Bitter taste receptors as targets for tocolytics in preterm labor therapy

Abstract

Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and phospholipase C β2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca²⁺]_i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Keywords: G-protein coupled receptor; chloroquine; relaxation; uterine smooth muscle.

Figure 1.

Mouse and human myometrium express TAS2Rs. A) Tas2R expression pattern in myometrium from d 18 pregnant mice. B) TAS2R expression in myometrium from women in third trimester of pregnancy. C) TAS2R expression in hTERT-HM cells. Quantitative PCR primers for TAS2Rs are listed in Tables 1 and 2. To visualize differences among TAS2Rs, C_t values for each TAS2R was normalized against β-actin and then against Tas2R137 in mice and TAS2R5 in humans.

Figure 2.

Bitter tastants completely relax OT-induced contraction of uterine strips from d 18 pregnant mice. A) OT (100 nM)-induced sustained contraction of uterine strips from d 18 pregnant mice. B–D) Dose-dependent effects of ChQ (B), MgSO₄ (C), and albuterol (D) on 100 nM OT-induced mouse uterine strip contraction. E) Summarized results of experiments A–D, another 2 bitter tastants, denatonium (Denat) and 1, 10-phenanthroline (Phen), nifedipine (Nif), and indomethacin (Indom). Data are means ± sem; n = 4–6 mice. Strips were placed in KPS containing 1.16 mM MgSO₄ (i.e., final concentration of MgSO₄ was 2.16 mM in C, and also in experiments in Fig. 4); and that to avoid potential relaxing effects of solvent (i.e., ethanol for nifedipine and DMSO for indomethacin), higher levels of nifedipine and indomethacin were not examined. Relaxation calculation is described in Materials and Methods.

Figure 3.

ChQ relaxes KCl- and PGF2α-induced contraction of myometrial strips from d 18 pregnant mice. A–C) Force response to 40 mM KCl alone (A), relaxation caused by 1 mM ChQ (B), and summarized results (C) (means ± sem; n = 5 for each group; KCl alone vs. KCl + ChQ, unpaired Student’s t test). D–F) Force response to 5 µM PGF2α alone (D), relaxation caused by 1 mM ChQ (E), and summarized results (F) (means ± sem; n = 5 for each group; PGF2α alone vs. PGF2α + ChQ, unpaired Student’s t test). ****P < 0.0001.

Figure 4.

Bitter tastants relax human uterine strips more completely than currently used tocolytics. A) Representative tension response to 100 nM OT in uterine strips from women in late pregnancy. B–D) Tension responses caused by 1 mM ChQ (B), 1 mM MgSO₄ (C), and 10 nM albuterol (D) on 100 nM OT-precontracted uterine strips from women in late pregnancy. E) Summarized results of experiments A–D. Data are means ± sem; n = 5–6 strips. F) Representative tension response to 60 mM KCl in uterine strips from women in late pregnancy. G–I) Tension responses caused by 1 mM ChQ (G), 1 mM MgSO₄ (H), and 10 nM albuterol (I) on 60 mM KCl-precontracted uterine strips from women in late pregnancy. J) Summarized results of experiments F–I. Data are means ± sem; n = 4–7 strips. ****P < 0.0001, uterotonic alone vs. uterotonic + tocolytic, unpaired Student’s t test.

Figure 5.

ChQ activates TAS2R signaling to modestly raise [Ca²⁺]_i without changes in force generation in myometrium from d 18 pregnant mice. A) Representative traces showing [Ca²⁺]_i after 1 mM ChQ, or 1 mM ChQ + 1 µg/ml PTX pretreatment. Arrow indicates time when ChQ was applied to cells. PTX and other inhibitors were applied as pretreatment as described in text. B) Effects of inhibitors of TAS2R signaling pathway on ChQ-induced rise in [Ca²⁺]_i. ChQ causes oscillations in [Ca²⁺]_i; peak [Ca²⁺]_i in A was calculated for comparison; n = 9 cells for ChQ (1 mM) alone, n = 16 for PTX (1 µg/ml), n = 14 for galleon (20 µM), n = 15 for U73122 (3 µM), and n = 14 for 2-aminoethoxydiphenyl borate (2-APB) (50 µM). ChQ alone vs. ChQ + inhibitor, unpaired Student’s t test. C) Representative trace showing that ChQ alone does not affect myometrial contraction. D) Summarized results of experiments in C; n = 5. ****P < 0.0001.

