TAS2R activation promotes airway smooth muscle relaxation despite β(2)-adrenergic receptor tachyphylaxis

Abstract

Recently, bitter taste receptors (TAS2Rs) were found in the lung and act to relax airway smooth muscle (ASM) via intracellular Ca(2+) concentration signaling generated from restricted phospholipase C activation. As potential therapy, TAS2R agonists could be add-on treatment when patients fail to achieve adequate bronchodilation with chronic β-agonists. The β(2)-adrenergic receptor (β(2)AR) of ASM undergoes extensive functional desensitization. It remains unknown whether this desensitization affects TAS2R function, by cross talk at the receptors or distal common components in the relaxation machinery. We studied intracellular signaling and cell mechanics using isolated human ASM, mouse tracheal responses, and human bronchial responses to characterize TAS2R relaxation in the context of β(2)AR desensitization. In isolated human ASM, magnetic twisting cytometry revealed >90% loss of isoproterenol-promoted decrease in cell stiffness after 18-h exposure to albuterol. Under these same conditions of β(2)AR desensitization, the TAS2R agonist chloroquine relaxation response was unaffected. TAS2R-mediated stimulation of intracellular Ca(2+) concentration in human ASM was unaltered by albuterol pretreatment, in contrast to cAMP signaling, which was desensitized by >90%. In mouse trachea, β(2)AR desensitization by β-agonist amounted to 92 ± 6.0% (P < 0.001), while, under these same conditions, TAS2R desensitization was not significant (11 ± 3.5%). In human lung slices, chronic β-agonist exposure culminated in 64 ± 5.7% (P < 0.001) desensitization of β(2)AR-mediated dilation of carbachol-constricted airways that was reversed by chloroquine. We conclude that there is no evidence for physiologically relevant cross-desensitization of TAS2R-mediated ASM relaxation from chronic β-agonist treatment. These findings portend a favorable therapeutic profile for TAS2R agonists for the treatment of bronchospasm in asthma or chronic obstructive lung disease.

Fig. 1.

Dynamic changes of cell stiffness in response to a full β₂-adrenergic receptor (β₂AR) agonist isoproterenol (Iso) and a selective bitter taste receptor (TAS2R) agonist chloroquine (Chloro) in human airway smooth muscle (ASM), as assessed by Magnetic Twisting Cytometry. Primary human ASM cell cultures were established from 5 separate lung donors. Isolated human ASM cells were untreated (A and B) or treated for 18 h with 1 μM albuterol (C and D). Cells were contracted with 10 μM methacholine (MCh) and then relaxed with 10 μM Iso (A and C) or 1 mM Chloro (B and D). For each cell, changes in stiffness in response to Iso or Chloro were normalized to its respective MCh-contracted stiffness. Values are presented as median (A: n = 53–266; B: n = 62–281; C: n = 58–248; D: n = 70–233 individual cell measurements).

Fig. 2.

Maximum stiffness reduction of MCh-contracted human ASM induced by Iso and Chloro in untreated (A) and albuterol-exposed (B) cells. Values are means ± SE (A: n = 53–281; B: n = 58–248 individual cell measurements). *P < 0.01; #P < 0.00005.

Fig. 3.

Dynamic changes of cell stiffness in response to 1 mM Chloro in untreated and albuterol-exposed human ASM. To control random effects due to multiple cell measurements from the same donor, the nested regression was used for group comparisons. Values are means ± SE (n = 775–859 individual cell measurements from 5 lung donors).

Fig. 4.

β₂AR desensitization and intracellular signaling in isolated human ASM cells. A: cultured primary human ASM cells were incubated for 18 h with ascorbic acid (AA) or β-agonist (Iso), and cAMP was measured by CatchPoint assay (Molecular Devices). Iso-mediated accumulation of cAMP (15 min) is abrogated in human ASM cells that were pretreated for 18 h with β-agonist, but not in untreated cells (means ± SE, n = 4 experiments). NS, nonsignificant. *P < 0.01. B: human ASM cells were loaded with Fluo-4 AM, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) release evoked by TAS2R activation was measured. Arrow indicates the time of Chloro addition. [Ca²⁺]_i transient curves to 1 mM Chloro is unabated in human ASM cells that were pretreated for 18 h with β-agonist. Results shown are from a single representative experiment of 3 performed.

Fig. 5.

TAS2R activation evokes ASM relaxation under conditions of β₂AR desensitization. Intact mouse trachealis were studied in the absence (Control; A and B) or after 18 h of exposure to the β-agonist Iso (Post-Iso treatment; C and D). Rings were contracted with 0.1 mM acetylcholine (ACh) and relaxed with 10 μM Iso [Iso1 (A), Iso2 (C)]. After each Iso treatment, the ring was washed and then rechallenged with the same dose of ACh, followed by 1 mM Chloro addition [Chloro1 (B), Chloro2 (D)]. ACh was maintained in the bath when Iso and Chloro were added. Results shown are from a single representative experiment of at least 4 performed.

Fig. 6.

TAS2R activation is highly efficacious in relaxing ASM under conditions of β₂AR desensitization. As described above in Fig. 4, intact mouse trachealis were studied before (Control) or after treatment with the β-agonist Iso (Post-Iso treatment). Subsequent Iso and Chloro responses were measured in rings contracted with ACh. Values are means ± SE (n = 4 experiments). *P < 0.01.

Fig. 7.

TAS2R activation dilates intact human small airways under conditions of β₂AR desensitization. Human precision-cut lung slices (PCLSs) were incubated for 18 h in the absence (Control) or presence of long-acting β-agonist salmeterol (Post-Sal treatment), washed, and constricted with carbachol. Carbachol-constricted airways were treated sequentially with 1 μM Iso and 50 μM Chloro. Values are means ± SE (obtained using 102 PCLSs derived from 4 donors). *P < 0.005.