Gastric body cholinergic contractile signal transduction in M2 and M3 receptor knockout mice

Although most smooth muscles express a greater density of M2 than M3 muscarinic receptors, based on the potency of subtype selective muscarinic receptor antagonists, the M3 subtype predominantly mediates contraction. The effect of inhibitors of putative contractile signal transduction pathway enzymes on carbachol-induced contractions was determined in wild-type (WT) mice and mice lacking either the M2 (M2KO) or the M3 (M3KO) receptor subtype. Contractile responses to KCl, then increasing carbachol concentrations in the presence and absence of enzyme inhibitors was determined. The KCl-induced contraction was not different between strains. The carbachol response was unaffected in the M2KO strain but decreased 42% in M3KO mice (p < 0.01). Darifenacin potency was high in both WT and M2KO strains, indicating M3-mediated contractions, and low in the M3KO strain, suggesting M2-mediated contractions. The phosphatidyl inositol-specific phospholipase C (Pi-PLC) inhibitor ET-18-OCH3 had no effect. Inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) and sphingomyelin synthase with D609 decreased maximal contraction in all strains. M3-mediated contractions in the M2KO strain were decreased 54% by the protein kinase C (PKC) inhibitor chelerythrine. M2-mediated contractions in the M3KO and WT strains were decreased by the Rho kinase (ROCK) inhibitor Y27632 as well as the ROCK, PKA and PKG inhibitor H89. The M3 subtype activates PKC and either PC-PLC or sphingomyelin synthase, while the M2 subtype activates ROCK and either PC-PLC or sphingomyelin synthase. These studies suggest that multiple parallel pathways mediate cholinergic contractions in stomach body smooth muscle.