M₂ and M₃ muscarinic receptor‐mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice

Abstract

1 Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M 2 or M 3 muscarinic receptors, in order to characterize the mechanisms involved in M 2 and M 3 receptor-mediated contractile responses. 2 Single applications of carbachol (0.1-100 mM) produced concentration-dependent contractions in preparations from M 2 -knockout (KO) and M 3 -KO mice, mediated via M 3 and M 2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M 2 -preferring antagonist methoctramine (300 nM) or the M 3 -preferring antagonist 4-DAMP (30 nM). 3 The M 2 -mediated contractions were mimicked in shape by submaximal stimulation with high K þ concentrations (up to 35 mM), almost abolished by voltage-dependent Ca 2 þ channel (VDCC) antagonists or depolarization with 140 mM K þ medium, and greatly reduced by pertussis toxin (PTX) treatment. 4 The M 3 -mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K þ -depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K þ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca 2 þ . 5 The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. 6 The results suggest that the M 2 receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca 2 þ entry, especially associated with action potential discharge, and that the M 3 receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca 2 þ entry and intracellular Ca 2 þ release. In intact tissues coexpressing M 2 and M 3 receptors, M 2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

“…Using muscarinic receptor mutant mice, we previously demonstrated that activation of either M 2 or M 3 receptor subtypes produces contraction in intestinal smooth muscle [30]. Moreover, we found that the M 2 -induced contraction largely depends on Ca sensitization as well.…”

“…Rho, via acting on Rho kinase, causes phosphorylation of MLCP to inhibit its activity and thereby induces Ca 2+ sensitization of contractile proteins. Various muscarinic responses of intestinal smooth muscles have been characterized by using muscarinic receptor subtype-KO mice [19,30,31]. Although mRNA for all five muscarinic receptor subtypes (M 1 -M 5 ) has been found in gut smooth muscles [20], only M 2 and M 3 receptors have been shown to mediate muscarinic responses in gut smooth muscles so far.…”

“…Food (MF, Oriental Yeast Co., Tokyo, Japan) and water were supplied ad libitum. The two WT strains of mouse were used without distinction for experiments [30,31], because there were no significant differences in any of the pD 2 , maximum responses and Hill coefficient of the concentration-response relationships for carbachol-induced contractions in the two WT strains [30].…”

“…However, such pharmacological studies often remain inconclusive because of the poor subtype selectivity of available muscarinic antagonists. For example, pharmacological studies suggested that carbacholevoked contractions are exclusively mediated by M 3 receptors, whereas recent studies using muscarinic receptor knockout mice implicated both M 3 and M 2 receptors in this response [30,31].…”

“…However, such pharmacological studies often remain inconclusive because of the poor subtype selectivity of available muscarinic antagonists. For example, pharmacological studies suggested that carbacholevoked contractions are exclusively mediated by M 3 receptors, whereas recent studies using muscarinic receptor knockout mice implicated both M 3 and M 2 receptors in this response [30,31].Therefore, in the present study, using M 2 -knockout (KO) or M 3 -KO or M 2 /M 3 -double KO mice and wild type (WT) mice, we examined the activity of carbachol in inducing Ca 2+ sensitization in -toxin-permeabilized smooth muscles of the small intestine. In general, M 2 and M 3 muscarinic receptors are coupled preferentially with G i/o -and G q/11 -type G-proteins, respectively.…”