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Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor–transmembrane protein 16A–voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma

  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Pei Wang
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Wei Zhao
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Jie Sun
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Tao Tao
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Xin Chen
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Yan-Yan Zheng
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China

    College of Life Science, Nanjing Normal University, Nanjing, China
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  • Cheng-Hai Zhang
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Zhong Chen
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Yun-Qian Gao
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Fan She
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Ye-Qiong Li
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Li-Sha Wei
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China
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  • Ping Lu
    Affiliations
    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Mass
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  • Cai-Ping Chen
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China

    Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing, China
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  • Ji Zhou
    Affiliations
    Department of Respiratory, Jiangsu Province Hospital, Nanjing, China
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  • Da-Quan Wang
    Affiliations
    Department of Thoracic and Cardiovascular Surgery, Jiangsu Province Hospital, Nanjing, China
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  • Liang Chen
    Affiliations
    Department of Thoracic and Cardiovascular Surgery, Jiangsu Province Hospital, Nanjing, China
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  • Xiao-Hao Shi
    Affiliations
    Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, China
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  • Linhong Deng
    Affiliations
    Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, China
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  • Ronghua ZhuGe
    Affiliations
    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Mass
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  • Hua-Qun Chen
    Correspondence
    Hua-Qun Chen, PhD, College of Life Science, Nanjing Normal University, Nanjing 210023, China.
    Affiliations
    College of Life Science, Nanjing Normal University, Nanjing, China
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  • Min-Sheng Zhu
    Correspondence
    Corresponding author: Min-Sheng Zhu, PhD, Model Animal Research Center, Nanjing University, Nanjing 210061, China.
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center, Ministry of Education (MOE) Key Laboratory of Model Animal for Disease Study and the Medical School of Nanjing University, Nanjing, China

    Innovation Center for Cardiovascular Disorders, Beijing, China
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  • Author Footnotes
    ∗ These authors contributed equally to this work.

      Background

      Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness.

      Objective

      We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness.

      Methods

      We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model.

      Results

      Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity.

      Conclusions

      A G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.

      Graphical abstract

      Key words

      Abbreviations used:

      5-HT (5-Hydroxytryptamin), Ai38D (Cre-dependent GCaMP3 reporter mouse), ASM (Airway smooth muscle), ASMC (Airway smooth muscle cell), ClCa (Ca2+-activated Cl−), CTR (Control), GCaMP3 (Genetically encoded calcium indicator), GPCR (G protein-coupled receptor), IP3 (Inositol 1,4,5-triphosphate), IP3R (IP3 receptor), IT-CI (IP3/TMEM16A-mediated calcium influx), KO (Knockout), LTD4 (Leukotriene D4), MCh (Methacholine), mKrebs (Modified Krebs solution), MLCK (Myosin light chain kinase), NS (Not significant), OVA (Ovalbumin), PLC (Phospholipase C), ROCI (Receptor-operated Ca2+ influx), Rrs (Resistance of the respiratory system), SMC (Smooth muscle cell), SOCI (Store-operated Ca2+ influx), SR (Sarcoplasmic reticulum), T16Ainh-A01 (Specific inhibitor of TMEM16A), TMEM16A (Transmembrane protein 16A), TXA2 (Thromboxane A2), VDCC (Voltage-dependent Ca2+ channel), VOCI (Voltage-operated Ca2+ influx), ΔF/F0 (Fluorescence ratio)
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      and SKF96365, a nonselective antagonist of Ca2+ channels, markedly inhibits ASM contraction induced by many different inflammatory mediators.
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      Three patterns of Ca2+ influx, including receptor-operated Ca2+ influx (ROCI), store-operated Ca2+ influx (SOCI), and voltage-operated Ca2+ influx (VOCI) have been implicated in ASM contraction.
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      As ASM abundantly expresses transmembrane protein 16A (TMEM16A), a key molecule in Ca2+-activated Cl (ClCa) channels, we speculate that VOCI has an essential role in the contraction of mediators, because Cl channels can mediate depolarization currents in SMCs.
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      TMEM16A is a member of the ClCa channel family and is expressed in many different tissues.
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      Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle.
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      Studies on expression and function of the TMEM16A calcium-activated chloride channel.
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      by mediating VOCI, which may serve as a sensitive Ca2+ signal amplifier to participate in contraction in SMCs. In ASM and internal anal sphincter smooth muscle, even the intrinsic calcium spark may activate TMEM16A to a certain extent or even induce contraction.
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      Ca2+ sparks act as potent regulators of excitation-contraction coupling in airway smooth muscle.
      In this report, we demonstrated that some asthmatic inflammatory mediators may efficiently activate TMEM16A and thereby mediate ASM contraction. These contractile properties of ASM may be conferred by the abundant expression of TMEM16A and mediator receptors. Moreover, we suggest that inflammatory mediator-mediated contraction is regulated by a GPCR/IP3/Ca2+/TMEM16A/voltage-dependent Ca2+ channel (VDCC) signaling cascade, and this cascade may serve as a sensitive mechanism by which ASM responds to physiological and pathological stimuli. Our results emphasize the importance of inflammatory mediators in some types of asthmatic hyperresponsiveness.

      Methods

       Animals

      The animal experiments performed in this study were conducted in accordance with the guideline of the Animal Care and Use Committee of the Model Animal Research Center of Nanjing University (Nanjing, China). Establishment of the floxed allele of Tmem16a in mice has been described previously.
      • Zhang C.H.
      • Wang P.
      • Liu D.H.
      • Chen C.P.
      • Zhao W.
      • Chen X.
      • et al.
      The molecular basis of the genesis of basal tone in internal anal sphincter.
      Hartley guinea pigs were used in some tests, because histamine and leukotriene D4 (LTD4) are not potent bronchoconstrictors in mice,
      • Barnes P.J.
      Histamine and serotonin.
      • Yang G.
      • Haczku A.
      • Chen H.
      • Martin V.
      • Galczenski H.
      • Tomer Y.
      • et al.
      Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4.
      while guinea pig ASM shows the same response as the case with humans.
      • Drazen J.M.
      • Austen K.F.
      • Lewis R.A.
      • Clark D.A.
      • Goto G.
      • Marfat A.
      • et al.
      Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro.

       Western blot analysis

      Muscle strips were homogenized in 100 μL of lysis buffer as described by Davis et al.
      • Davis A.J.
      • Forrest A.S.
      • Jepps T.A.
      • Valencik M.L.
      • Wiwchar M.
      • Singer C.A.
      • et al.
      Expression profile and protein translation of TMEM16A in murine smooth muscle.
      The primary antibodies used for the Western blots are summarized in Table E1 in this article's Online Repository at www.jacionline.org. Enhanced chemiluminescence (ECL) reagents (SUDGEN Co, Ltd, Nanjing, China) was used to visualize the blots.

       Isolation of ASMCs

      Smooth muscle strips were prepared from the trachea and extrapulmonary bronchus of the TMEM16A control (CTR) and knockout (KO) mice (8-20 weeks, both sexes) or Hartley guinea pigs (250-300 g, male), and treated with papain and collagenase as previously described.
      • Zhuge R.
      • Bao R.
      • Fogarty K.E.
      • Lifshitz L.M.
      Ca2+ sparks act as potent regulators of excitation-contraction coupling in airway smooth muscle.

