Biol. Pharm. Bull. 30(12) 2328—2333 (2007)

choactive drugs are widely used by many people, particularly by the young. These drugs have been synthesized by minor modifications to the chemical structures of existing drugs and are called designer drug. For example, methylenedioxymethamphetamine (MDMA) was derived from amphetamine by altering the natural molecular structure. Although the use of MDMA is prohibited in many countries, new psychoactive chemicals are being constantly developed and can be easily obtained from individuals or via the Internet. The Tokyo Metropolitan Government in Japan enacted an “Ordinance concerning the abuse prevention of the psychoactive drugs” in April 2006 that prohibited the manufacture, cultivation, sales, possession, use, etc., of these drugs. Therefore, we are developing screening methods to rapidly elucidate the effects of these drugs on the central nervous system. Psychoactive drugs are roughly classified into phenethylamine, tryptamine, and piperazine derivatives. Many of the psychoactive drugs modify the monoamine neurotransmission systems including the dopaminergic, serotonergic, and adrenergic neuronal systems. We previously constructed a reproducible, simple, and small-scale in vitro determination method for the reuptake and release of monoamines (dopamine (DA), serotonin (5-HT), and norepinephrine (NE)) using rat brain membranes. This method was then applied to study the effects of designer drugs on monoamine reuptake and release. We have been investigating the effects of 21 designer drugs, some of which are known to be psychoactive in man. For example, 4-fluoroamphetamine (4-FMP), 1-(1,3-benzodioxol-5-yl)-2-butanamine (BDB), 2-methylamino-3,4-methylenedioxy-propiophenone (methylone), and a-methyltryptamine (AMT) showed strong reuptake inhibition and release acceleration of DA, 5-HT, and NE. Although many of drugs described in our previous report (for example, 3,4,5-trimethoxyamphetamine (TMA), 2,4,5trimethoxyamphetamine (TMA-2), and 2,4,6-trimethoxyamphetamine (TMA-6)) reveal no presynaptic effects, it is reported that these drugs elicit hallucinogenic effects in human. Thus, it is also possible that these designer drugs could modify brain activity by directly interacting with monoamine receptors. Many monoamine receptors, including DA, 5-HT, and NE receptors belong to the superfamily of seven-transmembrane-domain, guanine nucleotide-binding protein (G protein)-coupled receptors. G protein-coupled receptors play a crucial role in regulating physiological functions, and consequently are targets for the action of many classes of drugs. Because the [S]guanosine-5 -O-(3thio)-triphosphate ([S]GTPgS) binding assay is thought to be a relatively simple functional assay of receptor-mediated G protein activation, we tried to construct a simple and small-scale method for determining G protein binding using the rat brain membranes.