ATP and Odor Mixture Activate TRPM5-Expressing Microvillous Cells and Potentially Induce Acetylcholine Release to Enhance Supporting Cell Endocytosis in Mouse Main Olfactory Epithelium

Abstract

The main olfactory epithelium (MOE) functions to detect odor molecules, provide an epithelial surface barrier, and remove xenobiotics from inhaled air. Mechanisms coordinating the activities of different cell types within the MOE to maintain these functions are poorly understood. Previously, we showed that superficially located microvillous cells (MCs) in the MOE expressing transient receptor potential channel M5 (TRPM5) are cholinergic and chemoresponsive and that they play an important role in maintaining odor responses and olfactory-guided behavior under challenging chemical environment. Here we investigated TRPM5-MC activation and subsequent paracrine regulation. Ca2+ imaging showed that TRPM5-MCs dose-dependently increase their intracellular Ca2+ levels in response to ATP, an important signaling molecule for airway mucociliary movement, and to an odor mixture. Pharmacological examination showed that the ATP responses are primarily mediated by P2X purinergic receptors. Interestingly, using the endocytosis dye pHrodo Red dextran, we found that chemical-activated TRPM5-MCs significantly increase the number of pHrodo-labeled puncta compared to controls without stimulation and compared to cells that do not respond to ATP or to the odor mixture. These results indicate potential vesicle recycling after release of the signaling molecule acetylcholine (ACh). Interestingly, TRPM5 knockout (KO) results in a decrease in ATP-induced pHrodo internalization. We further investigated cholinergic regulation of neighboring supporting cells (SCs). We found that ACh strongly elevates intracellular Ca2+ and potentiates pHrodo endocytosis in SCs. The ACh effects are diminished in the presence of atropine or M3 muscarinic receptor antagonist and in SCs lacking M3 receptors. Collectively, these data suggest that TRPM5-MCs may regulate the MOE’s multicellular network activity via cholinergic paracrine signaling for functional maintenance and adaptive plasticity.

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling

“…2,6,[41][42][43] Indeed, nose SCC are commonly contacted by peptidergic (CGRP-positive, SP-positive) sensory nerve fibers, 2,7,22,44 whereas only 25% of tracheal BC are in close position to sensory nerve fibers. 2,6,[41][42][43] Indeed, nose SCC are commonly contacted by peptidergic (CGRP-positive, SP-positive) sensory nerve fibers, 2,7,22,44 whereas only 25% of tracheal BC are in close position to sensory nerve fibers.…”

“…There is evidence from studies of the nasal epithelium that SCC and microvillous cells respond to appropriate chemical stimuli by ACh-release, thereby activating neighboring and supporting cells, respectively, and evoking local responses and stimulation of sensory nerve fibers, initiating defense mechanisms. 2,6,[41][42][43] Indeed, nose SCC are commonly contacted by peptidergic (CGRP-positive, SP-positive) sensory nerve fibers, 2,7,22,44 whereas only 25% of tracheal BC are in close position to sensory nerve fibers. Stimulation of nasal SCC with denatonium causes SP-mediated neurogenic inflammation dependent on cholinergic transmission, 22 though the direct release of ACh from these cells was not shown.…”

Chemical Exposure-Induced Changes in the Expression of Neurotrophins and Their Receptors in the Main Olfactory System of Mice Lacking TRPM5-Expressing Microvillous Cells

“…The lack of TRPM5-MCs in Skn-1a −/− mice may diminish such a need for such NT-mediated regulation. Another possible explanation is that p75NTR expression level is regulated by activity of supporting cells which is modulated by acetylcholine as shown in our previous study [ 9 , 10 ]. p75NTR immunolabeling has been shown to present in supporting cells by Steuer et al [ 22 ] and our current study.…”

“…Another population of MCs, which we and other investigators previously characterized, expresses transient receptor potential channel M5 (TRPM5-MCs) [ 7 , 8 ]. Our subsequent study revealed that TRPM5-MCs are chemoresponsive and capable of synthesizing and releasing acetylcholine (ACh), which can modulate the activities of OSNs and supporting cells [ 9 , 10 ]. More recently, we identified an important role for TRPM5-MCs in maintaining olfactory function using mice deficient in the POU homeobox transcription factor gene Skn-1a (also known as Pou2f3 ; Skn-1a −/− mice), in which TRPM5-MCs in the MOE failed to differentiate [ 11 , 12 ].…”

“…Because TRPM5-MCs are not directly involved in olfactory signaling and information processing, it is expected that TRPM5-MCs modulate MOE network activity. In addition to the cholinergic modulation of OSNs and SCs [ 9 , 10 ], TRPM5-MCs may engage other mechanisms that play important roles in MOE maintenance. One such mechanism is adult neurogenesis, which involves globose and horizontal basal cells residing in the basal compartment of the olfactory epithelium [ 13 ].…”