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

[H-3]Aniracetam bound to specific and saturable recognition sites in m embranes prepared from discrete regions of rat brain. In crude membran e preparation from rat cerebral cortex, specific binding was Na+ indep endent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [H-3]aniracet am 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 prot ein. Specifically bound [H-3]aniracetam was not displaced by various m etabolites of aniracetam, nor by other pyrrolidinone-containing nootro pic drugs such as piracetam or oxiracetam. Subcellular distribution st udies showed that a high percentage of specific [H-3]aniracetam bindin g was present in purified synaptosomes or mitochondria, whereas specif ic binding was low in the myelin fraction. The possibility that at [ea st some [H-3]aniracetam binding sites are associated with glutamate re ceptors is supported by the evidence that specific binding was abolish ed when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapp ed in the membranes) and could be restored after addition of either gl utamate or pha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) b ut 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, cortic al, or cerebellar membranes, which contain a high density of excitator y amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, speci fically bound [H-3]- aniracetam could not be displaced by cyclothiazid e or GYKI 52466, which act as a positive and negative modulator of AMP A receptors, respectively.