Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization, Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells

Abstract

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca2+]i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca2+]i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca2+]i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca2+]i increases from histamine, but failed to inhibit [Ca2+]i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca2+]i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca2+]i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca2+]i inhibition and TAS2R subtype, nor whether [Ca2+]i was increased by Gq- or Gi-coupled GPCRs. Selected studies revealed a correlation between [Ca2+]i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca2+]i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca2+]i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca2+]i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca2+]i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca2+]i inhibition limb.

Fig 1. Biphasic effect of the TAS2R10 agonist CQ on HASM cell [Ca²⁺]_i mobilization.

(A) Representative dose-response and time course of [Ca²⁺]_i in cells treated with 3 μM histamine and the indicated concentrations of CQ. (B) Biphasic [Ca²⁺]_i response to CQ in HASM concomitantly treated with 3 μM histamine. (C) Inhibitory limb of the [Ca²⁺] response to CQ in HASM concomitantly treated with 3 μM histamine. (D) Stimulation of [Ca²⁺] by CQ in HASM. There is no co-treatment with histamine in this experiment. Shown are representative results (mean ± SE of quadruplicates) of 3–5 independent experiments performed.

Fig 2. Effects of TAS2R31 agonists on histamine-stimulated [Ca²⁺] in HASM cells.

Effect of varying concentrations of AA (A) or SAC (B) on 3 μM histamine-mediated [Ca²⁺]_i increase. Shown are representative results (mean ± SE of quadruplicates) of 3–5 independent experiments performed.

Fig 3. Inhibition of ET-1 stimulated [Ca²⁺]_i in HASM cells differs based on TAS2R agonist.

(A) Inhibition of ET-1 stimulated [Ca²⁺] by AA. (B) AA stimulates [Ca²⁺]_i in the absence of ET-1. (C) CQ has a minimal inhibitory effect on ET-1 stimulated [Ca²⁺] (compared to histamine stimulated effect in Fig 1). Shown are representative results (mean ± SE of quadruplicates) of 3–5 independent experiments performed.

Fig 4. Quantitative effect of TAS2R agonists on [Ca²⁺]_i in HASM stimulated by five GPCR agonists.

(A-E) Cells were treated with the 3 μM histamine, 1 μM ET-1, 5 mM BK, 1 mM ACh, or 100 μM Ang II in the absence (buffer) or presence of 50 μM of the indicated TAS2R agonists. Statistical analysis was not performed on co-treatments that resulted in [Ca²⁺]_i enhancement. Large positive values were truncated to allow visualization of the smaller values. *, P<0.05; # P<0.005 vs control (buffer). N = 5–8 experiments.

Fig 5. Heat map of the relative effects of TAS2R agonists on [Ca²⁺] stimulated by the indicated procontractile CPCR agonists.

Data from the experiments in Fig 4 were normalized to maximal stimulation (red) or inhibition (blue). TAS2R agonists are on the top row and their subtype specificity shown. Procontractile GPCR agonists are listed in the first column. N/A, not applicable.

Fig 6. TAS2R inhibition of stimulated [Ca²⁺]_i is not dependent on G_i or G_q coupled receptor stimulation.

(A) HASM were co-treated with G_q-coupled HRH1 agonists NMH or 2,3 TFMP, or the G_i-coupled HRH3 agonist immethridine and 50 μM CQ. (B) Somatostatin (G_i-coupled) stimulated [Ca²⁺]_i is inhibited by QUI and AA. *, P<0.05, N = 4 experiments.

Fig 7. Heterogeneity of HASM membrane potential responses evoked by procontractile agonists co-treated with TAS2R agonists.

Cultured HASM cells were studied using a fluorescence-based membrane potential dye. The indicated agents were added singly, or in combination, at the 16 sec time point. The final concentrations were: Hist 3 μM, CQ 50 μM, SAC 50 μM, ET-1 1 μM, AA 50 μM. Results are mean ± SE of triplicate determinations from a single representative experiment of 2–5 performed.

Fig 8. The heterogeneity of TAS2R inhibition of [Ca²⁺]_i is recapitulated in HASM physiological responses.

(A, B) Isolated HASM in culture were studied using magnetic twisting cytometry. Cells were treated with 3 μM histamine or 1 μM ET-1 alone or together with the indicated TAS2R agonists (50 μM). * P<0.01 vs control (histamine or ET-1 with buffer). N = 303–400 cells per condition.

Fig 9. TAS2R inhibition of contracted human bronchi.

Results are from experiments performed on 10–15 airway rings from three different donated lungs. The concentrations of drugs were: histamine 10 μM, ET-1 1 μM, AA 100 μM, CQ 100 μM. *, P<0.001; † P<0.05, compared to histamine or ET-1 alone.

Fig 10. Model of TAS2R signaling to relaxation of HASM.

Stimulation of GPCR-A results in an increase in [Ca²⁺]_i, depolarization, and contraction (red). GPCR-A is not influenced by TAS2R interaction, thus [Ca²⁺]_i mobilization and ultimately contraction is not affected. Activation of certain GPCRs (GPCR-B) also promote contraction by increasing [Ca²⁺]_i, but are influenced (green dotted lines) by TAS2R agonists (acting at TAS2R-X). This results in a decrease in [Ca²⁺]_i, which opposes depolarization and relaxes HASM (green). This GPCR-B/TAS2R-X interaction could be in proximal (i) components or other downstream components (ii). TAS2R-Y does not specifically interact with a procontractile GPCR, but relaxes HASM by a calcium-dependent transducer, such as BK_Ca, that hyperpolarizes the membrane (blue).