Figure 6.

ChQ activates TAS2R-coupled G protein gustducin in myometrial cells. A, B) Effects of 1 mM ChQ on 600 nM OT-induced Ca²⁺ rise in d 18 pregnant mouse myometrial cells. Fluo-3 fluorescent images (A) show changes in [Ca²⁺]_i displayed as ΔF/F₀ and were taken at time indicated by black arrows and roman letters on time course (B) of [Ca²⁺]_i (represented as ΔF/F₀ integrated over entire cell). Transient rise in [Ca²⁺]_i after ChQ was applied is likely due to activation of TAS2R signaling pathway, resulting in IP₃ generation (Supplemental Fig. 3). C, D) Effects of 1 mM ChQ on 600 nM OT-induced cell shortening in d 18 pregnant mouse myometrial cells. Changes in cell length (C, red lines) taken at time indicated on time course (D) of length change. E) Comparison of OT-induced change in [Ca²⁺]_i in myometrial cells from WT mice and Gnat3^−/− mice. Experiments were performed as in A; values were calculated on basis of ΔF/F₀ at time points marked as i and ii in A and B. F) Summarized results showing percentage reversal by ChQ of OT-induced Ca²⁺ rise in WT cells with and without PTX (1 μg/ml) treatment, and in Gnat3^−/− cells. Note that percentage reversal is [100 × (iii − iv)/(iii − i)], where i, ii, and iii are marked in B. G) Comparison of OT-induced change in cell length of myometrial cells between WT mice and Gnat3^−/− mice. Experiments were performed as in C; values were calculated on basis of lengths at time points marked i and ii in C and D. H) Summarized results showing percentage reversal by ChQ of OT-induced shortening of myometrial cells from WT and Gnat3^−/− mice. Percentage reversal is [100 × (ii − iii)/(ii − i)], where i, ii, and iii are marked in D. Means ± sem; n = 10–19 cells for (A–H). I) Relaxation by ChQ of 600 nM OT-induced contraction of myometrium from Gnat3^−/− mice is suppressed compared to that in strips from WT mice; n = 13 for WT and 9 for Gnat3^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student’s t test (E–H), and ANOVA and post hoc pairwise Student’s t tests (I).

Figure 7.

ChQ activates TAS2Rs in cultured human myometrial cells. A, B) Efficiency of shRNA knockdown targeting human TAS2R14 (A) and TAS2R10 (B) as assessed by qPCR. C, D) [Ca²⁺]_i rise induced by 10 mM ChQ is significantly suppressed by shRNA knockdown targeting human TAS2R14 (C), but not TAS2R10 (P = 0.75) (D). E, F) shRNA knockdown targeting human TAS2R14 (E), but not TAS2R10 (F), inhibits reversal by 1 mM ChQ of 600 nM OT-induced Ca²⁺ rise. Data are means ± sem; n = 30–73 cells. *P < 0.05, **P < 0.01; scrambled vs. shRNAi, unpaired Student’s t test.

Figure 8.

ChQ, via gustducin, rescues LPS- or RU-486-induced preterm labor better than MgSO₄ and albuterol. A) Mean cumulative labor induced by LPS followed by treatment with saline, ChQ, MgSO₄, or albuterol in WT mice, or followed by treatment with ChQ in Gnat3^−/− mice. LPS (10 µg, i.p.) was administered on d 16. Lines for WT-saline and WT-MgSO₄ are overlapped. P < 0.05 saline vs. ChQ; P < 0.05 saline vs. albuterol; P < 0.01 WT ChQ vs. Gnat3^−/− ChQ; n = 11 for each group and P value from χ² test. B) Mean cumulative labor induced by RU-486 followed by intrauterine administration of saline, ChQ, MgSO₄, or albuterol in WT mice, or followed by treatment with ChQ in Gnat3^−/− mice. RU-486 (150 mg, s.c.) was administered on d 16. Lines for WT-saline and WT-albuterol are overlapped. P < 0.05 saline vs. ChQ, P < 0.05 WT ChQ vs. Gnat3^−/− ChQ; n = 10–11 for each group, χ² test.