[ 3 H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

[ 3 H]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°C), and underwent rapid dissociation. Scatchard analysis of [ 3 H]aniracetam binding revealed a single population of sites with an apparent K D value of ∼70 n M and a maximal density of 3.5 pmol/mg of protein. Specifically bound [ 3 H]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 [ 3 H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [ 3 H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°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 α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [ 3 H]aniracetam binding in unwashed membranes. High levels of [ 3 H]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 [ 3 H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.