       Patch-clamp recording

      All currents were recorded using a MultiClamp 700B amplifier, Digidata 1440A and pClamp 10 (Molecular Devices, Sunnyvale, Calif). The currents were filtered at 2 kHz and sampled at 10 kHz, and the data were analyzed using Clampfit 10. A whole-cell patch clamp was used to record the Cl currents. A focal pressurized perfusion (ALA Scientific Instruments, Farmingdale, NY) of 3 μmol/L 5-HT, 1 μmol/L U46619, 3 μmol/L histamine, 100 nmol/L LTD4, 3 μmol/L methacholine (MCh), and 10 mmol/L caffeine was administered through the application pipette to induce currents.

       Isometric contraction measurement

      Isometric contraction of mouse left extrapulmonary bronchi were measured by using a wire myograph (610-M; Danish Myo Technology, Aarhus, Denmark) with 37°C Krebs solution. Isometric contraction of guinea pig tracheal and human bronchial smooth muscle strips were measured by using a force transducer (MLT0201, AD Instruments, Bella Vista, New South Wales, Australia). Smooth muscle strips of guinea pig were incubated in a 37°C organ bath with modified Krebs solution (mKrebs),
      • Weichman B.M.
      • Muccitelli R.M.
      • Tucker S.S.
      • Wasserman M.A.
      Effect of calcium antagonists on leukotriene D4-induced contractions of the guinea-pig trachea and lung parenchyma.
      while human bronchial strips were measured in Krebs. Human bronchial smooth muscle strips were isolated from lung biopsies of pulmonary cancer patients, from the Department of Thoracic and Cardiovascular Surgery of Jiangsu Province Hospital.

       Measurement of Ca2+ signals

      The Tmem16aSMKO and CTR mice were crossed with Cre-dependent GCaMP3, a genetically encoded calcium indicator, reporter mice (Ai38D).
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      • Borghuis B.G.
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      • Madisen L.
      • Tian L.
      • De Zeeuw C.I.
      • et al.
      A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.
      The GCaMP3 fluorescence of left extrapulmonary bronchial smooth muscle was excited using a 488-nm argon laser, and the images were recorded every 2.5 seconds using a confocal microscope (FV-1000, Olympus, Tokyo, Japan). The dynamics of the Ca2+ signals were determined by the percentage of fluorescence ratio (ΔF/F0).

       Measurement of respiratory resistance

      We used the flexiVent system (SCIREQ, Montreal, Quebec, Canada) to measure respiratory resistance. The ventilator was set to generate a tidal volume of 10 mL/kg at a frequency of 150 breaths per minute. Resistance of the respiratory system (Rrs) was measured by a perturbation of SnapShot (SCIREQ, Montreal, Quebec, Canada).

       Statistical analysis

      The data are presented as the means ± SEMs. Differences among the groups were determined using an unpaired 2-tailed Student t test or a 2-way ANOVA. A paired 2-tailed t test was also used to evaluate differences in some experiments (labeled in the text). The significance levels were set as follows: not significant (NS) P > .05, *P < .05, **P < .01, ***P < .001, ****P < .0001.
      For detailed descriptions of materials and methods, please see the Methods section in the Online Repository at www.jacionline.org.

      Results

       Inflammatory constrictors induce ClCa currents through TMEM16A in ASMCs

      Many inflammatory mediator receptors belong to the GPCR family, and their activation generates ClCa currents to depolarize the membrane in ASMCs.
      • Janssen L.
      • Sims S.
      Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes.
      • Janssen L.
      • Sims S.
      Histamine activates Cl-and K+ currents in guinea-pig tracheal myocytes: convergence with muscarinic signalling pathway.
      To determine the molecular nature of these currents, we performed whole cell voltage clamp recording (Fig 1, A) in ASMCs from TMEM16A KO mice and their CTRs. Before the tests, we measured the KO efficiency of TMEM16A protein and ClCa currents in ASM (see Fig E1 in the Online Repository at www.jacionline.org). In CTR ASMCs, 3 μmol/L 5-HT elicited inward currents in 11 of 26 SMCs (Fig 1, B and Fig E2, A in the Online Repository at www.jacionline.org). Similar currents were also detected when cells were treated with 1 μmol/L U46619, a stable derivative of thromboxane A2 (TXA2)
      • Coleman R.A.
      • Humphrey P.P.
      • Kennedy I.
      • Levy G.P.
      • Lumley P.
      Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H2 and thromboxane A2 on some isolated smooth muscle preparations.
      • Takami M.
      • Tsukada W.
      Correlative alteration of thromboxane A2 with antigen-induced bronchoconstriction and the role of platelets as a source of TXA2 synthesis in guinea pigs: effect of DP-1904, an inhibitor of thromboxane synthetase.
      (Fig 1, C and Fig E2, B). We found that 3 μmol/L MCh also efficiently induced inward currents, with apparent outward currents (Fig 1, D and Fig E2, C). Strikingly, deletion of TMEM16A essentially eliminated the inward currents induced by all 3 constrictors (Fig 1, B-D and Fig E2, A and C). This effect was highly unlikely to be secondary to TMEM16A deletion because the expression of the constrictor receptors remained unchanged in the mutant ASM (see Fig E3 in this article's Online Repository at www.jacionline.org). These data indicate that both cholinergic and inflammatory constrictors share a common mechanism by activating TMEM16A to elicit ClCa currents.
      Figure thumbnail gr1
      Fig 1TMEM16A mediates agonist-induced inward currents in ASMCs. A, Recording configuration. B-D, Left, Representative traces of 5-HT-induced (B), U46619-induced (C), and MCh-induced (D) currents in ASMCs from CTR and TMEM16A KO mice. Right, Quantification of the results (5-HT: n = 26 cells from 4 mice of each genotype; U46619: n = 20 cells from 4 mice for CTR, n = 22 cells from 4 mice for KO; MCh: n = 12 cells from 3 mice per genotype). E, F, Left, Representative traces of histamine-induced (E) and LTD4-induced (F) currents in dimethyl sulfoxide (DMSO) and T16Ainh-A01 preincubated guinea pig ASMCs. Right, Quantification of the results (histamine: n = 14 cells from 3 guinea pigs per group; LTD4: n = 21 cells for the DMSO group and n = 23 cells for the T16Ainh-A01 group from 4 guinea pigs each). The holding potential in all recordings was -60 mV. The blue blocks indicate the peak values of the outward currents, and the pink circles indicate the peak values of the inward currents. The bars represent the means ± SEMs; *P < .05, **P < .01, ***P < .001, ****P < .0001; 2-tailed Student t test; blue “*” for outward currents and red “*” for inward currents.
      We wondered whether TMEM16A also underlies ClCa currents evoked by other inflammatory mediators. We selected guinea pigs to test the effects of histamine and LTD4, because these 2 mediators are not potent in mice,
      • Barnes P.J.
      Histamine and serotonin.
      • Yang G.
      • Haczku A.
      • Chen H.
      • Martin V.
      • Galczenski H.
      • Tomer Y.
      • et al.
      Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4.
      and guinea pig ASM shows the same response as the case with humans.
      • Drazen J.M.
      • Austen K.F.
      • Lewis R.A.
      • Clark D.A.
      • Goto G.
      • Marfat A.
      • et al.
      Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro.
      Fig 1, E and F show that histamine and cysteinyl leukotrienes generated inward currents similar to the ClCa current induced by 5-HT and U46619 in mice ASMCs, although the amplitudes varied. These ClCa currents result from the opening of TMEM16A channels, because 10 μmol/L T16Ainh-A01, a specific inhibitor of TMEM16A,
      • Namkung W.
      • Phuan P.W.
      • Verkman A.S.
      TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells.
      essentially blocked these currents (Fig 1, E and F and Fig E2, D and E). In sum, TMEM16A underlies inflammatory mediator-induced ClCa channel activity in ASM cells from different species.

