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

摘要

Although most smooth muscles express a greater density of M_2 than M_3 muscarinic receptors, based on the potency of subtype selective muscarinic receptor antagonists, the M_3 subtype predominantly mediates contraction. The effect of inhibitors of putative contractile signal transduction pathway enzymes on carbachol-induced contractions was determined in wildtype (WT) mice and mice lacking either the M_2 (M_2KO) or the M_3 (M_3KO) receptor subtype. Contractile responses to KCI, then increasing carbachol concentrations in the presence and absence of enzyme inhibitors was determined. The KCI-induced contraction was not different between strains. The carbachol response was unaffected in the M_2KO strain but decreased 42% in M_3KO mice (p<0.01). Darifenacin potency was high in both WT and M_2KO strains, indicating M_3-mediated contractions, and low in the M_3KO strain, suggesting M_2-mediated contractions. The phosphatidyl inositol-specific phospholipase C (Pi-PLC) inhibitor ET-18-OCH_3 had no effect. Inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) and sphingomyelin synthase with D609 decreased maximal contraction in all strains. M_3-mediated contractions in the M_2KO strain were decreased 54% by the protein kinase C (PKC) inhibitor chelerythrine. M_2-mediated contractions in the M_3KO and WT strains were decreased by the Rho kinase (ROCK) inhibitor Y27632 as well as the ROCK, PKA and PKG inhibitor H89. The M_3 subtype activates PKC and either PC-PLC or sphingomyelin synthase, while the M_2 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.