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

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In the present study, we have characterized muscarinic receptor subtypes that mediate carbachol-induced Ca²⁺ sensitization of contraction in intestinal smooth muscle, using mutant mice lacking M₂ or M₃ muscarinic receptors or both receptor subtypes. In α-toxin-permeabilized muscle strips from wild-type (WT) mice, isometric tension responses to Ca²⁺ applied cumulatively (pCa 7.0-5.0) were increased when the muscarinic agonist carbachol (100 μM) was added to the medium, as judged from shifts of pCa-tension curves in both 50% effective concentration (EC₅₀) and maximum response (E_max) of pCa-tension curve. In preparations from M₂-knockout (KO) mice, pCa-tension curves were also shifted by carbachol (100 μM), and the extents of the EC₅₀ and E_max changes resembled those observed in preparations from WT mice. In preparations from M₃-KO or M₂/M₃-double KO mice, however, no significant changes in pCa-tension curves were obtained after carbachol application. The G_q/11-type G-protein inhibitor YM-254890 (1 μM) completely blocked the Ca²⁺ sensitization of contraction induced by carbachol in M₂-KO or WT preparations. The results strongly support the idea that the muscarinic activation of Ca²⁺ sensitization in intestinal smooth muscles is mediated by the M₃ muscarinic receptor coupled to G_q/11-type G-proteins, without any significant involvement of the other muscarinic receptor subtypes including M₂.