NEUROCHEMICAL MECHANISM OF NARCOTIC ACTION

在这项提案中，两个独立但相关的项目将是 其中结合描述了我的一些主要研究活动 实验室。1)阿片受体的分子特征和 2)耐受神经化学机制的确定 和依赖。第一个项目将使用一种MU型阿片类药物 我们最近将受体纯化成均一种，并将 包括：a)阿片结合和阿片介导重建 功能进入膜环境；b)培养单克隆性和 该受体的多克隆抗体，并使用它们来映射 受体在大脑中的分布，决定了不同的作用 部分受体的结合和功能，以及用于克隆 受体；c)阿片类药物结合的分子特征 纯化的受体，包括负协作性测试， 激动剂和拮抗剂结合作用的热力学分析 离子、鸟嘌呤核苷酸和脂类的结合和试验 将Mu受体相互转化为其他类型；以及d)克隆 通过合成寡脱氧核苷酸探针来分离受体 受体mRNA，并插入克隆载体。 阿片耐受/依赖的研究将采用NG108-15 阿片类药物介导的阿片结合的杂交细胞模型 功能(抑制腺苷环化酶)和一种耐受性样 慢性治疗过程中的适应过程。在以前的工作中， 我们的实验室表明，慢性阿片类激动剂治疗会诱导 这些细胞有三种截然不同的适应过程：1)受体 脱敏，或与腺苷环化酶解偶联；2)受体 下调，或从细胞表面消失；3)一个 戒断或停药后腺苷环化酶活性升高 慢性激动剂的慢性拮抗作用。我们建议研究 详细介绍每个流程，并确定它们与 哺乳动物大脑中的耐受/依赖。我们会努力证明 A)脱敏是由于 受体；b)在下调期间，受体沿 类似于下调的其他基因所经过的通路 受体；我们还将确定内化的动力学， 以及启动它的信号；c)研究Ca++的参与 和钙结合蛋白在腺苷环化酶中的升高 活动。要使NG细胞模型更类似于 大脑，我们将诱导它们分化，并比较慢性 这些细胞与未分化细胞的作用。 最后，我们将表征阿片受体下调在 大脑，这是我们最近报道的。

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.