[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 omega-phosphonate analogues of D-2-amino-5-phosphonopentanoate and by inorganic phosphate but not by L-glutamate or any other omega-carboxylates, omega-sulphinates or omega-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 (KD, 0.53 microM; Bmax, 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 Ki values as inhibitors of [3H]D-2-amino-5-phosphonopentanoate binding and their KD 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 (Ki 0.9 microM) 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-D-aspartate receptors physiologically. A study of the regional distribution of [3H]D-2-amino-5-phosphonopentanoate binding sites showed the hippocampus and cerebral cortex to have the highest density of these sites, while the cerebellum and spinal cord had the lowest.(ABSTRACT TRUNCATED AT 400 WORDS)