Significance We report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system. These cells exhibit structural markers of respiratory chemosensory cells (“brush cells”). They use the classical taste transduction cascade to detect potential hazardous compounds (bitter, umami, uropathogenic bacteria) and release acetylcholine in response. They lie next to sensory nerve fibers that carry acetylcholine receptors, and placing a bitter compound in the urethra enhances activity of the bladder detrusor muscle. Thus, monitoring of urethral content is linked to bladder control via a previously unrecognized cell type.

Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). Here, we assessed the specificity of MR antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and 8 of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at 4 antibody dilutions at minimum, so that finally at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabeled in the preabsorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice, even when the preabsorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localization of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localization in tissue sections of peripheral organs is limited to the M2R subtype utilizing the most commonly used methodological approaches.

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.

BACKGROUND AND PURPOSERecent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells. EXPERIMENTAL APPROACHRT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. KEY RESULTSTranscripts encoding ChAT and OCT1-3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC50: 23.3 mM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na CONCLUSIONS AND IMPLICATIONSThe presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways.

Cultured normal mammalian melanocytes exposed to a variety of antioxidants in the presence of millimolar concentrations of 4-hydroxyanisole exhibit dose-dependent modifications of cytotoxicity. While some antioxidants reduced the extent of damage produced by 4-hydroxyanisole, others appeared to increase it. Similar effects were found in a model system using lysis of human erythrocytes as an index of cell damage. Estimations on rat liver microsomes in the presence of tyrosinase and 4-hydroxyanisole showed increased peroxidation only at low substrate concentrations.