The bitter taste receptor agonist denatonium influences mouse tracheal epithelial ion transport

Abstract

In the airway epithelium, mucociliary clearance is strictly regulated by ciliary beat and transepithelial ion transport to provide homeostasis. During recent years it was shown that ciliated cells and specialized chemosensory cells in the airway epithelium express bitter taste receptors (Tas2R). We have previously shown that the Tas2R agonist denatonium stimulates particle transport speed in murine trachea via acetylcholine (ACh) release from brush cells. However, it remains to be elucidated if Tas2R stimulation also influences transepithelial ion transport processes. To address this question, we here performed measurements of the transepithelial ion current (ISC) of murine tracheas in Ussing chamber experiments. Apical application of denatonium changed ISC in a dose dependent manner (EC50 132.6 µM). Application of the Gai subunit inhibitor pertussis toxin (500 ng/ml) significantly reduced the denatonium-effect (1 mM), while the Gbg subunit inhibitor gallein (50 µM) had no effect. In the presence of TPPO (100 µM) an inhibitor of the transient receptor potential melastatin-5 (TRPM5) ion channel, a member of the canonical bitter signalling cascade, and in TRPM5 knock-out mice, the denatonium-effect was significantly reduced. Inhibition of an increase of intracellular Ca2+ with the phospholipase C inhibitor U-73122 (10 µM) did not change the denatonium-effect. Furthermore, the denatonium-effect was not dependent on cholinergic signalling, as blocking ACh receptors with mecamylamine (100 µM) and atropine (50 µM) had no influence. Interestingly application of the pannexin1 cannel inhibitor probenecid (1 mM) also reduced the denatonium-effect, pointing towards a release of ATP via pannexin channels. A decrease in intracellular ATP-levels leads to an activation of KATP potassium channels as shown with the inhibitor glibenclamide (100 µM). Additionally, blocking ATP-receptors with suramin (100 µM) significantly reduced the denatonium-effect, indicating that the released ATP acts in a paracrine way on the airway epithelial cells. Taken together we showed that activation of Tas2R in brush cells might represent important regulators of ion transport activity via ATP-dependent signalling. Selectively targeting Tas2R in the trachea might represent a useful target for stimulation of the mucociliary clearance.