Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice

Abstract

Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M₂ and M₃ subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca²⁺ mobilisation, myofilament Ca²⁺ sensitisation, generation of non-selective cationic and chloride currents, K⁺ current modulation, inhibition or potentiation of voltage-dependent Ca²⁺ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.

Keywords: gastrointestinal tract; knockout mouse; muscarinic receptor subtypes; non-selective cationic channels; signal transduction pathways; smooth muscle.

Figure 1

Three distinct muscarinic signalling pathways leading to transient receptor potential (TRP)-like cationic channel opening in ileal myocytes (redrawn from Tanahashi et al. 2020 [39]). The M₃/G_q/11/phospholipase C (PLC) pathway activates brief opening states of 70-pS and 120-pS cationic channels and concurrently evokes InsP₃-induced Ca²⁺ release. Opening of the lower conductance channel is induced by relief from PIP₂ inhibition following PLC-mediated hydrolysis, while the higher conductance channel is activated by PLC-generated diacylglycerol (DAG). The M₂ pathway transmits M₂ signals via G_i/o proteins to the 70-pS channel, which shifts the gating state from the brief to a longer opening mode, and also inhibits adenylyl cyclase. The M₂/M₃ pathway transmits M₂ signals via G_o protein, and M₃ signals via G_q/11/PLC, to the 70-pS cationic channel, resulting in channel gating with a much longer open mode. This pathway is the major contributor to the generation of mIcat, but is inactive when either the M₂ or M₃ receptor is absent, or when either G_o, G_q/11 or PLC is inactivated. In other words, the activity of this pathway is conditional, occurring only when both M₂/G_o and M₃/G_q/11 signalling pathways are activated. Studies of mAChR-KO mice [23,44] and TRPC-mutant mice [42] indicate that these 70-pS and 120-pS cationic channel activities are mediated by TRPC4 and TRPC6, respectively. The M₂/M₃ pathway, but not the M₂ or M₃ pathway, involves a signalling step in which Ca²⁺ has a potentiating effect on TRPC channel activation, suggesting that the M₃ pathway may facilitate M₂/M₃ pathway function through InsP₃-induced Ca²⁺ release. Whether the M₂/M₃ pathway has a significant role in stimulating InsP₃/DAG formation or inhibiting cAMP accumulation is currently unclear. One study suggested that G_o protein is not involved in adenylyl cyclase inhibition by M₂ receptors of intestinal smooth muscle [24]. These three pathways may also converge on voltage-dependent Ca²⁺ channels (VDCCs) to suppress Ca²⁺ influx via the same G-protein pathways mediating cationic channel activation (see Figure 10B in Tanahashi et al., 2009 [49]).

Figure 2

Signal transduction mechanisms underlying muscarinic contraction of mouse intestinal smooth muscle. The M₃ pathway activates multiple intracellular Ca²⁺ mobilisation events, including Ca²⁺ influx via VDCCs and voltage-independent Ca²⁺-permeable channels and intracellular Ca²⁺ release. Voltage-dependent Ca²⁺ influx is initiated by depolarisation from opening of TRPC4 and TRPC6 cationic channels, opening of Ca²⁺-activated Cl⁻ channels and inhibition of K_ATP channels, while voltage-independent Ca²⁺ entry is mediated by opening of TRPC6 cationic channels and Ca²⁺ store-operated Ca²⁺ channels. In addition to Ca²⁺ mobilisation, Ca²⁺ sensitisation of contractile proteins is elicited, thereby increasing the efficiency of contraction–[Ca²⁺]_i coupling. The M₂ or M₂/M₃ pathways induce contraction through a simple Ca²⁺ mobilisation mechanism in which Ca²⁺ entry via VDCCs is activated by TRPC4 channel-induced depolarisation. These pathways play a major role in mediating muscarinic contraction, with the M₂/M₃ pathway making a relatively greater contribution at low agonist concentrations. The M₂ pathway also induces contraction indirectly by inhibiting cAMP-dependent relaxation in response to adenylyl cyclase-activating agonists.

Figure 3

Effects of nicardipine on carbachol-evoked contractions in a wild-type mouse ileal longitudinal muscle strip. Carbachol was applied for 3 min at the indicated concentrations before (A) and after nicardipine treatment to block VDCCs (B). In (C), the recording traces in A and B are superimposed. Note that when muscarinic stimulation is weak, contraction is relatively more dependent on voltage-dependent Ca²⁺ entry and is more sensitive to PTX. This finding indicates that the M₂/M₃ (and M₂) pathway has a major role in mediating the contractile response to weak muscarinic stimulation. When receptor stimulation is stronger, the M₃ pathway predominates by activating multiple mechanisms for Ca²⁺ mobilisation as well as myofilament Ca²⁺ sensitisation.