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

Abstract

Background and purpose: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca 2 þ 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 þ ] 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 þ concentration [Ca 2 þ ] i was monitored using the Fluo-4 fluorophore. Existence and localization of M 3 receptors and the a subunit of G q/11 (Ga q/11 ) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. Key results: Both phasic and tonic components of contractions evoked by 2 mM CCh were inhibited by YM (3-10 mM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 mM) evoked an NSCC current as well as an elevation of the [Ca 2 þ ] i . Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 mM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M 3 receptors and Ga 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.

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

“…YM-254890 has been reported to block agonist-induced activation of G q/11 -type G-proteins in various tissues or cells [17,23,27,34]. We confirmed this blocking activity in preparations used for the present study, where YM-254890 abolished carbachol-evoked contractions mediated by intracellular Ca 2+ release that is known to involve G q/11 -type Gproteins [3,12,13].…”

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

“…FR and YM, two naturally occurring cyclic depsipeptides, are invaluable pharmacological tools for probing Gq-mediated cellular responses. Because of their specificity, they have become instrumental in defining and diagnosing the contribution of Gq proteins to complex biological processes in vitro and ex vivo (33–39, 52–59). FR and YM share a common mechanism of G protein inhibition: they act as guanine nucleotide dissociation inhibitors that preserve GDP-bound heterotrimers in their inactive state (19, 33).…”

Phospholipase C families: Common themes and versatility in physiology and pathology

“…Notably, cyclic peptides YM-254890 and FR900359 J o u r n a l P r e -p r o o f disrupt the interaction and activation of PLC isoforms by Gq while M119 and gallein, structurally related to fluorescein, similarly affect regulation of these isoforms by G [445,[458][459][460]. In addition to their use as pharmacological tools, their potential use to treat various diseases, including melanoma and opioid analgesia, is being assessed in preclinical studies [446,[461][462][463][464]. Although all these inhibitors bind to the G-protein subunits and not to PLC enzymes, they provide proof of principle for targeting regulatory protein-protein interactions that control PLC activity.…”