       TMEM16A is required for contractile responses to inflammatory constrictors

      We sought to determine the role of TMEM16A in constrictor-mediated contraction using TMEM16A KO and T16Ainh-A01-treated ASM ex vivo. We first examined the morphology of airways from KO and CTR mice. As shown in Fig 2, A and B, no apparent morphological difference is noticed, and no apparent change in muscle thickness can be detected in KO bronchus (P > .05, vs CTR, unpaired 2-tailed Student t test). We then compared the contractile responses to different stimuli in ASM from the KO (flox/flox; Cre+) and control mice (flox/+; Cre+ or flox/flox; Cre-). On stimulation with 60 mmol/L KCl or 10 μmol/L MCh, the KO ASM developed a force that was comparable to that of the control muscles (Fig 2, C and D and Fig E4, A and B in the Online Repository at www.jacionline.org), suggesting that these muscles had a normal contractile apparatus. Interestingly, when the KO muscle was treated with MCh at a concentration as low as 300 nmol/L, the evoked force was significantly reduced (KO: 2.58 ± 0.68 mN vs CTR: 5.66 ± 0.95 mN, P < .05, unpaired 2-tailed Student t test) (see Fig E5 in this article's Online Repository at www.jacionline.org). This result indicates that TMEM16A may be necessary for the contractile response to mild cholinergic stimulation. U46619 induced ASM contraction in a dose-dependent manner (Fig 2, E and Fig E4, C), and the maximal force induced was comparable to that achieved with MCh (∼12 mN for MCh, ∼10 mN for U46619). After TMEM16A deletion, 34% to 76% of the force was significantly inhibited (P < .0001 by 2-way ANOVA) (Fig 2, E), and the extent of inhibition varied with the dosage, as TMEM16A contributed more contraction at low U46619 concentration. We found that 5-HT also evoked CTR ASM contraction in a dose-dependent manner, the maximal force developed was ∼5 to 6 mN, which was only one-half of the force induced by U46619 or MCh (Fig 2, F). To our surprise, TMEM16A-deficient ASM displayed an almost abolished contractile response to various doses of 5-HT (P < .0001, 2-way ANOVA) (Fig 2, F and Fig E4, D), indicating that the force induced by 5-HT was primarily mediated by TMEM16A. This effect could be further confirmed in vivo, as the elevated Rrs of the mice challenged with 5-HT was significantly inhibited by TMEM16A deletion (P < .001, 2-way ANOVA) (Fig 2, G).
      Figure thumbnail gr2
      Fig 2TMEM16A is required for contraction in response to constrictors in mouse ASM. A, Representative hematoxylin and eosin staining of extrapulmonary bronchi from CTR and TMEM16A KO mice. Scale bars = 200 μm. B, TMEM16A deletion did not alter the thickness of the extrapulmonary bronchus. The bars represent the means ± SEMs; NS, P > .05; 2-tailed Student t test. C-F, Dose-response curves of TMEM16A KO (flox/flox Cre+), CTR (flox/+ Cre+) and (flox/flox Cre-) bronchial smooth muscle contraction in response to high K+ and different agonists. The bars indicate the means ± SEMs; the sample sizes (n) are indicated in the panels; ****P < .0001 comparing KO (flox/flox Cre+) with CTR (flox/+ Cre+) by ANOVA. G, In vivo dose-response curve of the Rrs of TMEM16A KO and CTR mice in response to 5-HT. The bars represent the means ± SEMs; the sample sizes (n) are indicated in the panel; ***P < .001; ANOVA.
      We also used ASM from guinea pigs to measure the contractile responses to histamine and LTD4. Both constrictors generated force in a dose-dependent manner, as previously reported (Fig 3, C and D).
      • Drazen J.M.
      • Austen K.F.
      • Lewis R.A.
      • Clark D.A.
      • Goto G.
      • Marfat A.
      • et al.
      Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro.
      Next, we used the inhibitor, T16Ainh-A01, to assess the role of TMEM16A in histamine- and LTD4-induced contraction. The specificity of this inhibitor in ASM was first confirmed using Tmem16aSMKO mice (Fig 3, A and B). Preincubating with T16Ainh-A01 largely dropped the basal tone of guinea pig ASM and decreased the capacity of histamine and LTD4 to induce contraction (Fig 3, C and D and Fig E6, A-C in the Online Repository at www.jacionline.org). In particular, the addition of T16Ainh-A01 almost completely abolished the force induced by low doses of histamine (10-300 nmol/L) and LTD4 (0.01-0.3 nmol/L) (Fig 3, C and D and Fig E6, A, and B). As the doses increased, the inhibition due to T16Ainh-A01 was rapidly reduced. When the histamine and LTD4 doses reached 3 μmol/L and 10 nmol/L, respectively, T16Ainh-A01 showed no apparent inhibition effect. Inhibition of TMEM16A shifts the half maximal effective concentration for histamine from 0.18 ± 0.07 μmol/L to 1.54 ± 0.45 μmol/L (P < .05, unpaired 2-tailed Student t test, n = 5) and for LTD4 from 69.09 ± 18.76 pmol/L to 2.58 ± 0.85 nmol/L (P < .05, unpaired 2-tailed Student t test, n = 5). These results suggest that TMEM16A is required for the sensitized response to weak stimulation with histamine and LTD4. We also tested T16Ainh-A01 activity on human bronchial smooth muscle. The addition of 10 μmol/L T16Ainh-A01 did not alter the basal tone, but it significantly inhibited the force induced by histamine (Fig 3, E). Taken together, the genetic deletion or pharmacological inhibition of TMEM16A inhibits the force evoked by inflammatory constrictors, suggesting that TMEM16A is required for the contraction.
      Figure thumbnail gr3
      Fig 3T16Ainh-A01 suppressed contractile response to constrictors in ASMs from guinea pigs and humans. A, Preincubation with 10 μmol/L T16Ainh-01 suppressed 3 μmol/L 5-HT induced left extrapulmonary bronchi contraction in CTR but not TMEM16A KO (CTR: n = 6, KO: n = 7). B, Preincubation with 10 μmol/L T16Ainh-01 did not suppress 3-μmol/L MCh–induced contraction in both CTR and KO (CTR: n = 5, KO: n = 5). Bars represent means ± SEMs; NS, P > .05, **P < .01 by 2-tailed Student t test. Dose-response curves of DMSO-preincubated and T16Ainh-A01-preincubated guinea pig tracheal smooth muscle contraction in response to histamine (C) and LTD4 (D). All force values are expressed as percentages of the reference contraction induced by 60 mmol/L KCl. The bars represent the means ± SEMs; the sample sizes (n) are indicated in the panel; ****P < .0001; ANOVA. E, Dose-response curve of DMSO-preincubated and T16Ainh-A01-preincubated human bronchial smooth muscle contraction in response to histamine. (Please see recording traces of each sample in .) All force values are expressed as percentages of the reference contraction induced by 60 mmol/L KCl. The bars represent the means ± SEM; the sample sizes (n) are indicated in the panel; *P < .05; ANOVA.

