NEUROCHEMICAL MECHANISM OF NARCOTIC ACTION

在这份建议书中，将有两个独立但相关的项目， 结合其中描述了一些主要的研究活动，在我的 实验室 1)阿片受体的分子表征和 2)确定耐受的神经化学机制 和依赖。 第一个项目将利用mu型阿片类药物 受体，我们最近纯化的同质性，并将 包括a）阿片样物质结合重建和阿片样物质介导的 功能进入膜环境; B）产生单克隆和 受体的多克隆抗体，并使用它们来绘制 受体在脑内的分布，决定了不同受体的作用 受体的结合和功能部分，并用于克隆 受体; c）阿片样物质结合的分子表征 纯化的受体，包括负协同性试验， 激动剂和拮抗剂结合的热力学分析，作用 离子、鸟嘌呤核苷酸和脂类结合的影响， mu受体与其它类型的相互转化;和d）克隆 受体的寡脱氧核苷酸探针的合成，分离 受体mRNA，并插入克隆载体中。 阿片类药物耐受性/依赖性研究将采用NG 108 -15 表现出阿片样物质结合、阿片样物质介导 功能（腺苷酸环化酶的抑制），以及耐受性样 慢性治疗过程中的适应过程。 在以前的工作中， 我们的实验室已经表明，慢性阿片类激动剂治疗诱导 这些细胞中有三种不同的适应过程：1）受体 脱敏或与腺苷酸环化酶解偶联; 2）受体 下调或从细胞表面消失;和3） 停药后腺苷酸环化酶活性增加，或 慢性激动剂的慢性拮抗作用。 我们建议研究 这些过程中的每一个细节，并确定其相关性， 哺乳动物大脑中的耐受性/依赖性。 我们将努力展示 a）减敏是由共价键的改变引起的， 受体; B）在下调期间，受体沿沿着a 类似于其他下调的途径 受体;我们还将确定内化的动力学， 以及启动它的信号;以及c）研究Ca++的参与 和Ca++结合蛋白在腺苷酸环化酶增加中的作用 活动 为了使NG细胞模型更类似于 大脑，我们将诱导他们的分化，并比较慢性 这些细胞与未分化细胞中的细胞的作用。 最后，我们将描述阿片受体下调， 大脑，我们最近报道。

In this proposal, two independent but related projects will be combined which describe some main research activities in my laboratory. 1) Molecular characterization of opioid receptors and 2) determination of the neurochemical mechanisms of tolerance and dependence. The first project will utilize a mu type opioid receptor which we recently purified to homogeneity, and will include a) reconstitution of opioid binding and opioid-mediated function into a membrane environment; b) raising monoclonal and polyclonal antibodies to the receptor, and using them to map the receptor's distribution in brain, determine the role of different portions of the receptor in binding and function, and for cloning the receptor; c) molecular characterization of opioid binding to the purified receptor, including tests of negative cooperativity, thermodynamic analysis of agonist and antagonist binding, effects of ions, guanine nucleotides and lipids on binding, and tests for interconversion of mu receptors to other types; and d) cloning the receptor by synthesis of oligodeoxynucleotide probes, isolation of receptor mRNA, and insertion into a cloning vector. Studies of opioid tolerance/dependence will employ NG108-15 hybrid cells model which exhibit opioid binding, opioid-mediated function (inhibition of adenylate cyclase), and a tolerance-like adaptation process during chronic treatment. In previous work, our lab has shown that chronic opioid agonist treatment induces three distinct adaptation processes in these cells: 1) receptor desensitization, or uncoupling from adenylate cyclase; 2) receptor down-regulation, or disappearance from cell surface; and 3) an increase in adenylate cyclase activity following withdrawal or antagonism of chronic of chronic agonist. We propose to study each of these processes in detail and determine their relevance to tolerance/dependence in mammalian brain. We will try to show that a) desensitization results from a covalent change in the receptor; b) during down-regulation, receptors move along a pathway similar to that traversed by other down-regulated receptors; we will also determine the kinetics of internalization, and the signal initiating it; and c) study the involvement of Ca++ and Ca++-binding proteins in the increase in adenylate cyclase activity. To make the NG cells model more similar to these in brain, we will induce differentiation in them and compare chronic effects of these cells with those in undifferentiated cells. Finally, we will characterize opioid receptor down-regulation in brain, which we have recently reported.