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 (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]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.
© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.