Abstract

Identification of the specific muscarinic acetylcholine receptor (mAChR) subtypes mediating stimulation of salivary secretion is of considerable clinical interest. Recent pharmacological and molecular genetic studies have yielded somewhat confusing and partially contradictory results regarding the involvement of individual mAChRs in this activity. In the present study, we re-examined the roles of M₁ and M₃ mAChRs in muscarinic agonist-mediated stimulation of salivary secretion by using M₁ and M₃ receptor single-knockout (KO) mice and newly generated M₁/M₃ receptor double-KO mice. When applied at a low dose (1 mg/kg, s.c.), the muscarinic agonist pilocarpine showed significantly reduced secretory activity in both M₁ and M₃ receptor single-KO mice. However, when applied at higher doses, pilocarpine induced only modestly reduced (5 mg/kg, s.c.) or unchanged (15 mg/kg, s.c.) salivation responses, respectively, in M₁ and M₃ receptor single-KO mice, indicating that the presence of either M₁ or M₃ receptors is sufficient to mediate robust salivary output. Quantitative reverse transcriptase-polymerase chain reaction studies with salivary gland tissue showed that the inactivation of the M₁ or M₃ mAChR genes did not lead to significantly altered mRNA levels of the remaining mAChR subtypes. Strikingly, the sialagogue activity of pilocarpine was abolished in M₁/M₃ receptor double-KO mice. However, salivary glands from M₁/M₃ receptor double-KO mice remained responsive to stimulation by the β-adrenergic receptor agonist, (S)-isoproterenol. Taken together these studies support the concept that a mixture of M₁ and M₃ receptors mediates cholinergic stimulation of salivary flow.