Unravelling the Role of Post-Junctional M2 Muscarinic Receptors in Cholinergic Nerve-Mediated Contractions of Airway Smooth Muscle

Abstract

It has long been recognised that airway smooth muscle cells (ASMCs) possess an abundance of M2 muscarinic receptors (M2Rs). However, the contribution of postjunctional M2Rs to contractions of airway smooth muscle (ASM) induced by the release of acetylcholine (ACh) from parasympathetic nerves was thought to be minimal. Instead, it was believed that these responses were exclusively mediated by activation of M3Rs. However, evidence is emerging that postjunctional M2Rs may have a greater role than previously realised. In this review, we discuss ACh signalling in airways, highlighting the well-established autoinhibitory role of prejunctional M2Rs and the putative roles of postjunctional M2Rs to cholinergic contractions of ASM. The cellular mechanisms that underpin M2R-dependent contractions of ASM are reviewed, with a particular emphasis on the role of ion channels in these responses. The regulation of M2R signalling pathways by β-adrenoceptor activation is also considered, along with the potential involvement of postjunctional M2Rs in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD).

Keywords: COPD; airways; cholinergic; contraction; muscarinic; smooth muscle.

Figure 1

(A) Representative trace showing contractions of murine bronchial rings evoked by EFS at 100 and 10 s intervals. Reducing the stimulus interval to 10 s enhanced contraction amplitude. (B) Representative trace showing the effect of reducing the stimulus interval from 100 to 10 s on a bronchial ring taken from a M2R KO mouse. (C,D) Effect of the M2R antagonists, methoctramine (C) and AFDX116 (D) on contractions of bronchial rings from wild-type mice evoked at 10 s intervals. Adapted from Alkawadri et al. (2021) [53].

Figure 2

(A,B) Representative traces showing the effects of nifedipine on contractions of murine bronchial rings evoked by EFS at 100 s (A) and 10 s intervals (B). (C) Effect of nifedipine on contractions of bronchial rings evoked by the cholinergic agonist carbachol (CCh) in the presence of the M3R antagonist, 4-DAMP. Adapted from Ghosh et al. (2025) [77].

Figure 3

(A,B) Representative traces showing the effects of Ani9 on contractions of murine bronchial rings evoked by EFS at 100 s (A) and 10 s intervals (B). (C) Effect of Ani9 on contractions of bronchial rings evoked by the cholinergic agonist carbachol (CCh) in the presence of the M3R antagonist 4-DAMP. Adapted from Ghosh et al. (2025) [77].

Figure 4

Model showing cellular mechanism underlying contractions of ASM induced by EFS at 100 and 10 s intervals. At 100 s intervals, EFS-evoked contractions of ASM are mediated by the activation of M3Rs leading to the release of Ca²⁺ from the sarcoplasmic reticulum via IP₃Rs. Ca²⁺ influx via L-type Ca²⁺ channels (LTCCs) is buffered by the activity of SERCA pumps on the peripheral sarcoplasmic reticulum. Reducing the stimulus interval to 10 s leads to the activation of M2Rs, which inhibits SERCA activity, resulting in reduced buffering in Ca²⁺ influx via LTCCs. Increased Ca²⁺ levels in the subsarcolemmal space activates Ano1 Ca²⁺-activated Cl⁻ channels leading to Cl⁻ efflux, membrane depolarization, and further activation of LTCCs. Increased cytosolic Ca²⁺ levels increase contraction amplitude. Created with BioRender.com. Adapted from Ghosh et al. (2025) [77].

Figure 5

Representative trace showing that CCh (10 nM) enhanced the amplitude of EFS-evoked contractions of ASM (2 Hz, 100 s intervals) and that this effect was reversed by methoctramine (A). (B) is a representative trace showing that CCh (10 nM) did not affect EFS-evoked contractions of ASM taken from M2R KO mice. (C) is a representative trace showing that methoctramine (100 nM) inhibited contractions of ASM induced by the acetylcholinesterase inhibitor neostigmine (1 µM). Adapted from Alkawadri et al. (2021) [53].

Figure 5

Representative trace showing that CCh (10 nM) enhanced the amplitude of EFS-evoked contractions of ASM (2 Hz, 100 s intervals) and that this effect was reversed by methoctramine (A). (B) is a representative trace showing that CCh (10 nM) did not affect EFS-evoked contractions of ASM taken from M2R KO mice. (C) is a representative trace showing that methoctramine (100 nM) inhibited contractions of ASM induced by the acetylcholinesterase inhibitor neostigmine (1 µM). Adapted from Alkawadri et al. (2021) [53].