Cholinergic Stimulation of Salivary Secretion Studied with M1 and M3 Muscarinic Receptor Single- and Double-Knockout Mice

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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.