Roles of M₂ and M₄ Muscarinic Receptors in Regulating Acetylcholine Release From Myenteric Neurons of Mouse Ileum

Abstract

We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1-M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1-M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.

M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: findings obtained with muscarinic-receptor knockout mouse

“…9A). As shown in our previous report, a strong immunoreactivity to an anti-M 2 receptor antibody was observed in the smooth muscle in both circular and longitudinal layers and the myenteric plexus (31). Double staining with anti-M 2 receptor and anti-SP antibodies produced a yellow signal in the myenteric plexus (Fig.…”

“…When we examined the coexpression of M 2 receptors and SP in the mouse ileum, an involvement of the M 2 receptor in SP release was revealed. Previously, the specificity of anti-M 2 -receptor antibody has been confirmed in the mouse ileum because the immunoreactivity of this antibody was not observed in M 2 -receptor KO mice (31). In the present study, localization of M 2 receptors on the SP-containing neurons was observed in the myenteric plexus of the mouse ileum.…”

“…An immunohistochemical study was carried out by the method described previously (31). Briefly, the intestine was isolated after the wild-type mice were deeply anesthetized with pentobarbital sodium (50 mg/kg ip), and the intestine was fixed by transcardiac perfusion.…”

“…M 2 receptors are distributed in smooth muscle cells and enteric neurons in the gastrointestinal tract (13,31). To clarify the site of action of M 2 -receptor inhibition of EFSinduced sustained contraction, the responses to SP, added exogenously, were compared in ileal preparations of wild-type and M 2 -and M 3 -receptor KO mice.…”

Evidence for M2 and M3 Muscarinic Receptor Involvement in Cholinergic Excitatory Junction Potentials Through Synergistic Activation of Cation Channels in the Longitudinal Muscle of Mouse Ileum

“…It is unlikely that the smaller size of cholinergic EJP in different KO preparations is caused by decreases in the amount of ACh released from the enteric nerves because the amount of released ACh in response to electrical stimulation does not significantly differ among M 2 -KO, M 3 -KO, and wild-type ileal preparations (28). Thus, the data indicate that the wild-type cholinergic EJP is not a simple mixture of M 2 and M 3 responses but may rather arise through a synergy between M 2 and M 3 receptors.…”