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

Journal name:
Nature Medicine
Volume:
16,
Pages:
1299–1304
Year published:
DOI:
doi:10.1038/nm.2237
Received
Accepted
Published online

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+ (BKCa) 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.

At a glance

Figures

  1. Bitter tastants of diverse structures evoke increases in [Ca2+]i in human airway smooth muscle cells.
    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 (a) and chloroquine (b). The mean ± s.e.m. dose-response curves were from five or six experiments, and the transients shown are representative of single experiments. (c) Maximal [Ca2+]i responses to 1.0 mM of the bitter tastants aristocholic acid (aristo), chloroquine, colchicine, denatonium, quinine, saccharin, salicin, strychnine and yohimbine and the bronchoconstrictive Gq-coupled agonists bradykinin (0.01 mM) and histamine (0.1 mM). Results are means ± s.e.m. from four to six experiments. *P < 0.01 versus basal; #P < 0.05 versus denatonium. (d) The [Ca2+]i response to bitter tastants 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.

  2. Bitter tastants induce relaxation of intact mouse tracheas in a non-cAMP-dependent manner.
    Figure 2: Bitter tastants induce relaxation of intact mouse tracheas in a non–cAMP-dependent manner.

    (a) Dose-response curves of relaxation for the β-adrenergic 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) Relaxation by chloroquine and quinine of intact mouse tracheas contracted by 1.0 mM serotonin (n = 4 experiments). (c) cAMP production in cultured human ASM cells incubated with 1.0 mM chloroquine for the indicated times, or for 15 min with 30 μM isoproterenol, as determined by radioimmunoassay. There was no evidence for chloroquine-induced cAMP accumulation (n = 3 experiments). Inset, immunoblot of VASP and phosphorylated VASP (P-VASP) in cultured human ASM cells exposed to 1.0 mM chloroquine or saccharin (sacc), or 10 μM forskolin (forsk). Forskolin, which stimulates cAMP production, resulted in phosphorylation of VASP as indicated by the upper band. (d) Bitter tastant reversibility and additivity studies with intact mouse tracheas. 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 exposure to chloroquine alone, the rings were washed and then rechallenged with the same dose of acetylcholine. *P < 0.05 versus acetylcholine alone; #P < 0.01 versus acetylcholine + isoproterenol, or chloroquine alone. Results are from four experiments. Data are presented as means ± s.e.m.

  3. Isolated airway smooth muscle responses to bitter tastants as assessed by single cell mechanics and membrane potentials.
    Figure 3: Isolated airway smooth muscle responses to bitter tastants as assessed by single cell mechanics and membrane potentials.

    (a) Cell stiffness of isolated ASM cells in response to 10 μM isoproterenol (iso), 1.0 mM chloroquine (chloro), 1.0 mM saccharin (sacc) or 1.0 μM histamine (hist). (b) Relaxation of isolated ASM cells in response to 1.0 mM saccharin in the presence of 1 μM of the PLCβ inhibitor U73122, 10 nM of the BKCa antagonists iberiotoxin (IbTx) and charybdotoxin (ChTx) or 100 nM of the PKA inhibitor H89. (c) The relaxation response to 1.0 mM chloroquine in isolated mouse airway contracted by 10 μM methacholine (Mch) in the absence or presence of 100 nM of the BKCa antagonist IbTx. Results are representative of five to eight experiments. (d) Membrane potential effects of saccharin and chloroquine. ASM cells loaded with a fluorescence-based membrane potential-sensitive dye were exposed to 1.0 mM chloroquine or saccharin, 1.0 μM histamine or 60 mM KCl (representative of four experiments). A decrease in relative fluorescence units (RFU) indicates hyperpolarization. (e) Effects of the BKCa antagonist IbTx (100 nM) on chloroquine and saccharin-promoted ASM hyperpolarization in intact ASM cells. Results represent the peak responses from four experiments. *P < 0.01 vs. vehicle control. Data are presented as means ± s.e.m.

  4. Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells.
    Figure 4: Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells.

    (a,b) Sequential confocal images of Fluo-3–loaded cells showing localized [Ca2+]i increases in the cell upon exposure of ASM cells to 0.3 mM saccharin (a). The images represent Fluo-3 fluorescence after background subtraction and baseline normalization (F / F0) with intensity encoded by pseudocolor. The arrows highlight local [Ca2+]i 'hot-spots'. The numbers over areas of the cells represent regions of interest (ROI) which correspond to the numbered intensity tracings (b). (c,d) [Ca2+]i images (c) and intensity tracings of ROIs (d), in ASM cells (loaded as in a and b) in response to exposure to 1.0 μM histamine. (e) Confocal line-scan imaging showing 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 at left. Arrows indicate several local [Ca2+]i events that occurred before the more defined increase within the isolated region. At the bottom is the spatially averaged normalized fluorescence signal (F / F0) generated from the line scan. Results are from single experiments representative of five performed.

  5. Bitter taste receptor agonists attenuate bronchoconstriction in a mouse model of asthma.
    Figure 5: Bitter taste receptor agonists attenuate bronchoconstriction in a mouse model of asthma.

    (a,b) Photomicrographs from sections of control (a) and ovalbumin-challenged (b) mouse lungs showing eosinophilic inflammation of the airway, epithelial hyperplasia and basement membrane thickening in ovalbumin challenged airways (H&E stain). Br, bronchus; Bm, basement membrane; Eo, eosinophil; Ep, epithelium; Bl, blood vessel. (c,d) Airway resistance in control (c) and ovalbumin-challenged (d) mice measured at baseline, in response to aerosolized methacholine (mch) and in response to single doses of quinine 150 μg or the β-agonist albuterol (3 μg) given during the bronchoconstrictive phase (n = 5 experiments). The studies were carried out with a dose of methacholine that resulted in a four- to five-fold increase in airway resistance over baseline (≥16 mg ml−1 in control mice and 8 mg ml−1 in ovalbumin-challenged mice). *P < 0.01 versus methacholine; #P < 0.05 versus methacholine. Data are presented as means ± s.e.m.

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Author information

Affiliations

  1. Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Deepak A Deshpande,
    • Wayne C H Wang,
    • Elizabeth L McIlmoyle,
    • Kathryn S Robinett,
    • Rachel M Schillinger &
    • Stephen B Liggett
  2. Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

    • Steven S An
  3. Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

    • James S K Sham
  4. Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Stephen B Liggett

Contributions

D.A.D., single-cell mechanics and imaging, data analysis and manuscript preparation; W.C.H.W., expression studies, gene knockdown, airway physiology, data analysis and manuscript preparation; E.L.M., calcium signaling and data analysis; K.S.R., intact airway studies, expression studies, data analysis and manuscript preparation; R.M.S., airway physiology; S.S.A., single cell mechanics, data analysis, manuscript preparation; J.S.K.S., confocal calcium imaging, data analysis, manuscript preparation; S.B.L. directed all studies, data analysis and interpretation and is the primary author of the manuscript.

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The authors declare no competing financial interests.

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