       Inflammatory constrictors activate GPCR-TMEM16A-VDCC signaling axis, leading to ASM contraction

      How do inflammatory constrictors activate TMEM16A, leading to ASM contraction? Based on the results in Figs 1 and 2 that inflammatory mediators induce ClCa currents and that the reversal potential for Cl in smooth muscle is approximately -30 to -20 mV,
      • Chipperfield A.
      • Harper A.
      Chloride in smooth muscle.
      we hypothesized that inflammatory mediators activate GPCR to release Ca2+ from SR, which in turn opens TMEM16A and subsequently VDCCs, leading to Ca2+ influx and contraction. To test this hypothesis, we examined the changes in Ca2+ signal by interfering with this signaling pathway. To measure the intracellular calcium concentration, we crossed Tmem16aSMKO mice with Ai38D mice, a line that expresses the GCaMP3 calcium indicator.
      • Zariwala H.A.
      • Borghuis B.G.
      • Hoogland T.M.
      • Madisen L.
      • Tian L.
      • De Zeeuw C.I.
      • et al.
      A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.
      In ASM from Tmem16aSMKO, 60 mmol/L KCl induced a robust calcium elevation similar to the CTR muscle (Fig 4, A and Video E1 in the Online Repository at www.jacionline.org). Also, 3 μmol/L MCh induced an increase in calcium, with an average peak ΔF/F0 value of 39.67 ± 3.44%, which did not differ from the CTR (P = .167) (Fig 4, I and Video E2 in the Online Repository at www.jacionline.org). These calcium elevation patterns appeared to be consistent with the corresponding contractile responses (Fig E5). Because 5-HT is a primary and typical inflammatory constrictor in mice, we measured the substantial calcium-releasing effect of 5-HT. We found that 300 nmol/L 5-HT induced a smaller rise in the ΔF/F0 peak value in KO ASM compared with the CTR (Tmem16aSMKO: 6.56 ± 1.02% vs CTR: 13.20 ± 2.15%; P < .05, unpaired 2-tailed Student t test) (Fig 4, C and Video E3 in this article's Online Repository at www.jacionline.org). When the muscle was treated with a relatively high dose of 5-HT (3 μmol/L), the ΔF/F0 peak value was 23.00 ± 3.29%, which was also significantly lower than the CTR (34.35 ± 4.31%) (P < .05, unpaired 2-tailed Student t test) (Fig 4, D and Video E4 in the Online Repository at www.jacionline.org). The ΔF/F0 value in the sustained phase was significantly reduced in 5-HT-treated KO muscle (P < .001, 2-way ANOVA) (Fig 4, C and D). We also measured the calcium release induced by other constrictors in Tmem16aSMKO smooth muscles and observed similar trends (data not shown). These results showed that TMEM16A was required for the calcium elevation in response to constrictors.
      Figure thumbnail gr4
      Fig 4KO of TMEM16A-suppressed Ca2+ signal. The Ca2+ elevation dynamics (left) and peak values (right) of 60 mmol/L KCl–induced (A), 3 μmol/L MCh–induced (B), 300 nmol/L 5-HT–induced (C), and 3 μmol/L 5-HT–induced (D) Ca2+ signaling in CTR and KO bronchial smooth muscle. The bars represent the means ± SEMs; the sample sizes (n) are indicated in the panel; NS, P > .05, *P < .05 by 2-tailed Student t test, ****P < .0001 by ANOVA.
      To determine whether this calcium release was mediated by VDCCs, we examined the effect of nifedipine, a specific inhibitor of l-type Ca2+ channels
      • Stork A.P.
      • Cocks T.M.
      Pharmacological reactivity of human epicardial coronary arteries: phasic and tonic responses to vasoconstrictor agents differentiated by nifedipine.
      on constrictor-induced rise of contraction. We found that 1 μmol/L nifedipine reduces both contraction induced by MCh and 5-HT, though to a much greater extent in 5-HT. However, in the KO bronchial smooth muscles, 1 μmol/L nifedipine did not alter the dose-response curves of either MCh or 5-HT (Fig 5, A and B). Although a previous report
      • Danielsson J.
      • Perez-Zoghbi J.
      • Bernstein K.
      • Barajas M.B.
      • Zhang Y.
      • Kumar S.
      • et al.
      Antagonists of the TMEM16A calcium-activated chloride channel modulate airway smooth muscle tone and intracellular calcium.
      suggested that TMEM16A may also regulate Ca2+ release from SR, we did not observe a greater inhibition effect in TMEM16A KO ASM compared with nifedipine-treated CTR ASM (Fig 5). Collectively, the results suggested that both inflammatory constrictors and a low dose of MCh might regulate ASM contraction through the GPCR-TMEM16A-VDCC axis.
      Figure thumbnail gr5
      Fig 5TMEM16A mediates contraction through VDCCs. Effect of preincubation with nifedipine on the dose-response curves of CTR and KO bronchial smooth muscle contraction in response to MCh (A) and 5-HT (B). Left, Representative traces; right, quantitative results. The bars represent the means ± SEMs; the sample sizes (n) are indicated in the panel; ****P < .0001 CTR + nifedipine compared with CTR; ANOVA.

       Inflammatory constrictors augment the cholinergic response through TMEM16A and are required for asthmatic hypersensitivity

      In asthma, the ASM becomes more sensitive to cholinergic stimulation, which reflects airway hyperresponsiveness. We hypothesized that TMEM16A activation by inflammatory constrictors might be involved in this process. Thus, we measured the ability of different inflammatory constrictors to augment MCh-induced responses. We pretreated mouse ASM with low doses of the constrictors (5-HT: 100 nmol/L; U46619: 30 nmol/L), and found no apparent or weak contraction. The ASM was then treated with low doses of MCh (30 nmol/L or 100 nmol/L), which also evoked no apparent or weak contraction (see Fig E7 in this article's Online Repository at www.jacionline.org). But the combination of inflammatory constrictors and MCh led to a significant increase in the evoked force (Fig 6, A and Fig E7). These augmented effects were significantly inhibited by TMEM16A deletion (P < .05, unpaired 2-tailed Student t test) (Fig 4, A). The percentage of inhibition of 5-HT and U46619 in the KO was 42.17 ± 10.51% and 60.34 ± 4.56%, respectively. These results show that the inflammatory constrictors potentiate ASM contraction in response to cholinergic stimulation through TMEM16A.
      Figure thumbnail gr6
      Fig 6TMEM16A is required for airway hyperresponsiveness. A, TMEM16A deletion attenuated the ability of 5-HT and U46619 to augment MCh stimulation. The bars represent the means ± SEM; the sample sizes (n) are indicated in the panel; ##P < .01, ####P < .0001 comparing the groups indicated by the lines using a paired 2-tailed Student t test; *P < .05, **P < .01 compared with CTR using an unpaired 2-tailed Student t test. B, Allergen (OVA) inhalation induced Rrs elevation in the asthmatic (OVA groups) and nonasthmatic (PBS groups) CTR and KO mice. The bars represent the means ± SEMS; the sample sizes (n) are indicated in the panel; ****P < .0001 CTR OVA versus CTR PBS by ANOVA; ####P < .0001 KO OVA versus CTR OVA by ANOVA. C, MCh gradient inhalation induced Rrs elevation in the asthmatic (OVA groups) and nonasthmatic (PBS groups) CTR and KO mice. Inset, Percentage of inhibition of ΔRrs by TMEM16A KO; the values were normalized by comparing them with the mean ΔRrs value of the corresponding MCh doses. The bars represent the means ± SEMS; the sample sizes (n) are indicated in the panel; *P < .05, **P < .01 compared with CTR PBS by the 2-tailed Student t test; #P < .05 compared with CTR OVA by the 2-tailed Student t test.
      To determine the possible contribution of this augmented effect in asthmatic constriction, we established an acute asthma model induced by chicken ovalbumin (OVA). Both the CTR and KO asthmatic mice displayed similar inflammatory infiltration and epithelial swelling (see Fig E8 in this article's Online Repository at www.jacionline.org). The CTR asthmatic animals that were challenged with the allergen (5% OVA) showed an elevated Rrs, as expected, whereas the KO asthmatic animals had a significantly lower Rrs (P < .001, 2-way ANOVA) (Fig 6, B). Moreover, after challenge with various doses of MCh, the KO animals also displayed reduced airway constriction (Fig 6, C), but the extent of inhibition varied with the MCh dose. When the MCh dose was <30 μg per mouse, the ΔRrs was inhibited by 40% in the KO mice, but no inhibition was measured when the doses were increased to 100 to 300 μg per mouse (Fig 6, C inset). Collectively, these results suggest that inflammatory constrictors may increase the cholinergic response through TMEM16A, thereby regulating asthmatic constriction.

