M₃ muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice

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.