Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of β-arrestin in HSY human parotid cells.

Objectives Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and β-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor–β-arrestin complexes in the HSY human parotid cell line. Methods Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and β-arrestin were examined using a β-arrestin GPCR assay kit. Results Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 μM, respectively, for 50 μM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of β-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. Conclusion Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of β-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.