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Functional Coupling between Metabotropic Glutamate Receptors and G‐Proteins in Rat Cerebral Cortex Assessed by Guanosine‐5′‐ O ‐(3‐[ 35 S]thio)triphosphate Binding Assay
Author(s) -
Odagaki Yuji,
Kinoshita Masakazu,
Toyoshima Ryoichi
Publication year - 2011
Publication title -
basic and clinical pharmacology and toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.805
H-Index - 90
eISSN - 1742-7843
pISSN - 1742-7835
DOI - 10.1111/j.1742-7843.2011.00705.x
Subject(s) - metabotropic receptor , metabotropic glutamate receptor , metabotropic glutamate receptor 1 , metabotropic glutamate receptor 5 , glutamate receptor , guanosine , gtp' , ionotropic effect , chemistry , g protein , pharmacology , receptor , biochemistry , medicine , biophysics , biology , enzyme
Stimulation of specific guanosine‐5′‐ O ‐(3‐[ 35 S]thio)triphosphate ([ 35 S]GTPγS) binding by l ‐glutamate was pharmacologically characterized in rat cerebral cortical membranes. Optimization of the experimental conditions with respect to the concentrations of GDP, MgCl 2 and NaCl in assay buffer prompted us to adopt the incubation of rat cerebral cortical membranes with 0.2 nM [ 35 S]GTPγS at 30°C for 60 min. in the presence of 20 μM GDP, 5 mM MgCl 2 and 100 mM NaCl as a standard condition. Specific [ 35 S]GTPγS binding was stimulated by l ‐glutamate in a concentration‐dependent manner but not by ionotropic glutamate receptor agonists. The stimulatory responses were also elicited by many agonists for metabotropic glutamate (mGlu) receptor, with (−)‐2‐oxa‐4‐aminobicyclo[3.1.0]hexane‐4,6‐dicarboxylic acid (LY379268) being the most potent. l ‐glutamate‐stimulated [ 35 S]GTPγS binding was inhibited by several mGlu antagonists, with (2 S )‐2‐amino‐2‐[(1 S ,2 S )‐2‐carboxycycloprop‐1‐yl]‐3‐(xanth‐9‐yl) propanoic acid (LY341495) being the most potent. The pharmacological properties of a series of agonists and antagonists indicated the involvement of group II mGlu receptors, especially mGlu2. Supportive of this notion was the finding that l ‐glutamate‐stimulated specific [ 35 S]GTPγS binding was augmented by 2,2,2‐trifluoro‐ N ‐[4‐(2‐methoxyphenoxy)phenyl]‐ N ‐(3‐pyridinylmethyl)ethanesulphonamide hydrochloride (LY487379), a reportedly selective allosteric positive modulator for mGlu2, by means of upward and leftward shift of the concentration–response curve. In addition, LY487379 per se stimulated [ 35 S]GTPγS binding, though, through a mechanism different from the stimulation by l ‐glutamate. Pre‐treatment of the membranes with N ‐ethylmaleimide (NEM) cancelled l ‐glutamate‐stimulated [ 35 S]GTPγS binding in a concentration‐ and incubation time‐dependent manner. Taken altogether, l ‐glutamate‐stimulated [ 35 S]GTPγS binding serves as a useful functional assay for the activation of NEM‐sensitive G i/o ‐mediated group II mGlu receptors in rat cerebral cortical membranes.