白鼠腦의 Subcellular 分劃에 對한 D-LSD 및 Serotonin 誘導體들의 통합에 관한 실질적 연구

BINDING OF D-LSD AND SEROTONIN DERIVATIVES TO THE SUBCELLVLLAR FRACTIONS OF THE RAT BRAIN

Although the existence of serotonergic neurons in the brain has been confirmed by both electrophysiologically and the distribution of a ㅣarge amount of serotonin (5-hydroxy tryptamine) in the brain , the ultimate neurochemical approach lies in the identification ofthe serotonin rebeptor to which serotonin, its agonists and antagonists bind in a very specific manner. The criteria for the specific binding of serotonin derivatives to the receptor are : (1) the binding must be saturable ; (2) stereospecific ； (3) of high-affinity and ; (4) the displacement of the serotonin binding by other serotonergic ligands should reflect the physiological and pharmacological Potencies of these drugs. Serveral laboratories including ours have identified the serotonin receptor by use of tritiated lysergic acid diethylamide (L SD ). The general procedure consists of (1) incubation of H3-LSD with subellular fragments of different degree of purities, (2) filtra tio n of the incubates on a glase filte r G-F/B, and counting of the radioactivity of the bound LSD in a scintillation counter. The displacement study, of the LSD binding by various ligands involves incubation of LSD and subcellular fragments with adding the ligands of different concentration. The results show that LSD binds to various subcellular fractions with a dissociation constant (Kd) of 11.0xl0~9M. The binding is saturable and stereospecific in the sense that 1-LSD, a physiologically inactive optical isomer of d-LSD， does not interfere the specific binding. The max imum specific binding is observed with a disrupted P4 microsomal membrane fraction. Binding study using different fractions of the rat brain show that the dissociation constant does not vary from fraction to fraction. However, there is a great variation in the maximum specific binding. Maximum specific binding of the crude homogenate is only 0.27 picom이/ mg protein, whereas in the case of the vigorously dsurupted P4 membrane fraction the m aximum sipecific binding is 2.3 picomole/mg protein. The specific binding can be displaced by the various tryptam ine derivatives. The DC50, the concentration at 50% displacement, of try ptamine derivatives has the following order ： 5-0H = 5-MeO> 4-OH> 5-CH3> F > 5,6-(OH)2> 5-F>7-0 H4-NH2〉7-OH>6-ᄋH〉7-MeO. This order is in agreement w ith the potencies of these drugs in contracting the smooth muscle investigated by John Vane. Amphetamine and mescaline derivatives are usually 100 times less potent than the tryptamine derivatives. Likewise, the binding of d-LSD does not seem to be associated with the hallucinogenic activity of this drug, because 2-Bromo-LSD, a non-hallucinogen, is as potentas d-LSD in the displacement of the d-LSD binding. Another serotonin antagonist cyproheptadine, but a nonhallucinogen, shows asimilar displacement. It appears that the serotonin receptor, so identified, is not a receptor which is responsible for the hallucinogenic activity. The membrane destabilizing factors such as hypoosmotic shock, treatment with phospholipase Cor EGTA, increase the m axim um LSD binding. Treatment with trypsin or protease decreases greatly the specific binding demonstrating that the serotonin receptor is a protein imbedded in a phospholipid matrix of the synaptic membranes