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 β₂-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 β₂-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, β₂-adrenergic receptor agonists noncompetitively suppressed muscarinic contraction. In concentration-inhibition curves for β₂-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. β₂-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 β₂-adrenergic action via allosteric sites in β₂-adrenergic receptors (synergism). In conclusion, KCa channels and allosterism may be novel targets of bronchodilator therapy for diseases such as asthma and COPD.
Keywords: COPD; G protein; L-type voltage-dependent Ca2+ channels; asthma; large-conductance Ca2+-activated K+ channels; muscarinic receptor antagonists; synergistic effects; β2-adrenoceptor agonists.