Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Abstract

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca²(+)](i)) in ASM in a Gβγ-, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP₃) receptor-dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca²(+)](i) response at the cell membrane, which opens large-conductance Ca²(+)-activated K(+) (BK(Ca)) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Figure 1

Bitter tastants of diverse structures evoke increases in [Ca²⁺]_i in human airway smooth muscle cells. Studies were performed with cultured primary ASM cells loaded with Fluo-4 AM. (a, b) [Ca²⁺]_i transients and dose response curves to saccharin and chloroquine from 5−6 experiments. (c) Maximal [Ca²⁺]_i responses to 1.0 mM of the bitter tastants aristocholic acid, chloroquine, colchicine, denatonium, quinine, saccharin, salicin, strychnine and yohimbine, and the bronchoconstrictive G_q-coupled agonists histamine (0.1 mM) and bradykinin (0.01 mM). Results are from 4−6 experiments. *, P < 0.01 vs. basal; #, P < 0.05 vs. denatonium. (d) The [Ca²⁺]_i response to bitter tastant is ablated by the PLC inhibitor U73122 and the βγ antagonist gallein, and attenuated by the IP₃ receptor antagonist 2APB. These studies were performed in the absence of extracellular calcium. Results shown are from a single representative experiment of at least three performed.

Figure 2

Bitter tastants evoke bronchodilatation in a non-cAMP dependent manner. (a) Dose-response curves of relaxation for the β-agonist isoproterenol (iso) and the bitter taste receptor agonists chloroquine (chloro), denatonium (denat), and quinine, derived from intact mouse tracheas contracted with 1.0 mM acetylcholine (n = 7 experiments). (b) Chloroquine and quinine relax intact mouse airway tracheas contracted by 1.0 mM serotonin (n = 4 experiments). (c) Cultured human ASM cells were incubated with 1.0 mM chloroquine for the indicated times, or for 15 min with 30 µM isoproterenol, and cAMP measured by radioimmunoassay. There was no evidence for chloroquine-promoted cAMP accumulation (n = 3 experiments). Inset: Cultured human ASM cells were exposed to 1.0 mM chloroquine or saccharin (sacc), or 10 µM forskolin (forsk), and cell extracts were immunoblotted to ascertain PKA-mediated VASP phosphorylation (upper band), a cAMP promoted event. Forskolin, which stimulates cAMP production, resulted in phosphorylation of VASP as indicated by the upper band. Neither chloroquine nor saccharin promoted VASP phosphorylation, consistent with the cAMP measurements. (d) The airway relaxation response to isoproterenol and chloroquine are additive. Intact mouse tracheas were contracted with 1.0 mM acetylcholine (ach) which was maintained in the bath when chloroquine (200 µM) or isoproterenol (30 µM), or both drugs, were added. After chloroquine exposure, the rings were washed and then rechallenged with the same dose of acetylcholine. *, P < 0.05 vs. acetylcholine alone; #, P < 0.01 vs. acetylcholine + isoproterenol, or chloroquine alone. Results are from four experiments.

Figure 3

Isolated airway smooth muscle responses to bitter tastants as assessed by single cell mechanics and membrane potentials. (a) isoproterenol (iso), chloroquine (chloro) and saccharin (sacc) relax, while histamine (hist) contracts, isolated ASM cells. (b) The relaxation responses in isolated ASM cells to 1 mM saccharin are inhibited by the PLCβ inhibitor U73122 (1 µM), 10 nM of the BK_Ca antagonists iberiotoxin (IbTx) and charybdotoxin (ChTx), but are unaffected by 100 nM of the PKA inhibitor H89. (c) The relaxation response to 1 mM chloro in isolated mouse airway is inhibited by 100 nM IbTx. Results are representative of 5−8 experiments performed. (d) ASM cells loaded with a fluorescence-based membrane potential-sensitive dye reveal hyperpolarization in response to 1.0 mM chloroquine and saccharin (representative of four experiments performed). (e) Chloroquine and saccharin-promoted ASM hyperpolarization is ablated by preincubation with 100 nM IbTx. Results represent the peak responses from four experiments. *, P < 0.01 vs. vehicle control.

Figure 4

Saccharin preferentially triggers localized [Ca²⁺]_i responses in ASM cells. (a,c) Sequential confocal images of Fluo-3 loaded cells shows activation of localized [Ca²⁺]_i increases in the cell periphery upon exposure of ASM cells to 0.3 mM saccharin, and a generalized increase in [Ca²⁺]_i with exposure to 1.0 µM histamine. The images are Fluo-3 fluorescence after background subtraction and baseline normalized (F/F₀) with intensity encoded by pseudo-color. The arrows highlight local [Ca²⁺]_i “hot-spots”. (b,d) Local [Ca²⁺]_i transients measured in regions of interest (ROI). Saccharin activated a rapid rise of Ca²⁺ in the peripheral end (ROI 1), but a smaller and gradual increase of [Ca²⁺]_i in the central regions (ROI 2,3) of the cell. The histamine response (ROI 4–7) was asynchronous and was observed throughout the cells. (e) Confocal linescan imaging shows spatially and temporally resolved local [Ca²⁺]_i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 µm parallel to the cell membrane at one end of an elongated ASM cell as shown in the left panel. Arrows indicate several local [Ca²⁺]_i events that occur prior to the more defined increase within the isolated region. The bottom panel is the spatially averaged normalized fluorescence signal (F/F₀) generated from the linescan. Results are from single experiments representative of five performed.

Figure 5

Bitter taste receptor agonists relieve bronchoconstriction in a mouse model of asthma. (a,b) Photomicrographs from sections of control and ovalbumin challenged mouse lungs shows eosinophilic inflammation of the airway, epithelial hyperplasia and basement membrane thickening in ovalbumin challenged airways (hematoxylin and eosin stain). Br, bronchus; Bm, basement membrane; Eo, eosinophil; Ep, epithelium; Bl, blood vessel. Airway resistance in control (c) and ovalbumin challenged mice (d) was measured at baseline, and in response to aerosolized methacholine (mch), and to quinine or the β-agonist albuterol given during the bronchoconstrictive phase (n = 5 experiments). The studies were carried out with a dose of methacholine which resulted in a 4−5-fold increase in airway resistance over baseline (≥ 16 mg/ml in control mice and 8 mg/ml in ovalbumin-challenged mice). *, P < 0.01 vs. methacholine; #, P < 0.05 vs. methacholine.