Expression of muscarinic and nicotinic acetylcholine receptors in the mouse 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.
References (30)
- et al.
Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents
Neuron
(2002) Biological functions of keratinocyte cholinergic receptors
The Journal of Investigative Dermatology Symposium Proceedings
(1997)- et al.
Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin
The Journal of Investigative Dermatology
(2004) - et al.
Functional characterization of the epidermal cholinergic system in vitro
The Journal of Investigative Dermatology
(2006) - et al.
Coexpression of α9 and α10 nicotinic acetylcholine receptors in rat dorsal root ganglion neurons
Neuroscience
(2002) - et al.
Localization of M2 and M3 muscarinic receptors in human bladder disorders and their clinical correlations
Journal of Urology
(2006) - et al.
Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis
The Journal of Investigative Dermatology
(1998) - et al.
Novel human α9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity
American Journal of Pathology
(2000) - et al.
Synergistic control of keratinocyte adhesion through muscarinic and nicotinic acetylcholine receptor subtypes
Experimental Cell Research
(2004) - et al.
Alterations of M(2)-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder
Neuroscience Letters
(2006)
Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor
Journal of Urology
Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging
Urology
Non-neuronal cholinergic system in human bladder urothelium
Urology
Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence
Pharmacological Reviews
Central role of α7 nicotinic receptor in differentiation of the stratified squamous epithelium
Journal of Cell Biology
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These authors contributed equally to this study.