{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T14:08:19Z","timestamp":1789999699762,"version":"4.0.1"},"reference-count":55,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2018,7,9]],"date-time":"2018-07-09T00:00:00Z","timestamp":1531094400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>To advance the development of bronchodilators for asthma and chronic obstructive pulmonary disease (COPD), this study was designed to investigate the mechanism of functional antagonism between \u03b22-adrenergic and muscarinic M2 receptors, focusing on allosteric effects and G proteins\/ion channels coupling. Muscarinic receptor antagonists (tiotropium, glycopyrronium, atropine) synergistically enhanced the relaxant effects of \u03b22-adrenergic receptor agonists (procaterol, salbutamol, formoterol) in guinea pig trachealis. This crosstalk was inhibited by iberitoxin, a large-conductance Ca2+-activated K+ (KCa) channel inhibitor, whereas it was increased by verapamil, a L-type voltage-dependent Ca2+ (VDC) channel inhibitor; additionally, it was enhanced after tissues were incubated with pertussis or cholera toxin. This synergism converges in the G proteins (Gi, Gs)\/KCa channel\/VDC channel linkages. Muscarinic receptor antagonists competitively suppressed, whereas, \u03b22-adrenergic receptor agonists noncompetitively suppressed muscarinic contraction. In concentration-inhibition curves for \u03b22-adrenergic receptor agonists with muscarinic receptor antagonists, EC50 was markedly decreased, and maximal inhibition was markedly increased. Hence, muscarinic receptor antagonists do not bind to allosteric sites on muscarinic receptors. \u03b22-Adrenergic receptor agonists bind to allosteric sites on these receptors; their intrinsic efficacy is attenuated by allosteric modulation (partial agonism). Muscarinic receptor antagonists enhance affinity and efficacy of \u03b22-adrenergic action via allosteric sites in \u03b22-adrenergic receptors (synergism). In conclusion, KCa channels and allosterism may be novel targets of bronchodilator therapy for diseases such as asthma and COPD.<\/jats:p>","DOI":"10.3390\/ijms19071999","type":"journal-article","created":{"date-parts":[[2018,7,9]],"date-time":"2018-07-09T11:18:53Z","timestamp":1531135133000},"page":"1999","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Involvement of Allosteric Effect and KCa Channels in Crosstalk between \u03b22-Adrenergic and Muscarinic M2 Receptors in Airway Smooth Muscle"],"prefix":"10.3390","volume":"19","author":[{"given":"Hiroaki","family":"Kume","sequence":"first","affiliation":[{"name":"Department of Respiratory Medicine and Allergology, Faculty of Medicine, Kindai University, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Osamu","family":"Nishiyama","sequence":"additional","affiliation":[{"name":"Department of Respiratory Medicine and Allergology, Faculty of Medicine, Kindai University, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takaaki","family":"Isoya","sequence":"additional","affiliation":[{"name":"Department of Respiratory Medicine and Allergology, Faculty of Medicine, Kindai University, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Higashimoto","sequence":"additional","affiliation":[{"name":"Department of Respiratory Medicine and Allergology, Faculty of Medicine, Kindai University, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Tohda","sequence":"additional","affiliation":[{"name":"Department of Respiratory Medicine and Allergology, Faculty of Medicine, Kindai University, 377-2 Ohnohigashi, Osakasayama 589-8511, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukihiro","family":"Noda","sequence":"additional","affiliation":[{"name":"Division of Clinical Sciences and Neuropsychopharmacology, Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"11051","DOI":"10.1073\/pnas.89.22.11051","article-title":"Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins","volume":"89","author":"Kume","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","first-page":"L221","article-title":"Role of G proteins and KCa channels in the muscarinic and \u03b2-adrenergic regulation of airway smooth muscle","volume":"268","author":"Kume","year":"1995","journal-title":"Am. J. Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2876","DOI":"10.2174\/092986708786242831","article-title":"RhoA\/Rho-kinase as a therapeutic target in asthma","volume":"15","author":"Kume","year":"2008","journal-title":"Curr. Med. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.pharmthera.2015.09.004","article-title":"Research and development of bronchodilators for asthma and COPD with a focus on G protein\/KCa channel linkage and \u03b22-adrenergic intrinsic efficacy","volume":"156","author":"Kume","year":"2015","journal-title":"Pharmacol. Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1038\/40187","article-title":"Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension","volume":"389","author":"Uehata","year":"1997","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"L1218","DOI":"10.1152\/ajplung.2001.280.6.L1218","article-title":"Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle","volume":"280","author":"Ito","year":"2001","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.ejphar.2006.11.007","article-title":"Direct effects of hydrogen peroxide on airway smooth muscle tone: Roles of Ca2+ influx and Rho-kinase","volume":"556","author":"Kojima","year":"2007","journal-title":"Eur. J. Pharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1111\/j.1365-2222.2008.03164.x","article-title":"Role of Ca2+ mobilization and Ca2+ sensitization in 8-iso-PGF2\u03b1-induced contraction in airway smooth muscle","volume":"39","author":"Shiraki","year":"2009","journal-title":"Clin. Exp. Allergy"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"L947","DOI":"10.1152\/ajplung.90288.2008","article-title":"The contribution of Ca2+ signaling and Ca2+ sensitivity to the regulation of airway smooth muscle contraction is different in rats and mice","volume":"296","author":"Bai","year":"2009","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1136\/thx.2009.129296","article-title":"Ca2+ homeostasis and structural and functional remodeling of airway smooth muscle in asthma","volume":"65","author":"Mahn","year":"2010","journal-title":"Thorax"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sakuma, K. (2015). Ca2+ dynamics and Ca2+ sensitization in the regulation of airway smooth muscle tone. Muscle Cell and Tissue, INTECH.","DOI":"10.5772\/60969"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0163-7258(93)90064-K","article-title":"Potassium channels in airway smooth muscle: A tale of two channels","volume":"58","author":"Kotlikoff","year":"1993","journal-title":"Pharmacol. Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.pharmthera.2005.10.007","article-title":"Large-conductance calcium-activated potassium channels: Structural and functional implications","volume":"110","author":"Ghatta","year":"2006","journal-title":"Pharmacol. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Wang, Y.X. (2013). Large-conductance calcium-activated potassium channels. Calcium Signaling in Airway Smooth Muscle Cells, Springer.","DOI":"10.1007\/978-3-319-01312-1"},{"key":"ref_15","unstructured":"Kume, H. (2017). Role of large-conductance calcium-activated potassium channels on airway smooth muscle in physiological and pathological conditions. Potassium Channels in Health and Disease, Nova Science Publishers."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"C1151","DOI":"10.1152\/ajpcell.1997.272.4.C1151","article-title":"Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes","volume":"272","author":"Wang","year":"1997","journal-title":"Am. J. Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1165\/rcmb.2002-0197OC","article-title":"Involvement of Ca2+ mobilization in tachyphylaxis to \u03b2-adrenergic receptors in trachealis","volume":"29","author":"Kume","year":"2003","journal-title":"Am. J. Respir. Cell Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.pharmthera.2007.03.014","article-title":"Potassium channel diversity in the pulmonary arteries and pulmonary veins: Implications for regulation of the pulmonary vasculature in health and during pulmonary hypertension","volume":"115","author":"Bonnet","year":"2007","journal-title":"Pharmacol. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1038\/nm.2237","article-title":"Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction","volume":"16","author":"Deshpande","year":"2010","journal-title":"Nat. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.pharmthera.2016.02.001","article-title":"Recent advances in therapeutic strategies that focus on the regulation of ion channel expression","volume":"160","author":"Ohya","year":"2016","journal-title":"Pharmacol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.ejphar.2015.05.020","article-title":"Pharmacological interaction between LABAs and LAMAs in the airways: Optimizing synergy","volume":"761","author":"Calzetta","year":"2015","journal-title":"Eur. J. Pharmacol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1085\/jgp.201210876","article-title":"Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle","volume":"141","author":"Mukherjee","year":"2013","journal-title":"J. Gen. Physiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1085\/jgp.201210953","article-title":"A Ca2+- and PKC-driven regulatory network in airway smooth muscle","volume":"141","author":"Dixon","year":"2013","journal-title":"J. Gen. Physiol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Fukunaga, K., Kume, H., Oguma, T., Shigemori, W., Tohda, Y., Ogawa, E., and Nakano, Y. (2016). Involvement of Ca2+ signaling in the synergistic effects between muscarinic receptor antagonists and \u03b22-adrenoceptor agonists in airway smooth muscle. