PHARMACOLOGY INTERACTION OF PYRACETAM UITH SPECIFIC ~H-IMIPRAI{INE BINDING SITES AND GABA-BENZODIAZEPINE RECEPTOR CO}{PLEX OF BRAIN MEMBRANES V. V. Rozhanets, K. K. Chakhbra, N. D. Danchev, K. M. Malin, D. Yu. Rusakov, and A. V. Val'dman UDC 615.214.3:547.745]015.4: 612.822.014,467:[615.214. 32+615 KEY WORDS: pyracetam, 3H-imipramine, 3H-flunitrazepam, GABA-activated binding, anxiolytic effect. Despite the extensive clinical use of pyracetam* the neurochemical mechanisms of its action have not yet been explained. Available data on its effect on the protein-synthesizing and energy-producing systems of the brain do notexplain several of the specific effects of pyracetam and, in particular, its anxiolytic action, revealed in experiments on animals [3]. The ~iters showed previously [2] that pyracetam in ~i~o does not affect binding of the cor- responding ligands with benzodiazepine, imipramine, dopamine (D2), and serotonin ($I and $2) receptors, and also with ~- and ~-adrenoreceptors of the rat brain, if the concentrations of these ligands were close to the dissociation constant (Kd) of the ligand--receptor complex. This may signify absence of competition between pyracetam and the ligands studied, but detailed study of the action of pyracetam on the affinity and maximal concentration (Bmax) of these receptors has not been undertaken~ In the present investigation the effect of pyracetam was studied on parameters of specific binding of 3H-inipramine and GABA-activated binding of 311-flunitrazepam with rat brain membranes. EXPERIMENTAL METIIOD Experiments were carried out on noninbred male albino rats and guinea:pigs weighing 180- 200 and 270-300 g respectively, and kent under natural conditions of light and darkness, with free access to water and food. Membranes of the unpurified synaptosome fraction were isolated from whole rat brain [5], from washed rat hippocampal membranes [7], and from guinea pig cere- bellum [9] without modifications 9 Suspensions of rat brain membranes in appropriate buffer solutions were kept at -20~ for up to 3 weeks; guinea pig cerebellar membranes were used on the day of isolation. Specific binding of 3}l-imipramine by whole brain membranes [5] and GABA- activated binding of 3H-flunitrazepam by washed rat hippocampal membranes [7] were studied also without modifications. Specific binding of 3ll-myanserine by guinea pig cerebellar membranes [9] ~as studied by the use of 10 TM M myanserine or diphenhydramine as displacing agents. In all cases the reaction was stopped by diluting the incubation mixture tenfold with the cor- responding cold buffer, followed by rapid filtration of the mixture through GF/B glass fiber filters (from Whatman, England). The filters, washed and dried in air twice, were extracted overnight in 5 ml of Soviet Zhs-8 scintillator and the radioactivity of the samples was de- termined on an SL-4000 scintillation counter (from Intertechnique, France). 3H-Imipramine (21 Ci/mmole), ~H-myanserine (16.5 Ci/mmole), and 3H-flunitrazepam (89 Ci/mmole) were obtained from Amersham Corporation (England) Values of K~ and B were calculated on Scatchard plots 9 o max by means of an I~-33E microcalculator (USA). The significance of differences was estimated by Student's t test. The protein concentration was determined by the method in [i0]. EXPERIMENTAL RESULTS It will be clear from Table 1 that pyracetam, in a concentration of 10 -6 M reduced speci- fic binding of 3H-imipramine, lowering Bma x significantly, but did not reduce the affinity of the binding sites, i.e., it behaved as a noncompetitive inhibitor of 31!-imiDramine binding. These data can be regarded as evidence of the heterogeneity of 3H-imipramine binding sites, on the assumption that some of them, with high affinity for pyracetam, are completely blocked *Soviet GABA anal0g (~-pyrrolidone derivatives) Research Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. I01, No. i, pp. 40-42, January, 1986. Original article submitted February 6, 1985. 0007-4888/86/0001-0051512.50 9 1986 Plenum Publishing Corporation 51