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

Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB - Nat. Med. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Given that the increase in [Ca2+]i promoted by some of the bitter tastants in human ASM was similar in magnitude to that of ligands acting on bronchoconstrictive GPCRs, we initially assumed that bitter taste receptors evoked ASM contraction. However, in isolated intact mouse airways, chloroquine, denatonium and quinine caused a dose-dependent relaxation, with a maximal response of >90% loss of the active contraction evoked by acetylcholine or serotonin (Fig. 2a,b). The maximal relaxation response to the full β2AR agonist isoproterenol under these same experimental conditions was a 30 ± 9.2% reduction in active tension (Fig. 2a). Bitter tastants were also found to relax baseline tracheal ring tension (Supplementary Fig. 4). The relaxation response to bitter tastants was fully reversible, as washing tracheal rings while maintaining the presence of the contractile agent alone resulted in a return to contraction that was equivalent in magnitude to the control condition (Supplementary Fig. 5). In a limited number of studies in fourth order bronchi obtained from non-diseased portions of human lung tissues, we also observed relaxation from 50−80% from chloroquine or saccharin on acetylcholine contracted rings (Supplementary Fig. 6). Bitter tastant-mediated airway relaxation was not altered by the cyclooxygenase inhibitor indomethacin or the nitric oxide synthase inhibitor L-NAME (data not shown), suggesting a direct activation of ASM receptors rather than a secondary response generated from bronchoactive ligands generated from airway epithelial cells. Airway relaxation observed with β-agonists is due to β2AR coupling to an increase in cAMP with subsequent PKA activation2. However, we found no evidence for chloroquine-promoted increases in cAMP or PKA activation in intact cultured ASM cells, as assessed by a sensitive radioimmunoassay or by the PKA-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP), respectively (Fig. 2c). In a set of serial dosing and washout experiments with intact airways and a submaximal dose of chloroquine, we found that exposure to both chloroquine and isoproterenol resulted in relaxation that was greater than that found with either compound alone (Fig. 2d). These data indicate that bitter tastants evoke marked airway relaxation which is reversible, is not due to cell injury since ASM functional contraction and relaxation are not impaired after washout, is non-cAMP dependent, and is additive to that of a β-agonist.


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

Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB - Nat. Med. (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

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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.
Mentions: Given that the increase in [Ca2+]i promoted by some of the bitter tastants in human ASM was similar in magnitude to that of ligands acting on bronchoconstrictive GPCRs, we initially assumed that bitter taste receptors evoked ASM contraction. However, in isolated intact mouse airways, chloroquine, denatonium and quinine caused a dose-dependent relaxation, with a maximal response of >90% loss of the active contraction evoked by acetylcholine or serotonin (Fig. 2a,b). The maximal relaxation response to the full β2AR agonist isoproterenol under these same experimental conditions was a 30 ± 9.2% reduction in active tension (Fig. 2a). Bitter tastants were also found to relax baseline tracheal ring tension (Supplementary Fig. 4). The relaxation response to bitter tastants was fully reversible, as washing tracheal rings while maintaining the presence of the contractile agent alone resulted in a return to contraction that was equivalent in magnitude to the control condition (Supplementary Fig. 5). In a limited number of studies in fourth order bronchi obtained from non-diseased portions of human lung tissues, we also observed relaxation from 50−80% from chloroquine or saccharin on acetylcholine contracted rings (Supplementary Fig. 6). Bitter tastant-mediated airway relaxation was not altered by the cyclooxygenase inhibitor indomethacin or the nitric oxide synthase inhibitor L-NAME (data not shown), suggesting a direct activation of ASM receptors rather than a secondary response generated from bronchoactive ligands generated from airway epithelial cells. Airway relaxation observed with β-agonists is due to β2AR coupling to an increase in cAMP with subsequent PKA activation2. However, we found no evidence for chloroquine-promoted increases in cAMP or PKA activation in intact cultured ASM cells, as assessed by a sensitive radioimmunoassay or by the PKA-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP), respectively (Fig. 2c). In a set of serial dosing and washout experiments with intact airways and a submaximal dose of chloroquine, we found that exposure to both chloroquine and isoproterenol resulted in relaxation that was greater than that found with either compound alone (Fig. 2d). These data indicate that bitter tastants evoke marked airway relaxation which is reversible, is not due to cell injury since ASM functional contraction and relaxation are not impaired after washout, is non-cAMP dependent, and is additive to that of a β-agonist.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

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.

Show MeSH
Related in: MedlinePlus