The parasympathetic network penetrates deeply the airway wall and regulates bronchoconstriction. ACh is the predominant parasympathetic neurotransmitter. Although, ASM express both cholinergic receptors, nicotinic receptors (nAChRs) and mAChRs, the cholinergic effects seem to be mediated by muscarinic activation⁽¹¹⁷⁾. In asthma, cutting the parasympathetic supply of the airways (vagotomy) prevents an increased smooth muscle contraction⁽¹¹⁸⁾, and AHR induced by persistent parasympathetic activation has been shown⁽¹¹⁹⁾. The striking role of ACh in airway remodeling was highly commented in a recent article⁽⁸⁾, where authors proposed methacholine-induced bronchoconstriction as the main driver of epithelial hyperplasia and subepithelial collagen deposition, independently of inflammation, as chronic stretch and mechanotransduction pathways are well-known mechanisms of muscle differentiation. However, the cited study failed to conclusively demonstrate that only mechanical stress was responsible of all histopathological changes, because they did not assess the non-neuronal and non-contractile effects of cholinergic networks, a non-asthmatic group was not included (taking into account that inflammatory signatures on resident cells change their responses), and ASM layers were not assessed. Nonetheless, despite the limitations, the contribution of ACh in airway remodeling was uncovered.

Two ACh sources have been identified: 1. neural, along parasympathetic fibers from vagal nerve, and 2. non-neural, from airway epithelium and immune cells. Both are implicated in increased ASM thickening in asthma⁽¹²⁰⁾, which was prevented by M₃-specific antagonists such as tiotropium bromide⁽¹²¹⁾. Also, M₂ is expressed and its downstream signaling has been related to modulation of some ASMC functions, either promotion or inhibition⁽¹²²⁾. ACh, either neuronal or non-neuronal, regulates inflammatory cell responses that may explain the anticholinergic benefitsin asthma and COPD⁽¹²³⁾. Collectively, these findings are revealing new therapeutic targets, therefore, we chose the cholinergic signaling pathway to be explored deeply in this review as a prototype of GPCR agonism.

Muscarinic Receptor Signaling

The mAChR family consist of five receptor subtypes that belong to GPCRs. Mammal airways including humans express M₁, M₂, and M₃ subtypes; more precisely, epithelial cells (M₁–M₄), pulmonary vessel endothelial cells (M₁–M₅), mesenchymal cells, such as smooth muscle fibers (M₂, M₃) and fibroblasts (M₂> M₁> M₃> M₄), and lung-infiltrating immune cells, such as mononuclear leukocytes (M₁–M₅) ⁽¹²⁴⁾. The M₄ mRNA and protein have been reported in rabbit bronchiolar ASM, but not from humans. Although, pharmacological ligand binding studies showed a mixed population M₂:M₃ in a 4:1 ratio, respectively, a functional dominance of M₃ appears to mediate muscarinic effects under physiological circumstances⁽¹²⁵⁾. This predominance could be a consequence of receptor compartmentalization, facilitating or inhibiting signal transduction depending on accessibility to specific transducers or kinetic regulation. M₃-induced bronchoconstriction is mainly mediated by a caveolae-dependent system. M₂ contribution was mainly uncover in M₃^-/- knockout mice with cellular caveolae dissolution. Location of M₂ and M₃ in caveolae is dependent on Caveolin-1 (Cav-1) and Caveolin-3 (Cav-3) expression, respectively⁽¹²⁶⁾. Many processes involve coupling of mAChRs to their cellular effector systems, via heterotrimeric G proteins. These are composed of one α-, β- and γ-subunit and transduction signals depends on both the α-subunit and βγ-subunit. In ASMCs, muscarinic activation is classically considered as the main signal for muscle contraction. However, there is increasing evidence indicating that alterations in its downstream signaling pathways might be responsible for ASM remodeling and AHR⁽¹²⁷⁾.

