Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate.
The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate [( 3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and "granule" membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 microM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5-0.9 microM and Bmax 3.2-3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate greater than L-cysteate greater than L-cysteinesulphinate greater than L-serine-O-sulphate greater than ibotenate greater than L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Shea
R D
RD
University of Melbourne, Clinical Pharmacology and Therapeutics, Austin Hospital, Heidelberg, Victoria, Australia.
Manallack
D T
DT
Conway
E L
EL
Mercer
L D
LD
Beart
P M
PM
D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects; D002525:Cerebellar Cortex / Q000033:anatomy & histology / Q000378:metabolism; D002540:Cerebral Cortex / Q000033:anatomy & histology / Q000378:metabolism; D003523:Cycloserine / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism* / Q000493:pharmacokinetics; D018698:Glutamic Acid; D005998:Glycine / Q000378:metabolism* / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008024:Ligands; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D005971:Glutamates; D008024:Ligands; D017459:Receptors, Amino Acid; D011956:Receptors, Cell Surface; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D012694:Serine; D003523:Cycloserine; D007736:Kynurenic Acid; C057013:7-chlorokynurenic acid; D005998:Glycine