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.

摘要

Stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) 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]GTPgammaS at 30 degrees C for 60 min. in the presence of 20 muM GDP, 5 mM MgCl(2) and 100 mM NaCl as a standard condition. Specific [(35)S]GTPgammaS 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]GTPgammaS binding was inhibited by several mGlu antagonists, with (2S)-2-amino-2-[(1S,2S)-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]GTPgammaS binding was augmented by 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulphon amide 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]GTPgammaS 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]GTPgammaS binding in a concentration- and incubation time-dependent manner. Taken altogether, l-glutamate-stimulated [(35)S]GTPgammaS 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.