Elsevier

Life Sciences

Volume 80, Issues 24–25, 30 May 2007, Pages 2308-2313
Life Sciences

Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium

https://doi.org/10.1016/j.lfs.2007.01.046Get rights and content

Abstract

Acetylcholine (ACh) and its receptors play a crucial role in bladder physiology. Here, we investigated the presence of muscarinic receptor subtypes (MR) and nicotinic acetylcholine receptor (nAChR) α-subunits in the mouse urothelium by RT–PCR and immunohistochemistry. With RT–PCR, we detected mRNAs coding for all of the five different MR subtypes and for the nicotinic receptor subunits α2, α4, α5, α6, α7, α9 and α10, whereas the α3-subunit was not expressed. Using immunohistochemistry, we localised a panel of acetylcholine receptors in the different layers of the murine bladder urothelium, with predominant appearance in the basal plasma membrane of the basal cell layer and in the apical membrane of the umbrella cells. M2R and subunit α9 were observed exclusively in the umbrella cells, whereas the MR subtypes 3–5 and the nAChR subunits α4, α7 and α10 were also detected in the intermediate and basal cell layers. The subunit α5 was localised only in the basal cell layer. In conclusion, the murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since these receptors have different electrophysiological and pharmacological properties, and therefore are considered to be responsible for different cellular responses to ACh, this differential distribution is expected to confer cell type-specificity of cholinergic regulation in the bladder urothelium.

Introduction

Acetylcholine (ACh) is a major regulator of urinary bladder function, acting upon virtually every structural component of the bladder including the detrusor muscle, nerve terminals and the urothelium (Chess-Williams, 2002, Andersson and Wein, 2004, Birder, 2005, Hegde, 2006). There are two principal classes of ACh receptors, i.e. muscarinic receptors (MR) and nicotinic receptors (nAChR). Five subtypes of MR (M1R–M5R) are known, all of which are G-protein coupled receptors. In contrast, nAChR are ligand-gated ion channels. Outside the skeletal muscle motor endplate, functional nAChR are composed of homo- or heteropentamers of either α-subunits alone or α- and β-subunits. Subtype composition determines receptor characteristics such as preferred ligand affinity and cation permeability. The α-subunit carries the ligand binding site. Beside the α1-subunit that is localised at the motor endplate, α-subunits 2–7, 9 and 10 are expressed in various mammalian cells (Lukas et al., 1999, Elgoyhen et al., 2001).

Based on RT–PCR and Western blot data, both MR (rat: Tong et al., 2006; human: Mansfield et al., 2005, Tyagi et al., 2006) and nAChR (rat: Beckel et al., 2006) are expressed in the bladder mucosa, but very little is known as to which specific cell types carry the various MR and nAChR subtypes. In the epidermis, both MR and nAChR subtypes display a highly differentiated distribution pattern among the layers and subserve layer-specific functions such as regulation of proliferation, migration, and terminal differentiation and apoptosis (Grando, 1997, Ndoye et al., 1998, Arredondo et al., 2002, Nguyen et al., 2004, Kurzen et al., 2004). We hypothesized that, similarly, receptor distributions are also cell-type specific in the urothelium, and addressed this question by immunohistochemistry. We chose mice for this study in view of the increasing use of this species in elucidating bladder physiology and pathophysiology and the availability of MR and nAChR-subunit knockout mice for further functional investigations. Since data on the expression of cholinergic receptors in the murine bladder have not been reported previously, our immunohistochemical investigations were preceded by RT–PCR to support the antibody data with an independent technique.

Section snippets

RT–PCR

FVB mice were killed by inhalation of an overdose of isoflurane (Abbott, Wiesbaden, Germany). The bladder was carefully dissected, opened, and fixed in a Petri dish with the luminal surface facing upwards. A cotton-tipped applicator (Q-tip) was gently rubbed along the luminal surface and then placed in lysis buffer (RLT-buffer, Qiagen, Hilden, Germany). Further sample processing was as described in detail earlier (Lips et al., in press). Primer sequences are provided in Table 1.

Immunohistochemistry

Mice were killed

Muscarinic receptors

RT–PCR revealed expression of all five MR subtypes (M1R–M5R) in the mouse urothelium (Fig. 1A). M1R-immunoreactivity was distinct at the basal plasma membrane of the basal cells (Fig. 1C). In contrast, M2R-immunoreactivity was restricted to the umbrella cells (Fig. 1E). Immunolabelling of all epithelial cell layers was observed with antisera directed against subtypes M3R, M4R and M5R, with a slight predominance of M4R-immunolabelling in the basal cell layer (Fig. 1I). All these patterns of

Discussion

This is the first study reporting the occurrence of MR and nAChR subtypes in the urothelium of the murine urinary bladder. Previous immunohistochemical studies on the occurrence of cholinergic receptors in the urothelium of other species were restricted to MR and did not provide information as to a differential distribution among the different urothelial cell layers (rat: Saito et al., 2006; human: Mukerji et al., 2006). Our present data reveal that, as it is known from the stratified

Conclusion

The murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since various cholinergic receptor subtypes confer different functions in stratified epithelia, e.g. in skin, we propose that these selective distribution patterns serve to confer cell type-specificity of cholinergic regulation in the bladder urothelium.

Acknowledgements

The authors thank K. Michael for preparing the figures. This study was supported by the DFG, grant Li 1051/1–1 to K.S.L., and by a project bound donation of the Dr. R. Pfleger GmbH.

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