[H-3] ANIRACETAM BINDS TO SPECIFIC RECOGNITION SITES IN BRAIN MEMBRANES

[H-3]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [H-3]aniracetam binding revealed a single population of sites with an apparent K-D value of similar to 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [H-3]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [H-3]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at [east some [H-3]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [H-3]aniracetam binding in unwashed membranes. High levels of [H-3]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [H-3]- aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.