title: [3H]D-2-amino-5-phosphonopentanoate as a ligand for N-methyl-D-aspartate receptors in the mammalian central nervous system.
abstract: 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)
mesh_terms: D015763:2-Amino-5-phosphonovalerate; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D014633:Valine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D005971:Glutamates; D016194:Receptors, N-Methyl-D-Aspartate; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid; D015763:2-Amino-5-phosphonovalerate; D014633:Valine