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 (3mgkg(-1), i.p.) elicited a small increase in [Ca2+](i) in the SMG. Quantitative analyses demonstrated that resting [Ca2+](i) was approximate to 37nm, which was increased by pilocarpine (3mgkg(-1)) and Bet (10mgkg(-1)) to 44 and 69nm, 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.