[ 3H] D-2-amino-5-phosphonopentanoate as a ligand for N-methyl- d-aspartate receptors in the mammalian central nervous system

Tritiated d-2-amino-5-phosphonopentanoate has been prepared and evaluated as a radioligand for investigating N-methyl- d-aspartate receptors in rat brain membranes. A radioactive impurity, which was more acidic than 2-amino-5-phosphonopentanoate, interfered with the binding assay for [ 3H] d-2-amino-5-phosphonopentanoate in preliminary experiments and developed progressively with time of storage of the ligand, was isolated by ion-exchange purification and its binding site characterized. Binding of the 3H-impurity was increased in the presence of calcium ions, with a maximum effect at a concentration of 1–3 mM, but not by sodium, potassium or magnesium ions. It was inhibited by ω-phosphonate analogues of d-2-amino-5-phosphonopentanoate and by inorganic phosphate but not by L-glutamate or any other ω-car☐ylates, ω-sulphinates or ω-sulphonates tested. The site of binding for the 3H-impurity was not identified, but from its pharmacological profile it appears to be unrelated to any excitatory amino acid receptor so far described. Binding of purified [ 3H] d-2-amino-5-phosphonopentanoate to rat cerebral cortical membranes was saturable ( K D , 0.53 μM; B max , 4.3 pmol/mg protein), was maximal at pH 7.3, but was not particularly temperature sensitive. Dissociation of the receptor-ligand complex was very rapid. Magnesium ions had an inhibitory effect on the binding of [ 3H] d-2-amino-5-phosphonopentanoate, but the mechanism of this action was not clear. For a wide range of competitive excitatory amino acid antagonists with different potencies and receptor specificities there was a direct relationship between their K i values as inhibitors of [ 3H] d-2-amino-5-phosphonopentanoate binding and their K D values for antagonism of N-methyl- d-aspartate induced depolarizations. Thus, [ 3H] d-2-amino-5-phosphonopentanoate binds to electrophysiological N-methyl- d-aspartate receptors. Among endogenous agonists, l-glutamate had the highest affinity ( K i 0.9 μM) for the [ 3H] d-2-amino-5-phosphonopentanoate binding site; l-homocysteate and S-sulpho- l-cysteine also had high affinity. However, quinolinate and N-acetylaspartylglutamate had relatively low affinity. It is considered that l-glutamate is the most likely substance to be the transmitter activating, N-methyl- daspartate receptors physiologically. A study of the regional distribution of [ 3H] d2-amino-5-phosphonopentanoate binding sites showed the hippocampus and cerebral cortex to have the highest density of these