In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C β2 ) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.airway sensory innervation | respiratory epithelium | jugular-nodose ganglion | bitter-tasting substances

Background: A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far.

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It is pivotal to identify protein-protein interaction in situ to understand protein function. Conventional methods to determine the interaction of proteins destruct tissue or are applicable to cell culture only. To identify association of proteins in cells in tissue, we adapted indirect double-labeling immunofluorescence and combined it with conventional confocal laser scanning microscopy (CLSM) to measure fluorescence resonance energy transfer (FRET). As a model system, we chose caveolin-1alpha and caveolin-2, two major components of endothelial caveolae, and examined their interaction in the endothelium of vessels in fixed tissues of laboratory animals and human glomus tumors. Several methodological aspects were examined. Measuring the absolute increase in fluorescence (DeltaIF) was superior compared to determining the relative FRET efficiency, because it is more robust against small increases of fluorescence during measurements that results from unavoidable minimal crossreactivity of the secondary antibodies. Both, sequential and simultaneous incubation of secondary antibodies result in robust and reliable increases in DeltaIF. If incubated sequentially, however, the acceptor-labeled secondary antibody should be applied first. The size of the secondary reagent (F(ab')2 vs whole antibody) has no major influence. In conclusion, CLSM-FRET can measure close spatial association of proteins in situ and can be applied to human surgical material.