It is generally agreed that increased cholinergic tone is an important factor in bronchoconstriction in chronic obstructive airways disease (COPD).  Despite this, the mechanisms underlying acetylcholine-induced contractions are incompletely understood. Cholinergic contractions are widely thought to be mediated by postjunctional M3 muscarinic receptors (M3R) on airway smooth muscle (ASM), even though they are outnumbered 4:1 by postjunctional M2 receptors (M2R) in most species. M2R are also expressed prejunctionally on parasympathetic nerves, where they inhibit acetylcholinerelease¹. Pharma companies have therefore sought to develop M3R-selective anticholinergic drugs to treat COPD because of the risk that blocking prejunctional M2R could increase acetylcholine release and exacerbate bronchoconstriction¹.

Recently, we have discovered that M2R can contribute more to cholinergic contractions than previously thought⁴. When mouse bronchial rings were subjected to electrical field stimulation (EFS) at 100s intervals (2Hz, 1s trains), phasic contractions were evoked that were entirely blocked by the M3R blocker, 4-DAMP and were unaffected by M2R blockers. Surprisingly, when the stimulus interval was shortened to 10s, the contraction amplitude increased by 2-3-fold. Again, the contraction was completely blocked by 4-DAMP, but this time M2R blockers (methoctramine, AFDX-116), selectively blocked all of the increase in amplitude at 10s stimulus intervals (Fig.1). Taken together, this suggested that shortening the stimulus interval resulted in M2R sensitisation of M3R-mediated contractions. When similar experiments were carried out in M2R knockout mice, shortening the stimulus interval failed to enhance contraction amplitude. Interestingly, when a sub-threshold concentration of carbachol was applied to tissues receiving the stimulus trains at 100s intervals, there was a methoctramine-sensitive increase in the amplitude of the phasic contractions in wild-type mice, but not in M2R knockout mice. This is consistent with carbachol stimulating extra-junctional M2R.  

ASM cells possess L-type voltage-dependent Ca²⁺ channels (L-VDCC) and the means to regulate membrane potential via TMEM16A channels and various K⁺ channels. However, there is a lack of consensus as to the roles played by these channels in ASM contraction^2,3. We further explored the mechanisms underlying the M2R-mediated enhancement of EFS-evoked contractions using blockers of either L-VDCC (nifedipine, verapamil) or TMEM16A (Ani9, CaCC_inhA01). These drugs all failed to affect responses evoked at 100 s intervals, but completely reversed the enhancement seen on switching to 10s stimulation.  Thapsigargin, a SERCA pump blocker, enhanced EFS responses at 100s intervals in a similar manner to switching to 10s intervals, and this effect was reversed by nifedipine or Ani9. 

We propose a model whereby cholinergic stimulation results in L-VDCC activation by TMEM16A-mediated membrane depolarisation. In M3R-exclusive responses, the incoming Ca²⁺ is immediately taken up into sub-membrane sarcoplasmic reticulum via SERCA (the ‘superficial buffer barrier’ mechanism). When M2Rs (possibly located extra-junctionally) are co-activated, Gi protein-mediated suppression of cAMP production results in SERCA pump inactivation, which allows the incoming Ca²⁺ to reach the contractile proteins.  Since extra-neuronal production of acetylcholine has been proposed to contribute to increased cholinergic tone in COPD⁵, we suggest that this may stimulate M2R on ASM and contribute to the bronchoconstriction typical of this disorder.

All work was carried out in compliance with EU legislation.