No abstract

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(β2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCβ2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

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.

In the pulmonary vasculature of man, pig and guinea-pig, acetylcholine (ACh) exerts a relaxant effect by interacting with muscarinic receptors located on endothelial cells. The present experiments were designed to detect the endogenous source of ACh in the pulmonary vasculature. For this purpose, we investigated whether pulmonary artery endothelial cells contain elements of the "cholinergic gene locus", the ACh synthesising enzyme choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT). ChAT mRNA was detected by means of reverse transcription polymerase chain reaction (RT-PCR) in endothelial cells of porcine pulmonary arteries freshly isolated and after 7 days in culture. ChAT protein was demonstrated in endothelial cells in vitro and in situ. ChAT immunoreactivity was present in endothelial cells freshly isolated and after 2, 4, 7, and 9 days in culture. Tissue sections from extra-and intraparenchymal pulmonary arteries of man, pig and guinea-pig expressed a mosaic pattern of ChAT-positive and -negative endothelial cells. VAChT mRNA was detected by RT-PCR in rat pulmonary artery and in endothelial cells isolated from human and porcine pulmonary trunk. The detection of VAChT and ChAT mRNA and the demonstration of ChAT protein in vitro and in situ suggest that the endothelium is an endogenous source of ACh in the pulmonary vasculature.