Differential effects of SH-reactive agents on [³H](±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate and [³H]glutamate binding in brain synaptic membranes treated with triton X-100

Specific binding of [³H](±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an antagonist highly selective for the N-methyl-d-aspartate (NMDA)-sensitive subclass of the brain excitatory amino acid receptors, was examined in rat brain synaptic membranes treated with a low concentration of Triton X-100 using a filtration assay method. The binding was displaced by l-glutamic acid (Glu) and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists as well as competitive antagonists for the subclass invariably displaced the binding, while noncompetitive antagonists were ineffective as displacers of the binding. Agonists selective for the other subclasses did not affect the binding, d-2-Amino-5-phosphonovaleric and d-2-amino-7-phosphonoheptanoic acids exhibited more potent inhibition of the binding than their respective l-isomers, with the other aminophosphonates being inactive. Preincubation of synaptic membranes with different SH-reactive agents invariably resulted in a significant reduction of [³H]CPP binding, without significantly affecting NMDA-sensitive [³H]Glu binding. These results suggest that a filtration assay method is also applicable to detecting the binding of an antagonist highly selective for the NMDA-sensitive receptors to the NMDA recognition sites, in addition to a centrifugation assay method. Heterogeneity of the NMDA recognition sites is also suggested.