      Discussion

      In asthmatic airways, a large amount of inflammatory mediators are released from infiltrated immune cells and some directly cause smooth muscle spasms.
      • Barnes P.J.
      • Chung K.F.
      • Page C.P.
      Inflammatory mediators of asthma: an update.
      The molecular mechanism of inflammatory mediator-induced airway contraction remains incompletely understood. In this study, we found that inflammatory constrictors such as 5-HT, TXA2, LTD4, and histamine were capable of inducing ClCa currents in ASMCs by activating the TMEM16A Cl channel, the resultant activation of TMEM16A and calcium influx were necessary for their contractile responses. This regulatory mechanism was also predominantly adopted by a weak cholinergic stimulation in terms of contraction. Because of the abundant expression of TMEM16A in ASM, TMEM16A-based signaling may serve as a sensitive mechanism by which ASM responds to physiological and pathologic stimuli. Moreover, we found that TMEM16A was also required by the synergistic effect of inflammatory mediators and MCh both ex vivo and in asthmatic animals. This effect may well explain why the airway becomes more sensitive to neuronal stimuli during allergenic inflammation in asthma. As such a sensitivity is an essential factor for the formation of asthmatic responsiveness, our results suggest that ASM contractility mediated by the activation of TMEM16A may contribute significantly to asthmatic hyperresponsiveness.
      The receptors for inflammatory mediators mediate immune responses through multiple signaling cascades.
      • Barnes P.J.
      • Chung K.F.
      • Page C.P.
      Inflammatory mediators of asthma: an update.
      Among these cascades, calcium release from the SR is mediated by the sequential activation of Gq, PLC, and IP3/IP3 receptor (IP3R).
      • Billington C.K.
      • Penn R.B.
      Signaling and regulation of G protein-coupled receptors in airway smooth muscle.
      In this study, we show that in ASM cells that express both TMEM16A and constrictor receptors, the activation of Gq/PLC/IP3/IP3R signaling can also activate TMEM16A. Several lines of evidence from other reports also support such coupled activation of TMEM16A
      • Jin X.
      • Shah S.
      • Liu Y.
      • Zhang H.
      • Lees M.
      • Fu Z.
      • et al.
      Activation of the Cl− channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor.
      • Borchers M.T.
      • Biechele T.
      • Justice J.P.
      • Ansay T.
      • Cormier S.
      • Mancino V.
      • et al.
      Methacholine-induced airway hyperresponsiveness is dependent on Gαq signaling.
      by this signaling pathway. Thus, inflammatory constrictors may induce 1 more signaling pathway to promote calcium elevation in asthmatic airways, IP3/TMEM16A-mediated calcium influx (IT-CI). Based on our observation that TMEM16A inhibition did not always completely abolish the force induced by the constrictors, the functional contribution of IT-CI to the contractile responses may vary according to the constrictor, for example, the contribution to the 5-HT response is more profound than that to TXA2. Here, we propose a working model for the regulation of ASM contraction by constrictors (Fig 7). In this model, ASMC expresses abundant TMEM16A clusters, inflammatory constrictor receptors, and muscarinic receptors. Activation of their corresponding receptors produces IP3 and promotes calcium release from the SR. The elevated [Ca2+]i, in turn, causes TMEM16A activation to generate Cl current and VDCC activation, and VOCI, which activates MLCK. This signaling pathway primarily responds to inflammatory constrictors and cholinergic transmitters, particularly at low concentrations. If the signals triggered by these stimuli are strong enough, then the calcium release from SR or through ROCI and SOCI will be sufficient to directly activate MLCK to contract smooth muscle.
      Figure thumbnail gr7
      Fig 7Schematic showing the hypothesized TMEM16A-based signaling model. A, Inflammatory constrictors bind to their corresponding GPCRs and sequentially activate the Gq-PLC-IP3 axis. IP3 activates IP3Rs and induces Ca2+ release from SR. Ca2+ released from the SR can directly (IP-CR) or indirectly activate MLCK through the TMEM16A-VDCCs axis, mediate Ca2+ influx (IT-CI), amplify the cytosolic Ca2+ signal and activate more MLCK through this indirect pathway. B, Under stimulation of single constrictor, TMEM16A can be activated to depolarize plasma membrane. C, Under simultaneous stimulation of inflammatory constrictors and acetylcholine (ACh), ASMCs always show augmented contraction. This augmentation effect may be due to the interaction of the signaling network formed by different receptors. If the spatial distance between different GPCRs is small enough, the activation of these receptors may enhance local activation of IT-CI and hence augment contractile response. IP-CR, IP3-mediated calcium release from the SR.
      A unique property of asthma is that the ASM becomes more sensitive to cholinergic and other stimuli, which underlies 1 aspect of hyperresponsiveness.
      • Cockcroft D.W.
      • Davis B.E.
      Mechanisms of airway hyperresponsiveness.
      A synergistic effect of allergenic inflammatory mediators and a cholinergic transmitter is believed to be a possible cause of this effect.
      • Catalli A.
      • Janssen L.J.
      Augmentation of bovine airway smooth muscle responsiveness to carbachol, KCl, and histamine by the isoprostane 8-iso-PGE2.
      • Gerthoffer W.T.
      Agonist synergism in airway smooth muscle contraction.
      In this report, we indeed observed an apparent synergistic effect of inflammatory constrictors and MCh. Importantly, we found that this effect could be significantly blocked by TMEM16A deletion. Because the synergistic effect of the inflammatory constrictors and MCh is more profound at low concentrations,
      • Catalli A.
      • Janssen L.J.
      Augmentation of bovine airway smooth muscle responsiveness to carbachol, KCl, and histamine by the isoprostane 8-iso-PGE2.
      • Gerthoffer W.T.
      Agonist synergism in airway smooth muscle contraction.
      we suggest that the GPCR/TMEM16A/VDCCs cascade used by the constrictors may play an essential role in asthmatic hyperresponsiveness. The genesis of hyperresponsiveness may be due to the synergistic activation of TMEM16A cluster by combined stimulation of nerve transmitter and inflammatory constrictors that are overproduced in asthmatic airway (Fig 7). Considering that inhibition of TMEM16A activity is capable of not only directly relaxing ASM but also reducing hyperresponsiveness, we speculate that TMEM16A inhibitors might be an ideal strategy for asthma therapy. In addition, compared with antagonists of inflammatory mediators such as leukotriene antagonists, which have been approved and are effective asthma therapies,
      • Price D.
      • Musgrave S.D.
      • Shepstone L.
      • Hillyer E.V.
      • Sims E.J.
      • Gilbert R.F.T.
      • et al.
      Leukotriene antagonists as first-line or add-on asthma-controller therapy.
      an important advantage of TMEM16A inhibition is that it may block multiple constrictors simultaneously and provide improved efficacy.
      Key messages
      • TMEM16A activation depolarizes the membrane of ASM and causes calcium influx and contraction, which causes the airway to sensitively respond to several inflammatory mediators and cholinergic stimuli.
      • Synergistic activation of TMEM16A by allergenic inflammatory mediators and cholinergic transmitter contributes to asthmatic hyperresponsiveness.
      We would like to thank Dr Lawrence M. Lifshitz for his critical reading of the manuscript and constructive comments, and Dr Min-Min Luo for his advice on Ca2+ signal measurement and his donation of Ai38D mice.

