The M1 and M3 subtypes are the major muscarinic acetylcholine receptors in the salivary gland and M3 is reported to be more abundant. However, despite initial reports of salivation abnormalities in M3-knockout (M3KO) mice, it is still unclear which subtype is functionally relevant in physiological salivation. In the present study, salivary secretory function was examined using mice lacking specific subtype(s) of muscarinic receptor. The carbachol-induced [Ca2+]i increase was markedly impaired in submandibular gland cells from M3KO mice and completely absent in those from M1/M3KO mice. This demonstrates that M3 and M1 play major and minor roles, respectively, in the cholinergically induced [Ca2+]i increase. Two-dimensional Ca2+-imaging analysis revealed the patchy distribution of M1 in submandibular gland acini, in contrast to the ubiquitous distribution of M3. In vivo administration of a high dose of pilocarpine (10 mg kg-1, s.c.) to M3KO mice caused salivation comparable to that in wild-type mice, while no salivation was induced in M1/M3KO mice, indicating that salivation in M3KO mice is caused by an M1-mediated [Ca2+]i increase. In contrast, a lower dose of pilocarpine (1 mg kg-1, s.c.) failed to induce salivation in M3KO mice, but induced abundant salivation in wild-type mice, indicating that M3-mediated salivation has a lower threshold than M1-mediated salivation. In addition, M3KO mice, but not M1KO mice, had difficulty in eating dry food, as shown by frequent drinking during feeding, suggesting that salivation during eating is mediated by M3 and that M1 plays no practical role in it. These results show that the M3 subtype is essential for parasympathetic control of salivation and a reasonable target for the drug treatment and gene therapy of xerostomia, including Sjögren's syndrome.
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