while neurohypophysis is not labeled (Fig. 3c), adenohypophysis cells had cytoplasm rich in mRNA for ␣4 receptor subunit (Fig. 3c). In the digestive apparatus of T. carnifex ␣4 mRNA can be found in the stomach, expressed in the gastric mucosae cells (Fig. 3e) but not in the muscular tissue (Fig. 3e) or in the tubular glands (Fig. 3e). In the gut ␣4 mRNA has a distribution similar to that of ␣7: goblet cells are marked for ␣4 anti-sense probe (Fig. 3d). All controls treated with ␣4 sense probe have no signal (Fig. 3f and g). We summarized the degree of expression of ␣7 and ␣4 nicotinic receptors in each tissue in Table 1. All tissues express both subunits with substantial differences between different organs and with significant differences within the same organs.

Fig. 3. In situ hybridization performed with ␣4 riboprobes on several tissues of T. carnifex. 20 mm scale bar corresponds to: (a–b–d–g) 16.5 ␮m; (c–e–f) 6.5 ␮m. (a) Skin section showing no labeling for ␣4 antisense riboprobe, counterstained with nuclear fast red (Vector). (b) Sagittal brain section at the medulla oblongata level. Neuronal cell bodies (NC) are labeled for ␣4 while nervous fibres (NF) are without signal. (c) Hypophysis section. It is evident the contrast between the two parts of the gland: adenohypophysis (AH) strongly labeled and neurohypophysis (NH) not labeled. Note the absence of hybridization signal in the red blood cells (white arrows). (d) Stomach section. It should be noted that not all structures are labeled. Secreting cells in the gastric mucosae (MG) express the ␣4 nicotinic subunit while muscular cells (MC) and tubular glands (TG) are clearly without signal. (e) Gut section. Hybridization labeling arises from goblet cells. Note that nuclei are clear suggesting a high degree of specificity of the reaction. (f) Gut section. Using ␣4 sense riboprobe no signal is present in the goblet cells. (g) Stomach sagittal section treated with ␣4 sense riboprobe without any kind of signal.

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4. Discussion In a general perspective this study highlighted a different distribution pattern for the two subunits of nAChrs in tissues; we showed a wide expression of ␣7 and a more restricted localization for ␣4 mRNA in amphibian tissues with some interesting peculiarities. In the brain this difference is evident, probably due to the basic nervous functions of ␣7 in synaptic transmission. The limited expression of ␣4 in the brain could be explained with the presence of most homo-pentameric ␣7 receptors, even if we cannot exclude that other subunits not tested are coexpressed with ␣4; this model is also supported by what other authors found in the mammalian brain with most nAChrs containing either ␣4␤2 or ␣7 subunits (Schoepfer et al., 1990; Charpantier et al., 1998). Conversely, the clearly specific localization of the hybridization signal found in the hypophysis, with ␣4 mRNA expressed only in the adenohypophysis and the total absence of ␣7mRNA in both portions, suggests a particular role for subunit ␣4 but not for ␣7 in the adenohypophysis of T. carnifex; in the pars distalis of rat hypophysis previously the presence of AChE and the absence of ChAT activity has been shown, regard the occurrence of acetylcholine binding sites (Barron and Hoover, 1983). In this view we could postulate a non-neuronal hypothesis of control of the anterior lobe by the posterior lobe of the hypophysis also in this urodele, through a non-␣7 nAChr. It is plausible that, in the adenohypophysis, subunit ␣4 could set up heteropentameric receptors with other subunits like ␣10 as our further investigations indicate (data not published). Considering the pattern found in the integumentary system, it is noteworthy that the expression of nAChrs is limited to the ␣7 subunit only in a very restricted area, the epidermal glands in the dermis, probably due to ACh control of the physiological secretion process, via cholinergic innervation of these glands. Both ␣7 and ␣4 subunits seem to be highly expressed within the epithelium of the stomach of T. carnifex. Both in humans and rats it has been shown that ACh and its related enzymes are present in the luminal side of the stomach where they are important for many functions (Wessler et al., 1998) and our findings are consistent with this pattern. Also in the small intestine we have found that goblet cells, which are responsible for mucous production, are labeled for both mRNA subunits. In other species, it has been reported that these cells degranulate in response to ACh (Specian and Neutra, 1980; Neutra et al., 1982; Phillips et al., 1984). We would tend to exclude neuronal pathways of stimulation of goblet cells: although cholinergic neurons are present in the myenteric and submucosal plexus, so they can innervate intestinal epithelium stimulating enterocytes to secrete chloride, it should be noted that this process occurs via muscarinic receptors (Cooke, 1984; O’Malley et al., 1995); it is also important to note that luminal epithelial cells express ChAT, the enzyme that synthesizes ACh (Klapproth et al., 1997). We can postulate the hypothesis of paracrine control of epithelial cells on mucous-secreting cells, acting through the interaction of

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ACh produced by enterocytes with nAChrs exposed on goblet cell membranes. Even if further investigations should be made to verify these hypotheses, the different localization of mRNAs for subunits ␣7 and ␣4 shown in this study extends our knowledge of the neuronal distribution of nAChrs in lower vertebrates and supports the presence of non-neuronal nAChrs in the amphibian T. carnifex.

Acknowledgements We are indebted to Prof. Ballivet and Dr. Barabino for the plasmid gift and to Mr. Giuseppe Falcone for his contribution to the images elaboration.

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