      Methods

       Genetic mouse models

      TMEM16A CTR (Tmem16aflox/+; SMA-Cre+) and KO/Tmem16aSMKO (Tmem16aflox/flox; SMA-Cre+) mice of both sexes were subjected to a series of analyses at 8 to 20 weeks, including Western blot, immunofluorescence, patch-clamp, isometric tension, and respiratory resistance measurements. Tmem16aflox/flox; SMA-Cre- mice of both sexes were also used as 1 more control group in some isometric tension measurements as indicated in Fig 2. TMEM16A CTR and KO mice were crossed with Ai38D mice,
      • Zariwala H.A.
      • Borghuis B.G.
      • Hoogland T.M.
      • Madisen L.
      • Tian L.
      • De Zeeuw C.I.
      • et al.
      A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.
      and both sexes were used for the Ca2+ measurements at 8 to 20 weeks. All mice were in a mixed C57BL/6 and Sv/129 background.

       Western blot analysis

      Muscle strips isolated from the trachea and extrapulmonary bronchus of CTR and KO mice were homogenized in 100 μL of lysis buffer (20 mmol/L TRIS base, 137 mmol/L NaCl, 2 mmol/L EDTA, 1% Nonidet P 40 (NP-40), 10% glycerol, protease inhibitor cocktail (Roche, Mannheim, Germany) (pH = 8).
      • Davis A.J.
      • Forrest A.S.
      • Jepps T.A.
      • Valencik M.L.
      • Wiwchar M.
      • Singer C.A.
      • et al.
      Expression profile and protein translation of TMEM16A in murine smooth muscle.
      Next the homogenates were incubated on ice for 15 minutes and centrifuged at 3000g to remove tissue debris. Then 80 μL of each sample was transferred to a new tube. The protein concentrations were measured with a bicinchoninic acid kit (Pierce BCA protein assay kit, Pierce Biotechnology, Rockford, Ill). The proteins were mixed with 5× sample buffer (10% SDS, 20% glycerol, 0.05% bromophenol blue, 10 mmol/L β-mercapto-ethanol, 200 mmol/L TRIS-HCl, 8 mol/L urea) and then denatured at 95°C for 5 minutes. After resolution by SDS-PAGE, the proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% nonfat milk in RIS-buffered saline with Tween, followed by sequential incubation with primary and horseradish peroxidase–conjugated secondary antibodies. The primary antibodies used for the Western blots are summarized in Table E1. ECL (SUDGEN Co, Ltd, Nanjing, China) was used to visualize the blots.

       Immunofluorescence

      Paraffin slices (10-μm thick) of the left lobe of the mouse lung were used for immunofluorescence staining. Nonspecific binding of the primary antibodies was blocked by incubating the slices with PBS with Tween buffer containing 1% BSA and 5% nonimmune goat serum. A rabbit anti-TMEM16A polyclonal antibody (1:200 dilution, ab53212, Abcam, Cambridge, UK) and mouse anti-α-smooth muscle actin (SMA) monoclonal antibody (1:500 dilution, Clone 1A4, Thermo Fisher Scientific, Waltham, Mass) were used as primary antibodies. An Alexa Fluor 488-conjugated goat antirabbit antibody (Invitrogen, Thermo Fisher Scientific) diluted 1:250 and an Alexa Fluor 555-conjugated goat antimouse antibody (Invitrogen) diluted 1:250 were used as secondary antibodies. The cell nuclei were stained with 4′-6-diamidino-2-phenylindole, dihydrochloride. The images were captured using a confocal microscope (FV-1000, Olympus).

       Isolation of ASMCs

      Smooth muscle strips were prepared from the trachea and extrapulmonary bronchus of the TMEM16A CTR and KO mice (8-20 weeks, both sexes) or Hartley guinea pigs (250-300 g, male). The resulting tissues were dissected in dissociation medium (DM) (136 mmol/L NaCl, 5.36 mmol/L KCl, 0.44 mmol/L KH2PO4, 4.16 mmol/L NaHCO3, 0.34 mmol/L Na2HPO4, 20 mmol/L HEPES, 5.56 mmol/L glucose, 4.9 mmol/L MgCl2, pH 7.1). The first digestion was performed with MgCl2-free DM containing 30 U/mL papain, 0.2 mmol/L dithiothreitol, and 0.02 mmol/L EDTA, and the second digestion was performed with a solution containing 3 U/mL collagenase 1A, 1 mg/mL BSA, and 0.1 mg/ml deoxyribonuclease I in MgCl2-free DM. The tubes from the second digestion were shaken vigorously until most of the clumps disappeared (∼20 minutes). After centrifugation at 300g for 2 minutes, the collected cells were washed with MgCl2-free DM once by centrifugation at 300g for 2 minutes. The isolated cells were resuspended in DM, transferred to dishes, and stored at 4°C. DM was replaced with the bath solution for patch-clamp recording prior to use.

       Patch-clamp recording

      All currents were recorded using a MultiClamp 700B amplifier, Digidata 1440A and pClamp 10 (Molecular Devices). The currents were filtered at 2 kHz and sampled at 10 kHz, and the data were analyzed using Clampfit 10 (Molecular Devices).
      To record the Cl currents following stimulation with 600 nmol/L [Ca2+]i, the bath solution contained 150 mmol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L d-glucose, 10 mmol/L mannitol, and 10 mmol/L HEPES; the pH was adjusted to 7.4 with NaOH. The pipette solution contained 130 mmol/L CsCl, 10 mmol/L ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1 mmol/L MgCl2, 8 mmol/L CaCl2, 10 mmol/L HEPES, and 1 mg ATP; the pH was adjusted to 7.3 with NaOH.
      • Manoury B.
      • Tamuleviciute A.
      • Tammaro P.
      TMEM16A/anoctamin 1 protein mediates calcium-activated chloride currents in pulmonary arterial smooth muscle cells.
      ECl = 0 mV under these conditions. Whole-cell currents were recorded in freshly isolated SMCls in response to 1-second voltage pulses from −80 to +100 mV in 10-mV increments, followed by 700 ms pulses to -60 mV.
      To record the currents induced by the constrictors and caffeine, the bath solution contained 130 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 20 mmol/L HEPES, and 10 mmol/L d-glucose; the pH was adjusted to 7.4 with NaOH. The pipette solution contained 140 mmol/L KCl, 1 mmol/L MgCl2, 20 mmol/L HEPES, and 0.025 mmol/L ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid; the pH was adjusted to 7.2 with KOH. ECl = 0 mV under these conditions. Cells were held at -60 mV. A focal pressurized perfusion (ALA Scientific Instruments) of 3 μmol/L 5-HT, 1 μmol/L U46619, 3 μmol/L histamine, 100 nmol/L LTD4, 3 μmol/L MCh, and 10 mmol/L caffeine was administered through the application pipette to induce currents. The currents were recorded no longer than the fifth minute after breaking the patch of membrane under the pipette.

