BENZODIAZEPINE RECEPTOR REGULATION AND PURIFICATION

这项建议的目的是研究黄曲霉毒素的分子机制。 苯二氮类药物的作用及其调节机制 受体；纯化和鉴定BZ受体和内源性 调节器；并量化受体占有率之间的关系 和药理作用。我们早期关于结合的动力学研究 BZS对大脑皮层的作用为BZ受体提供了第一个证据 以两种可相互转换的构象存在。我们会进一步跟进 结合动力学，在各种已知影响BZ的条件下 结合，在不同的大脑区域使用三类放射性标记 配体(即BZ激动剂和拮抗剂以及Beta-Caroline 惊厥药)。对结合动力学的详细分析可能会产生信息 不适用于均衡约束性测量。补肾益气汤对小鼠心脏功能的影响 新型“温和剂”制备的突触体膜与GABA的结合 方法(即，用两种固有的细胞酶处理膜， GABA转氨酶和琥珀酸半醛脱氢酶，以去除 内源GABA)，将进行研究。我们将探讨这一关系，并 通过研究BZ与GABA受体的结合研究BZ与GABA受体的相互调节 已知慢性BZ治疗后不同脑区GABA的变化 导致BZ受体下调。慢性治疗的效果 还将研究与BZ拮抗剂对BZ与GABA结合的影响。 用不可逆的BZ研究BZ受体的周转率 配体、蛋白质合成抑制剂和轴浆流动抑制剂。 联合应用BZS及其受体测定BZ受体的占位率 相应的(~3H)BZS将与它们的抗惊厥药物和 反冲突活动。这样的研究也可能揭示出备用的存在 感受器。BZ受体和内源性调节剂将与 亲和纯化的非离子洗涤剂正辛基葡萄糖苷 色谱学、高效液相色谱仪及其他 分离技术及其生化和物理性质 特色化的。了解补肾益气汤的作用机制， 对BZ受体的调节及其特性将有助于我们理解 这类重要的药物以及焦虑和焦虑的潜在机制 癫痫发作障碍。

The objectives of this proposal are to study the molecular mechanisms of action of benzodiazepines (BZs) and mechanisms of regulation of BZ receptors; to purify and characterize BZ receptors and endogenous modulator; and to quantitate the relationship between receptor occupancy and pharmacological effects. Our earlier kinetic studies on the binding of BZs to cerebral cortices provided the first evidence that BZ receptors exist as two interconvertible conformations. We will further pursue the binding kinetics, under a variety of conditions that are known to affect BZ binding, in different brain regions using three classes of radiolabeled ligands (i.e., BZ agonists and antagonists, and Beta-carboline convulsants). Detailed analysis of binding kinetics may yield information not available from equilibrium binding measurements. The effects of BZs on the binding of GABA to a synaptosomal membrane prepared by a novel "gentle" method (i.e., treating the membrane with two intrinsic cellular enzymes, GABA transaminase and succinic semialdehyde dehydrogenase, to remove endogenous GABA), will be studied. We will explore the relationship and inter-regulation between BZ and GABA receptors by studying the binding of GABA to different brain regions after chronic BZ treatment which is known to cause BZ receptor down-regulation. The effects of chronic treatment with BZ antagonists on the binding of BZ and GABA will also be studied. The turnover rate of BZ receptors will be studied using irreversible BZ ligands, protein synthesis inhibitors, and an axoplasmic flow inhibitor. The occupancy of BZ receptors measured by co-administered BZs and their corresponding (3H)BZs will be correlated with their anticonvulsant and anticonflict activity. Such study may also reveal the presence of spare receptors. BZ receptors and endogenous modulator will be solubilized with the nonionic detergent, n-octyl glucoside, purified with affinity chromatography, high performance liquid chromatography (HPLC) and other separation techniques, and their biochemical and physical properties characterized. Understanding the mechanisms of action of BZs, the regulation of BZ receptors, and their properties will help us understand this important class of drugs and the underlying mechanisms of anxiety and seizure disorders.