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N. Ishii, M. Miyazu, A. Yoshida, A. Takai; Endothelin-1 Contracts Bovine Ciliary Smooth Muscle Evoked by Binding Type 1 Receptor Coupled With the Gq/11 Protein. Invest. Ophthalmol. Vis. Sci. 2010;51(13):991.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the effects of endothelin-1 (ET1) on the contractility of the ciliary muscle whose tonus is regarded as one of the most determinant factors regulating aqueous humor outflow.
Isometric tension was recorded in ciliary muscle bundles excised from fresh bovine eyes obtained from a local slaughterhouse. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp, and the intraocular free Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of endothelin receptors type A and B (ETRA and ETRB) were examined by RT-PCR and immunofluorescence microscopy.
ET1 (1-100 nM) evoked contraction in a dose-dependent manner (ED50=6±1 nM; number of experiments, n=4). Like contractions evoked by stimulation of M3¬muscarinic receptor (M3R) by carbachol (CCh; 2 µM), the ET1-evoked contraction showed a strong dependence on extracellular Ca2+ and was completely inhibited by YM-254890 (500 nM; n=4), a Gq/11-specific inhibitor. It was also partially inhibited by Y-27632 (20 µM; n=4), a Rho kinase inhibitor. Unlike CCh-evoked contraction, however, the ET1-evoked contraction had no rapid phasic component and tended to be washed out very slowly. Also the ET1-evoked response exhibited a higher sensitivity to inhibition by Ni2+ (1 mM) compared with CCh-evoked response. ETRA antagonists dose-dependently inhibited the ET1-evoked whereas ETRB antagonists were without effect. In whole-cell voltage clamp experiments ET1 (10-30 nM) showed an (albeit weak) activating effect on two types of non-selective cation channel which are known to be opened upon M3R stimulation. Immunostaining revealed a dense distribution of ETRA in the plasma membrane of ciliary myocytes.
The present results strongly indicate that ET1, as well as CCh, contracts bovine ciliary muscle by a Gq/11-coupled mechanism. The relatively weak potency of ET1 may suggest that ETRA activated by ET1 interacts with Gq/11 more loosely than does M3R activated by CCh.
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