       Histopathology

      The extrapulmonary bronchi and left lobes of the lung were immersed in 4% Paraformaldehyde (PFA) at 4°C overnight and dehydrated in a graded series of ethanol solutions. The tissue was embedded in paraffin, and 10-μm sections were prepared for standard hematoxylin and eosin staining. Standard hematoxylin and eosin staining was performed on paraffin slices (10-μm thick) of the extrapulmonary bronchi and left lobes of the lung as previously described.
      • Zhang W.C.
      • Peng Y.J.
      • Zhang G.S.
      • He W.Q.
      • Qiao Y.N.
      • Dong Y.Y.
      • et al.
      Myosin light chain kinase is necessary for tonic airway smooth muscle contraction.
      The thickness of the bronchial smooth muscle layer was measured from the innermost edge to the outermost edge using ImageJ software (National Institutes of Health, Bethesda, Md). The thickness of the smooth muscle layer of the extrapulmonary bronchus was assessed at 3 predetermined bronchiole sites (2 sites near each end close to the cartilage and 1 site in the middle of the smooth muscle layer). Three slices per mouse were measured.

       Isometric contraction measurement

      For the force measurements, the left extrapulmonary bronchi from TMEM16A CTR and KO mice were dissected from the surrounding tissues in ice-cold Krebs solution (118.07 mmol/L NaCl, 4.69 mmol/L KCl, 2.52 mmol/L CaCl2, 1.16 mmol/L MgSO4, 1.01 mmol/L NaH2PO4, 25 mmol/L NaHCO3, 11.1 mmol/L glucose), rings of approximately 1.5 mm in length were cut and mounted on a wire myograph (610-M, Danish Myo Technology), and the isometric tension was recorded using a PowerLab recording device (AD Instruments). The bronchus rings were kept in Krebs buffer and oxygenated with an O2-CO2 mixture (95% O2-5% CO2) at 37°C. A proper preload was administered to obtain an approximately 5 mN resting tension after a 60-mmol/L K+ stimulation and a 40-minute equilibrium. After another stimulation with 60 mmol/L K+, the contractile responses to other stimuli were measured.
      To measure the contraction of guinea pig trachea, the tracheas of male Hartley guinea pigs (250-300 g) were quickly removed and placed in ice-cold mKrebs solution (118 mmol/L NaCl, 4.6 mmol/L KCl, 0.5 mmol/L MgCl2, 1.8 mmol/L CaCl2, 1 mmol/L KH2PO4, 24.9 mmol/L NaHCO3, 11.1 mmol/L glucose),
      • Weichman B.M.
      • Muccitelli R.M.
      • Tucker S.S.
      • Wasserman M.A.
      Effect of calcium antagonists on leukotriene D4-induced contractions of the guinea-pig trachea and lung parenchyma.
      and dissected from the surrounding tissues. Rings comprising 2 tracheal cartilages were cut from the tracheas. To obtain strips, the cartilage opposite the muscle layer was cut. One cartilaginous end of the trachea strip was anchored to the bottom of the muscle bath chamber, and the other end was connected to a force transducer (MLT0201, AD Instruments). Isometric tension was measured using a PowerLab recording device. The trachea strips were maintained in mKrebs oxygenated with an O2-CO2 mixture (95% O2-5% CO2) at 37°C. The resting tension was adjusted to 1g. The strips were stimulated at least twice with 60 mmol/L KCl before subsequent experiments. The samples were preincubated with 10 μmol/L T16Ainh-A01 or dimethyl sulfoxide (DMSO) vehicle for 10 minutes before addition of the reagents.
      To measure human bronchial smooth muscle contraction, surgically resected samples from lung cancer patients were quickly incubated in ice-cold, oxygen-saturated HEPES-Tyrode buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, 5.6 mmol/L glucose, 10 mmol/L HEPES, pH 7.4). After transport to the laboratory, the surrounding tissues were cleaned, and the bronchial smooth muscle tissues were cut into 3-mm-wide strips. One end of the strip was anchored to the bottom of the muscle bath chamber, which was filled with Krebs solution oxygenated with an O2-CO2 mixture (95% O2-5% CO2) at 37°C. The other end was connected to a force transducer (MLT0201, AD Instruments). Because the diameter and thickness of the smooth muscle layer were different in different samples, the suitable resting tension was tested following stimulation with 60 mmol/L KCl. The samples were preincubated with 10 μmol/L T16Ainh-A01 or DMSO vehicle for 10 minutes before addition of the reagents.

       Measurement of Ca2+ signals in Ai38D reporter mice

      The Tmem16aSMKO and CTR mice were crossed with -reporter Ai38D mice. The left extrapulmonary bronchi of both the CTR and KO mice were mounted on a confocal wire myograph (120 cw; Danish Myo Technology) in HEPES-Tyrode buffer (137.0 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl2, 1.8 mmol/L CaCl2, 10 mmol/L HEPES, 5.6 mmol/L glucose, pH 7.4). The agonists were added using a self-made gravity perfusion system with a flow velocity of ∼10 mL/min. The GCaMP3 fluorescence was excited using a 488-nm argon laser, and the images were recorded every 2.5 seconds using a confocal microscope (FV-1000, Olympus). The average fluorescence intensity of the region of interest (bronchial smooth muscle range) was calculated with SV10-ASW software (Olympus), the average fluorescence intensity during the 1 to 2 minutes before stimulation was defined as the initial fluorescence (F0), and the dynamics of the Ca2+ signals were determined by % ΔF/F0.

       Sensitization with OVA

      Mice of both sexes were used at 8 to 10 weeks of age. On days 0 and 14, the mice were sensitized with 100 mg of OVA (Sigma-Aldrich, St Louis, Mo) dissolved in 4 mg of aluminium hydroxide (Imject Alum, Pierce) by intraperitoneal injection.
      • Zhang W.C.
      • Peng Y.J.
      • Zhang G.S.
      • He W.Q.
      • Qiao Y.N.
      • Dong Y.Y.
      • et al.
      Myosin light chain kinase is necessary for tonic airway smooth muscle contraction.
      The mice were then challenged with aerosolized 1% OVA in PBS for 60 minutes on days 24, 25, and 26. The mice that had received the last challenge within 24 hours were used as acute asthma models. Mice that received 4 mg of aluminium hydroxide diluted in PBS via intraperitoneal injection and were challenged with aerosolized PBS were used as a nonasthmatic control model.

