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Title: Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate.
Austin Authors: Cincotta, M;Summers, R J;Beart, P M
Affiliation: University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria, Australia
Issue Date: 15-Feb-1989
Publication information: Analytical Biochemistry; 177(1): 150-5
Abstract: 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.
Gov't Doc #: 2568102
Type: Journal Article
Subjects: Animals
Aspartic Acid.analogs & derivatives.metabolism
Binding, Competitive
Glutamates.diagnostic use
Glutamic Acid
Ibotenic Acid.analogs & derivatives.metabolism
Kainic Acid.metabolism
Radioligand Assay
Rats, Inbred Strains
Receptors, Glutamate
Receptors, Neurotransmitter.analysis
Tritium.diagnostic use
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
Appears in Collections:Journal articles

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