Specific High‐Affinity Binding of L‐[³H]Aspartate to Rat Brain Membranes

Abstract

The binding of L-[3H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na-independent binding was saturable, reversible, and optimal at 20 degrees C and at pHs in the neutral range. The dissociation constant (Kd) at 20 degrees C was about 200 nM. This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na-independent L-[3H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex greater than pons-medulla greater than spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na-independent binding in this region. The Na-dependent binding of L-[3H]-aspartate (Kd = 4894 nM) was strongly inhibited by D-aspartate, L-glutamate, D,L-aspartate beta-hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex greater than cerebellum = pons-medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

“…This is consistent with the observation made in the present study that neither [3H]aspartate nor [3H]glutamate binding was stimulated by sodium ions. This observation contrasts with observations in rat brain, in which [3H]aspartate binding was markedly enhanced in the presence of sodium chloride (Foster et al, 1981;Di Lauro et al, 1982).…”

“…Although Laspartate has higher affinity for NMDA receptors (Fagg and Matus, 1984), it is unlikely that [3H]aspartate labels NMDA receptors in human cerebellum, as NMDA itself and the selective: NMDA antagonists ~~-2-amino-5-phosphonovaleric acid (APV) and DL-2-amino-7-phosphonoheptanoic acid (APH) were inactive. Similarly, NMDA receptors do not appear to be stereoselective for the aspartic isomers (Watkins and Evans, 198 1;Fagg and Matus, 1984 It would seem likely therefore that the [3H]aspartate binding site is distinct from the recognised excitatory amino acid receptors, as has been suggested for rat brain (Sharif and Roberts, 1981; Di Lauro et al, 1982;Lane and Fagg, 1984). Much more experimentation is required to determine if this binding site represents a physiologically relevant aspartate receptor.…”

L‐[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

“…Its high affinity for L-aspartate suggests that it should be possible to label this site with ~-[~H]aspartate. Nevertheless the GLU B binding site apparently differs from binding sites other workers have characterized with this radioligand (Foster et al, 1981~;Sharif and Roberts, 1981;Di Lauro et al, 1982) and from a binding site with approximately equal affinities for L-glutamate and L-aspartate that has been studied by Michaelis et al (1981). Further investigation is required to explain this discrepancy.…”

Schwann cells exhibit excitotoxicity consistent with release of NMDA receptor agonists

“…Although DAAOx can metabolize glycine under some conditions (Wellner, 1970), we determined by thin-layer chromatography that it did not diminish glycine under our incubation conditions (Wu et al, 2004b). Other substrates for DAAOx include D-alanine and D-aspartate; however, D-alanine is far less potent than D-serine is (Chizhmakov et al, 1989;Wroblewski et al, 1989), and D-aspartate binds to the glutamate site (Di Lauro et al, 1982;Foster and Fagg, 1987). A requirement for a ligand at the D-serine site thus would remain.…”