       Measurement of respiratory resistance

      We used the flexiVent system (SCIREQ) to measure respiratory resistance. The OVA-sensitized mice were anesthetized with 250 to 300 mg/kg avertin 24 hours after receiving the last challenge of OVA or PBS. The anesthetized mice were cannulated with a blunt mouth 18-gauge metal needle, and the other end of the needle was connected to the rodent ventilator of the flexiVent system. The ventilator was set to generate a tidal volume of 10 mL/kg at a frequency of 150 breaths per minute. The Rrs was measured by a perturbation of SnapShot. The Newtonian resistance was measured by a perturbation of Quick prime-3 (SCIREQ, Montreal, Quebec, Canada). To measure the changes in respiratory resistance after induction with 5% OVA, Rrs values were recorded every minute for 45 minutes after the mice inhaled nebulized OVA for 5 minutes. To measure the changes in respiratory resistance in response to 5-HT challenge, sequential doses (37.5 μg, 75 μg, and 150 μg) of 5-HT were nebulized to challenge the airway, and the maximal Rrs was calculated for every dose within 3 minutes after challenge to generate dose-response curves. To measure the changes in respiratory resistance in response to MCh, sequential doses (10 μg, 30 μg, 100 μg, and 300 μg) of MCh were nebulized to challenge the airway, and the maximal Rrs were calculated for every dose within 3 minutes of challenge.
      Figure thumbnail fx2
      Fig E1Specific deletion of TMEM16A ClCa channels in ASM. A, Western blots (left) showed >90% KO efficiency (quantitative data [right]) in extrapulmonary bronchi. B, Representative immunofluorescence staining showed a high KO efficiency in smooth muscle layer of intrapulmonary bronchus. C, ClCa currents were significantly inhibited in KO ASMCs. Left, Representative recording traces; right, current-voltage curves of CTR and KO ASMCs. We set 600 nmol/L [Ca2+]i by pipette solution, ECl = 0 mV. Bars represent means ± SEMs, CTR: n = 10 cells from 3 mice, KO: n = 11 cells from 3 mice; ****P < .0001 by ANOVA. D, We eliminated 10 mmol/L caffeine-induced ClCa currents in the KO ASMCs. Left, Representative recording traces; right, quantitative results. Bars represent means ± SEMs; CTR: n = 49 cells from 10 mice, KO: n = 52 cells from 10 mice; ****P < .0001 by 2-tailed Student t test; blue * for outward currents and red * for inward currents.
      Figure thumbnail fx3
      Fig E2Representative recording traces of agonist-induced currents in ASMCs. A, Currents induced by 5-HT (3 μmol/L in application pipette) in mouse ASMCs. In CTR cells, on 5-HT stimulation, 11 of 26 cells exhibited typical inward currents, and 1 of 26 cells showed oscillated inward currents. In KO cells, only 2 of 26 cells exhibited weak inward currents. B, Currents induced by U46619 (1 μmol/L in application pipette) in mouse ASMCs. For CTR cells, 15 of 20 cells exhibited inward currents, and 2 of 20 cells simultaneously showed outward currents and 1 of 20 cells showed oscillated inward currents; for KO cells, none of total 22 cells exhibited inward currents, 8 cells exhibited outward currents. C, Currents induced by MCh (3 μmol/L in application pipette) in mouse ASMCs. For CTR, all 12 cells showed inward currents, among them, 5 cells exhibited outward currents and 1 cell showed oscillated inward currents; for KO, all 12 cells exhibited only outward currents. D, Currents induced by histamine (3 μmol/L in application pipette) in guinea pig ASMCs. For DMSO preincubated cells, all 14 cells showed inward currents; for 10 μmol/L T16Ainh-A01 preincubated cells, 11 of 14 cells showed reduced inward currents, and 3 of 14 cells did not show apparent currents. E, Currents induced by LTD4 (100 nmol/L in application pipette) in guinea pig ASMCs. For the DMSO group, 18 of 21 cells showed inward currents; for the T16Ainh-A01 group, 18 of 23 cells showed inward currents, but the amplitudes were suppressed.
      Figure thumbnail fx4
      Fig E3Expression of agonist receptors in TMEM16A KO ASM. A, Western blots assay for the expression of TMEM16A, mouse ACh receptor M3 (mAChR M3), 5-HT2A R, thromboxane receptor (TP R), and Flotillin-1 in TMEM16A KO and CTR extrapulmonary bronchial smooth muscle, in which β-actin was used as internal control. B, Quantitation of mAChR M3 (n = 6), 5-HT2A R (n = 3), TP R (n = 5), and Flotillin-1 (n = 6) in extrapulmonary bronchial smooth muscle. Bars represent means ± SEMs.
      Figure thumbnail fx5
      Fig E4Recording traces of ASM contraction and airway constriction in vivo. Typical contraction recording traces of TMEM16A KO and CTR bronchial smooth muscle contraction evoked by cumulative doses of KCl (A), MCh (B), U46619 (C), and 5-HT (D). E, Representative recording traces of 5-HT inhalation-induced airway constriction in CTR and KO mice.
      Figure thumbnail fx6
      Fig E5TMEM16A deletion inhibited ASM contraction induced by a low dose of MCh. A, The merged contraction tracking of bronchial smooth muscle. The forces induced by a low dose (300 nmol/L) and a relative high dose (3 μmol/L) of MCh were inhibited in TMEM16A KO muscle. The forces of the time points are represented as means ± SEMs. Sample sizes (n) were labeled in both panels. B, Quantitation for the maximal forces. Bars represent means ± SEMs; sample sizes (n) were the same as for A; *P < .05 by 2-tailed Student t test.
      Figure thumbnail fx7
      Fig E6Inhibitory effects of T16Ainh-A01 on the contractile response of guinea pig and human ASM to inflammatory mediators. A, Representative recording traces of DMSO or T16Ainh-A01 preincubated guinea pig trachea smooth muscle contraction induced by cumulative doses of histamine. B, Representative recording traces of DMSO or T16Ainh-A01 preincubated guinea pig trachea smooth muscle contraction induced by LTD4 gradients. C, 10 μmol/L T16Ainh-A01 induced relaxation of guinea pig trachea smooth muscle basal tone. Upper, Representative recording traces; lower, quantitative data. DMSO: n = 4, T16Ainh-A01: n = 6; **P < .01 by paired 2-tailed Student t test. D, Recording traces of 3 human bronchial smooth muscle samples showing inhibitory effects of T16Ainh-A01 on histamine-induced contraction.
      Figure thumbnail fx8
      Fig E7Representative recording traces of the augmentation effects. Extrapulmonary bronchial smooth muscles were treated with combined addition of 100 nmol/L 5-HT and 100 nmol/L MCh, or 30 nmol/L U46619 and 30 nmol/L MCh. The addition of agonists were represented by the color bars (100 nmol/L MCh: black, 100 nmol/L 5-HT: green, 30 nmol/L MCh: gray, 30 nmol/L U46619: blue).
      Figure thumbnail fx9
      Fig E8Histology of CTR and Tmem16aSMKO lungs with and without asthma. Arrows point positions with inflammatory cells infiltration. Scale bar = 100 μm (long) or 50 μm (short).
      Table E1Western blot primary antibodies
      Catalog no.TargetHost speciesVendorFinal concentration
      ab53212TMEM16ARabbitAbcam1:500
      ab134959TP RRabbitAbcam1:500
      sc-9108mAChR M3RabbitSanta Cruz1:200
      ab660495-HT2A RRabbitAbcam1:100
      sc-25506Flotillin-1RabbitSanta Cruz1:1,000
      a5441β-actinMouseSigma-Aldrich1:10,000

      Supplementary data

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