Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate.
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.
Cincotta
M
M
University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria, Australia.
Summers
R J
RJ
Beart
P M
PM
D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001345:Autoradiography; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007051:Ibotenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D007608:Kainic Acid / Q000378:metabolism; D007700:Kinetics; D008297:Male; D016202:N-Methylaspartate; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000032:analysis*; D012984:Software; D014316:Tritium; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D014316:Tritium; D007051:Ibotenic Acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid