Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein.
OBJECTIVE
In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM-254890 (YM), a Gq/11-specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh).


METHODS
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 Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of M3 receptors and the alpha subunit of Gq/11 (Galpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies.


RESULTS
Both phasic and tonic components of contractions evoked by 2 microM CCh were inhibited by YM (3-10 microM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 microM) evoked an NSCC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 microM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M3 receptors and Galpha(q/11).


CONCLUSIONS
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 Gq/11 is involved in a signal transduction system.
Department of Ophthalmology, Asahikawa Medical College, Hokkaido, Japan.
F
F
Yasui
M
M
Miyazu
A
A
Yoshida
K
K
Naruse
A
A
Takai
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000008:administration & dosage / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors*; D007473:Ion Channels / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D010456:Peptides, Cyclic / Q000008:administration & dosage / Q000494:pharmacology*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D007473:Ion Channels; D010456:Peptides, Cyclic; D043587:Receptor, Muscarinic M3; C475455:YM-254890; D002217:Carbachol; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium