Immunohistochemical localization of muscarinic receptors (M2) in the rat skin

Abstract.

Acetylcholine (ACh) produces pain when applied to human skin and excites cutaneous mechanoreceptors and nerve terminals. These effects are partially mediated by activation of muscarinic receptors. The expression of muscarinic receptor subtype M2 has been shown in sensory neurons of rat dorsal root ganglia using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry. The purpose of the present study was to determine whether these M2 receptors are targeted to the peripheral endings of sensory neurons in the rat skin. Double-staining histochemical procedures were employed using a specific antiserum to M2 receptors combined with either of the following neuronal markers: an antiserum to the neuropeptide substance P, an antiserum to the protein gene product 9.5, which is a marker for peripheral nerve fibres, and the histochemical marker of a subpopulation of unmyelinated C-fibre afferents, I-B4, the Bandeira simplicifolia-derived isolectin. The M2 receptor subtype was found on different populations of nerve fibres. In the nerve plexus at the epidermal-dermal junction, M2 receptors are mainly present on I-B4-positive axons but are absent on fibres with substance P immunoreactivity. Sweat glands receive M2-receptor-immunoreactive fibres that express neither I-B4 binding nor substance P immunoreactivity, whereas blood vessels of the deeper dermis are innervated by I-B4-positive nerve fibres that are immunoreactive for M2 receptors and substance P. In addition to axon profiles, keratinocytes and endothelial cells also exhibit M2 receptor immunoreactivity. The results show the presence of M2 receptors in neuronal and non-neuronal cells, suggesting multiple effects of acetylcholine in the skin.