Partial agonistic effects of pilocarpine on Ca2+ responses and salivary secretion in the submandibular glands of live animals

New Findings What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca2+‐mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca2+ responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca2+ responses and reduces the effects of other mAChR agonists on Ca2+ responses via its partial agonistic effects. These effects of pilocarpine on Ca2+ responses in the submandibular gland were further established in vivo with a novel Ca2+ imaging system and a genetically encoded Ca2+ indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca2+ responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca2+ responses and salivary secretion in the rat submandibular gland (SMG). In fura‐2‐loaded SMG cells, the maximal effect of pilocarpine on [Ca2+]i elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol‐ and bethanechol (Bet)‐induced [Ca2+]i increases, indicating that pilocarpine acts as a partial agonist for mAChR‐mediated Ca2+ responses. The partial agonistic effect of pilocarpine on Ca2+ dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca2+ indicator, YC‐Nano50. Administration of pilocarpine (3 mg kg−1, i.p.) elicited a small increase in [Ca2+]i in the SMG. Quantitative analyses demonstrated that resting [Ca2+]i was ∼37 nm, which was increased by pilocarpine (3 mg kg−1) and Bet (10 mg kg−1) to 44 and 69 nm, respectively. The inhibitory effects of pilocarpine on Bet‐induced Ca2+ responses were also elucidated in vivo. We further examined real‐time changes in pilocarpine‐induced SMG salivary secretion and showed that pilocarpine induced an extremely weak secretory response and reduced Bet‐induced secretion. Unlike Ca2+ responses, pilocarpine failed to reduce the effect of Bet on SMG blood flow. Our results demonstrate that pilocarpine acts as a partial agonist of mAChRs to induce weak salivary secretion that is correlated with small increases in [Ca2+]i. Furthermore, pilocarpine exhibits an antagonistic effect on mAChR‐induced Ca2+ responses and salivary secretion.