The cellular and molecular basis of bitter tastant-induced bronchodilation.

Bronchodilators are a standard medicine for treating airway obstructive diseases, and ß2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than ß2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular [Ca(2+)]i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cß [PLCß]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca(2+)]i induced by bronchoconstrictors, and this lowering of the [Ca(2+)]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in [Ca(2+)]i, and this inhibition can be prevented by pertussis toxin and G-protein ßγ subunit inhibitors, but not by the blockers of PLCß and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via Gßγ to increase [Ca(2+)]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca(2+)]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

Human airway contraction and formoterol-induced relaxation is determined by Ca2+ oscillations and Ca2+ sensitivity.

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca(2+) oscillations and Ca(2+) sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca(2+) signaling and Ca(2+) sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca(2+)-permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca(2+) concentration in the SMCs in the form of oscillatory Ca(2+) waves and an increase in Ca(2+) sensitivity. The frequency of the Ca(2+) oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca(2+) sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca(2+) oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca(2+) signaling and Ca(2+) sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca(2+) sensitivity and, subsequently, the frequency of the Ca(2+) oscillations of the airway SMCs.