Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the G _q/11 protein

Background and purpose: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca ²⁺ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M ₃ muscarinic receptors. We examined effects of YM-254890 (YM), a G _q/11-specific inhibitor, on contraction, NSCC currents and [Ca ²⁺] _i elevation induced by carbachol (CCh). Experimental approach: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca ²⁺ concentration [Ca ²⁺] _i was monitored using the Fluo-4 fluorophore. Existence and localization of M ₃ receptors and the α subunit of G _q/11 (Gα _q/11) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. Key results: Both phasic and tonic components of contractions evoked by 2 μM CCh were inhibited by YM (3-10 μM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 μM) evoked an NSCC current as well as an elevation of the [Ca ²⁺] _i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 μM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M ₃ receptors and Gα _q/11. Conclusions and implications: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G _q/11-coupled pathway. YM is a useful tool to assess whether G _q/11 is involved in a signal transduction system.