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17091590"},{"key":"ref_25","unstructured":"(2017, December 20). The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD). Available online: http:\/\/goldcopd.org."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.coph.2014.03.003","article-title":"The pharmacological rationale for combining muscarinic receptor antagonists and \u03b2-adrenoceptor agonists in the treatment of airway and bladder disease","volume":"16","author":"Dale","year":"2014","journal-title":"Curr. Opin. Pharmacol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/S2213-2600(13)70052-3","article-title":"Analysis of chronic obstructive pulmonary disease exacerbations with the dual bronchodilator QVA149 compared with glycopyrronium and tiotropium (SPARK): A randomised, double-blind, parallel-group study","volume":"1","author":"Wedzicha","year":"2013","journal-title":"Lancet Respir. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/S2213-2600(14)70065-7","article-title":"Efficacy and safety of umeclidinium plus vilanterol versus tiotropium, vilanterol, or umeclidinium monotherapies over 24 weeks in patients with chronic obstructive pulmonary disease: Results from two multicentre, blinded, randomised controlled trials","volume":"2","author":"Decramer","year":"2014","journal-title":"Lancet Respir. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1183\/09031936.00136014","article-title":"Tiotropium and olodaterol fixed-dose combination versus mono-components in COPD (GOLD 2-4)","volume":"45","author":"Buhl","year":"2015","journal-title":"Eur. Respir. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1038\/nrd2760","article-title":"Allosteric modulators of GPCRs: A novel approach for the treatment of CNS disorders","volume":"8","author":"Conn","year":"2009","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1016\/j.tips.2009.06.007","article-title":"\u20187TM receptor allostery: Putting numbers to shapeshifting proteins","volume":"30","author":"Kenakin","year":"2009","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1038\/nrd3954","article-title":"Signalling bias in new drug discovery: Detection, quantification and therapeutic impact","volume":"12","author":"Kenakin","year":"2013","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.ejphar.2014.10.025","article-title":"Pharmacological characterization of the interaction between aclidinium bromide and formoterol fumarate on human isolated bronchi","volume":"745","author":"Cazzola","year":"2014","journal-title":"Eur. J. Pharmacol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1111\/j.1365-2222.2006.02412.x","article-title":"Involvement of reduced sensitivity to Ca2+ in \u03b2-adrenergic action on airway smooth muscle","volume":"36","author":"Oguma","year":"2006","journal-title":"Clin. Exp. Allergy"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1124\/jpet.106.110718","article-title":"Sphingosine 1-phosphate causes airway hyper-reactivity by Rho-mediated myosin phosphatase inactivation","volume":"320","author":"Kume","year":"2007","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/341152a0","article-title":"Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation","volume":"341","author":"Kume","year":"1989","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1172\/JCI116969","article-title":"\u03b2-Adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms","volume":"93","author":"Kume","year":"1994","journal-title":"J. Clin. Investig."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"C1204","DOI":"10.1152\/ajpcell.1991.261.6.C1204","article-title":"Muscarinic inhibition of single KCa channels in smooth muscle cells by a pertussis-sensitive G protein","volume":"261","author":"Kume","year":"1991","journal-title":"Am. J. Physiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"L1125","DOI":"10.1152\/ajplung.00084.2002","article-title":"Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus","volume":"283","author":"Sarria","year":"2002","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1124\/jpet.102.044701","article-title":"Increased relaxant action of forskolin and isoproterenol against muscarinic agonist-induced contractions in smooth muscle from M2 