Mehta A K, Ticku M K
Department of Pharmacology, University of Texas Health Science Center, San Antonio 78284-7764.
J Neurochem. 1987 Nov;49(5):1491-7. doi: 10.1111/j.1471-4159.1987.tb01019.x.
The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.
在完整的原代培养脊髓神经元中研究了[3H]氟硝西泮的相互作用及其受各种药物的调节作用。在完整细胞中，[3H]氟硝西泮的结合迅速且具有饱和性。苯二氮䓬结合位点表现出高亲和力和饱和性，其表观解离常数KD为6.1±1.6 nM，最大结合容量Bmax为822±194 fmol/mg蛋白质。[3H]氟硝西泮结合的结合和解离呈现单指数动力学。特异性结合的[3H]氟硝西泮以浓度依赖的方式被氟硝西泮、氯硝西泮、地西泮、Ro 15-1788等苯二氮䓬类药物以及甲基-6,7-二甲氧基-4-乙基-β-咔啉-3'-羧酸酯等β-咔啉类药物取代。γ-氨基丁酸（GABA）激动剂以及促进GABA能传递的药物如乙唑酯、（+）依托咪酯和戊巴比妥以浓度依赖的方式增强了[3H]氟硝西泮与完整细胞的特异性结合。GABA激动剂的增强作用被荷包牡丹碱和印防己毒素拮抗。这些结果表明完整的培养脊髓神经元表现出苯二氮䓬-GABA受体-离子通道复合物的特性。由于这些细胞也可用于并行研究GABA诱导的36Cl内流的特性，因此它们为研究GABA突触药理学提供了理想的体外分析制备方法。