Muscarinic signaling can be divided in pathways for muscle contraction and those with non-contractile effects. In this way, the G_q-coupled M₃ activates phospholipase C (PLC), causing hydrolytic conversion of phosphatidylinositol 4,5-biphosphate (PIP₂) into inositol 1,4,5-trisphosphate (InsP₃) and sn-1,2-diacylglycerol (DAG). InsP3 is involved in Ca²⁺ mobilization from intracellular stores, which generates a rapid and transient increase for muscle contraction, while generated DAG activates PKC with subsequent triggering of mitogen-activated protein kinase (MAPK) signaling for non-contractile effects. Also, MAPK cascade can be activated through direct phosphorylation of Raf-1, independently of PKC⁽¹¹⁷⁾. β-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits of agonist-activated GPCRs. The third intracellular loop (i3) of M₃ is required for β-arrestin recruitment after homodimerization (M₃/M₃) and heterodimerization (M₂/M₃). Those macrocomplexes not only induce downregulation of mAChRs, but also act as scaffolds for components of the MAPK cascade, facilitating its activation⁽¹²⁸⁾. This is a third mechanism of M₃ signal transduction for cell growth and differentiation.

As a counterpart, G_i/o-coupled M₂ contributes to muscle contraction either affecting adenylyl cyclase in an inhibitory manner, or directly enhancing potassium and non-selective ion channels opening, and they both depend on the released βγ dimer. Additionally, M₂ modulates the relaxant effects of atrial natriuretic peptide (ANP), as it suppresses the ANP-induced activation of a membrane-spanning guanylyl cyclase via a pertussis toxin (PTX)-sensitive G protein⁽¹²⁹⁾. Since G_i/o is involved in muscarinic-induced actin reorganization, RhoA/Rho-kinase signaling pathway has been related to Ras and phosphoinositide 3-Kinase (PI3-K) activation, contributing to growth factors effects⁽¹²⁰⁾.

The complexity of non-canonical muscarinic signaling is illustrated by the ambiguity of their downstream pathways. The cGMP/PKG pathway is a well-documented mediator of muscle relaxation, and it has also anti-remodeling effects. Molecular evidence suggests it could depend on which compartment is activated and signal pattern. Some cascades activated by mAChRs are linked to second messengers such as cGMP by activation of two distinctive guanylyl cyclases. Muscarinic activation of tracheal ASM fragments is associated with contraction, but it also involves the generation of two cGMP signals, at 20-s and 60-s⁽¹³⁰⁾. These signals seem to be essential in reaching the contractile effects of several muscarinic agonists and might be relevant in ASM remodeling. The proposed model⁽¹³¹⁾ for this novel pathway emphasized that 20-s cGMP signal is linked to G_i/o coupled M₂ activation, inducing a massive and transient α₁β₁-NO-soluble guanylyl cyclase (sGC) translocation from cytoplasm to plasma membranes, whereas, the 60-s cGMP signal is associated with a natriuretic peptide receptor (NPR)-GC-B, activated by a G_q16-coupled M₃ sensitive to mastoparan. Those signals regulate the muscarinic signal transduction efficacy in response to agonists through phosphorylation events. In this way, M₃ phosphorylation by PKG-II may correlate to changes in the receptor affinity to agonists and antagonists. It has been proposed that cGMP induces PKG phosphorylation of the i3 loop that confers a potential feedback mechanism to terminate the cGMP-dependent muscarinic signal transduction cascades at the sarcolemma⁽¹³²⁾. Since phosphorylation of the same loop by others serine/threonine kinases downregulates the receptor by endocytosis and G protein uncoupling, indirect observations suggest that PKG-II phosphorylation of M₃ induces a mAChR dimer formation. Homodimer formation stabilizes or ‘freezes’ the M₃ population, in a refractory state to agonist activation, and prone to antagonist binding⁽¹³²⁾. We speculate in caveolae systems a higher density M₃ receptor population would support positive cooperativity through homodimer/heterodimer formation, which could enhance the signal transduction for MAPK activation and subsequent altered gene expression. Thus, cGMP produced in response to muscarinic agonists could be involved in growth promotion instead of classical cell arrest. Relevantly, we demonstrated that this pathway is still functional in sensitized ASMCs, while cGMP cascade induced by NPs and NO was downregulated⁽¹³¹⁾. A wide-spectrum of outcomes can result from diverse experimental designs. This complicates our understanding of the cholinergic signaling in human airway diseases. A brief scheme that will be discussed herein, includes effects related to remodeling promotion: 1. c-ASMC modulation, 2. s/p-ASMC proliferation synergism, and 3. synergism on h-ASMC induction, and alternatively possible actions for remodeling prevention like decreased s/p-ASMC proliferation. Although, in vivo relevance keeps hypothetic, this provide a coherent and useful picture for planning future research.