receptor knockout mice","volume":"305","author":"Matsui","year":"2003","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1803","DOI":"10.1113\/jphysiol.2010.204347","article-title":"BK channel \u03b21 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization","volume":"589","author":"Semenov","year":"2011","journal-title":"J. Physiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"149","DOI":"10.2174\/157015907781695973","article-title":"Allosteric theory: Taking therapeutic advantage of the malleable nature of GPCRs","volume":"5","author":"Kenakin","year":"2007","journal-title":"Curr. Neuropharmacol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1021\/jm201139r","article-title":"Allosteric modulation of seven transmembrane spanning receptors: Theory, practice, and opportunities for central nervous system drug discovery","volume":"55","author":"Melancon","year":"2012","journal-title":"J. Med. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1353","DOI":"10.1164\/rccm.2109060","article-title":"\u03b2-agonist intrinsic efficacy: Measurement and clinical significance","volume":"165","author":"Hanania","year":"2002","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"89","DOI":"10.2332\/allergolint.54.89","article-title":"Clinical use of \u03b22-adrenergic receptor agonists based on their intrinsic efficacy","volume":"54","author":"Kume","year":"2005","journal-title":"Allergol. Int."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1186\/s12931-016-0386-8","article-title":"Pharmacological characterisation of the interaction between glycopyrronium bromide and indacaterol fumarate in human isolated bronchi, small airways and bronchial epithelial cells","volume":"17","author":"Cazzola","year":"2016","journal-title":"Respir. Res."},{"key":"ref_47","first-page":"L556","article-title":"Inhibitory effects of Gs on desensitization of \u03b2-adrenergic receptors in tracheal smooth muscle","volume":"273","author":"Kume","year":"1997","journal-title":"Am. J. Physiol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1164\/ajrccm.159.2.9801006","article-title":"Inhibition of \u03b2-adrenergic desensitization by KCa channels in human trachealis","volume":"159","author":"Kume","year":"1999","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/S0031-6997(25)06847-4","article-title":"The search for synergy: A critical review from a response surface perspective","volume":"47","author":"Greco","year":"1995","journal-title":"Pharmacol. Rev."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1016\/j.tiv.2007.03.003","article-title":"A mathematical approach to study combined effects of toxicants in vitro: Evaluation of the bliss independence criterion and the loewe additivity model","volume":"21","author":"Goldoni","year":"2007","journal-title":"Toxicol. In Vitro"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1113\/jphysiol.1990.sp018076","article-title":"Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle","volume":"424","author":"Kume","year":"1990","journal-title":"J. Physiol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1111\/j.1476-5381.1995.tb15926.x","article-title":"Potassium currents in human freshly isolated bronchial smooth muscle cells","volume":"115","author":"Snetkov","year":"1995","journal-title":"Br. J. Pharmacol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.ejphar.2008.06.072","article-title":"Role of Ca2+ mobilization in desensitization of \u03b2-adrenoceptors by platelet-derived growth factor in airway smooth muscle","volume":"591","author":"Ikenouchi","year":"2008","journal-title":"Eur. J. Pharmacol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1164\/rccm.201410-1874OC","article-title":"Calcium channel blocker reduces airway remodeling in severe asthma: A proof-of-concept study","volume":"191","author":"Girodet","year":"2015","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_55","first-page":"116","article-title":"Research and development for anti-asthmatic agents with a focus on phenotype changing by Ca2+ signaling in airway smooth muscle cells","volume":"Volume 3","author":"Rahman","year":"2018","journal-title":"Frontiers in Clinical Drug Research\u2014Anti Allergy Agents"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/7\/1999\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:11:49Z","timestamp":1760195509000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/7\/1999"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,7,9]]},"references-count":55,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2018,7]]}},"alternative-id":["ijms19071999"],"URL":"https:\/\/doi.org\/10.3390\/ijms19071999","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,7,9]]}}}