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KMID : 0811720060100000329
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Korean Journal of Physiology & Pharmacology
2006 Volume.10 No. 0 p.329 ~ p.0
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EFFECTS OF A GQ/11-PROTEIN INHIBITOR, YM-254890, ON CARBACHOL-INDUCED RESPONSES OF BOVINE CILIARY MUSCLE
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Yasui Fuminori
Takai Yoshiko
Miyazu Motoi
Takai Akira
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Abstract
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In the ciliary muscle, a smooth muscle under parasympathetic control, contraction is initiated and sustained by stimulation
of M3-muscarinic receptors by the transmitter acetylcholine. It is believed that the initial phasic component of the contraction is triggered by Ca2+ release from intracellular stores mediated by Gq/11-linked signalling. The ensuing tonic component of the contraction is also highly dependent on Ca2+, but in this case, Ca2+ is provided by influx through receptor-operated cation channels (ROCs) rather than by release from stores [Takai et al. (2004) J Physiol 559, 899-922; Sugawara et al. (2006) Autonom Autacoid Pharmacol 26, 285-292]. However, little is known about the signalling mechanism involved in this Ca2+ influx. Here we have examined effects of YM-254890, a Gq/11-specific inhibitor on contraction, ROC currents and [Ca2+]i elevation induced by carbachol (CCh). Bovine eyes were obtained from a local slaughterhouse. Isometric tension was recorded from excised cilary muscle bundles using a strain-gauge transducer. For the other experiments, ciliary muscle cells were dispersed with collagenase and cultured for 1～5 days. Whole-cell currents were recorded by voltage clamp in the cultured using an Axopatch 200B amplifier (Axon Instruments). The intracellular free Ca2+ concentration [Ca2+]i was recorded using Fluo-4 (Invitrogen) as the indicator. In the immunostaining experiments, primary antibodies were visualised using secondary antibodies conjugated with AlexaFlor fluorescent dyes (Invitrogen). Relative concentrations of mRNA were estimated by real-time RT-PCR using a LightCycler System (Roche Diagnostics). In ciliary muscle bundles, superfusion of 2μM-CCh evoked a sustained contraction. Both phasic and tonic components of this contraction were inhibited by YM-254890 (3～10μM) in a dose-dependent manner. In the cultured cells, CCh (0.05～10μM) evoked a ROC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were also abrogated by YM-254890 (3～10μM). Immunostaining revealed a dense distribution of Gq/11 in the vicinity of the cytoplasmic side of the plasma membrane of the cultured cells. Real-time RT-PCR experiments estimated the ratio of cellular mRNA content of Gq to that of G11 to be 1 : 5. Gq/11 appears to be critically involved in Ca2+ mobilization in the tonic as well as phasic component of the contraction in the bovine ciliary muscle.
Source: Korean J Physiol Pharmacol.2006 Oct;10(Suppl II):240
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