Article date: 1989/2/15
PubMed ID: 2568102
Journal name: Analytical biochemistry (ISSN: 0003-2697)
A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.
Author List: Cincotta M, Summers R J, Beart P M
Publication Types: Journal Article; Research Support, Non-U.S. Gov't
Substances mentioned in the article: Glutamates; Receptors, Glutamate; Receptors, Neurotransmitter; Tritium; Ibotenic Acid; Aspartic Acid; Glutamic Acid; N-Methylaspartate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Kainic Acid;
Mesh terms: Animals; Aspartic Acid/analogs & derivatives; Autoradiography; Binding, Competitive; Brain/metabolism; Glutamates; Glutamic Acid; Ibotenic Acid/analogs & derivatives; Kainic Acid/metabolism; Kinetics; Male; N-Methylaspartate; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Glutamate; Receptors, Neurotransmitter/analysis; Software; Tritium; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid;