Biomaterial Database

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. 2011

Yuji Odagaki, and Masakazu Kinoshita, and Ryoichi Toyoshima

Department of Psychiatry, Faculty of Medicine, Saitama Medical University, Moroyama-machi, Iruma-gun, Japan. odagaki@saitama-med.ac.jp

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.

UI	MeSH Term	Description	Entries
D008297	Male		Males
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011869	Radioligand Assay	Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders).	Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D002462	Cell Membrane	The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.	Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D002540	Cerebral Cortex	The thin layer of GRAY MATTER on the surface of the CEREBRAL HEMISPHERES that develops from the TELENCEPHALON and folds into gyri and sulci. It reaches its highest development in humans and is responsible for intellectual faculties and higher mental functions.	Allocortex,Archipallium,Cortex Cerebri,Cortical Plate,Paleocortex,Periallocortex,Allocortices,Archipalliums,Cerebral Cortices,Cortex Cerebrus,Cortex, Cerebral,Cortical Plates,Paleocortices,Periallocortices,Plate, Cortical
D004305	Dose-Response Relationship, Drug	The relationship between the dose of an administered drug and the response of the organism to the drug.	Dose Response Relationship, Drug,Dose-Response Relationships, Drug,Drug Dose-Response Relationship,Drug Dose-Response Relationships,Relationship, Drug Dose-Response,Relationships, Drug Dose-Response
D005033	Ethylmaleimide	A sulfhydryl reagent that is widely used in experimental biochemical studies.	N-Ethylmaleimide,N Ethylmaleimide
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001667	Binding, Competitive	The interaction of two or more substrates or ligands with the same binding site. The displacement of one by the other is used in quantitative and selective affinity measurements.	Competitive Binding
D013462	Sulfur Radioisotopes	Unstable isotopes of sulfur that decay or disintegrate spontaneously emitting radiation. S 29-31, 35, 37, and 38 are radioactive sulfur isotopes.	Radioisotopes, Sulfur