OPIOID RECEPTOR MECHANISMS

阿片受体机制

• 批准号: 3209157
• 负责人: FEDOR MEDZIHRADSKY
• 金额: $ 18.93万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3209157
• 关键词: G protein; Pongidae; adenylate cyclase; astrocytes; biological signal transduction; brain metabolism; cell bank /registry; cell membrane; clone cells; drug addiction; drug addiction antagonist; drug tolerance; embryo /fetus; enzyme inhibitors; enzyme mechanism; fluorescent dye /probe; glia; laboratory rat; liposomes; membrane fusion; membrane lipids; membrane proteins; membrane reconstitution /synthesis; membrane structure; mixed tissue /cell culture; narcotic antagonists; neurons; neuropeptide receptor; neuropharmacology; opioid receptor; phospholipids; radiotracer; receptor coupling; receptor expression; stimulant /agonist; transport proteins

The overall goal of this proposal is to use techniques of high molecular resolution to characterize opioid receptor mechanisms as they occur in the membrane environment of intact neural cells in primary culture. Receptor occupancy and the efficacy of receptor-effector coupling in whole cells will be contrasted to those in isolated membranes from rat and monkey, considering the influence of unperturbed membrane structure, cytosolic factors, and ionic gradients across the plasma membranes. To study the kinetic properties of agonist and antagonist binding, purified GTP- regulatory protein will be incorporated by membrane fusion, and its concentration adjusted with pertussis toxin and alkaline treatment. Receptor-effector coupling will also be assessed following the transfer of opioid receptors from membranes to receptor-devoid astrocytes in primary culture. Fluorescent labeling of the mu-, delta-, and kappa-receptors will be carried out and the tagged receptors used to assess lateral membrane mobility as an essential process in their collision-coupling to the effectors, G-protein and adenylate cyclase. To describe the dependence of ligand-receptor-effector interactions on the chemical composition and/or physicochemical properties (fluidity and hydrophobicity) of the membrane, isolated lipid transfer proteins will be used to systematically alter the composition of neuronal cell membranes. By inducing fluorescent energy transfer between receptor and incorporated phospholipid, the structure of the functionally significant lipid boundary layer around the mu-, delta-, and kappa-receptor will be characterized. In neuronal cells and in neuron-glia co-cultures, chronically modified by specific lipid incorporation, receptor occupancy and coupling to G- protein/adenylate cyclase will be assessed focusing on the role of membrane modulation under conditions of cellular tolerance and dependence to opiates.

该提案的总体目标是使用高分子的技术 分辨率以表征阿片类药物受体机制发生在 原发性培养中完整神经细胞的膜环境。 受体 占用和受体效应偶联全细胞的功效 将与来自大鼠和猴子的孤立膜的人形成鲜明对比， 考虑到不受干扰的膜结构的影响，胞质 质膜的因素和离子梯度。 研究 激动剂和拮抗剂结合的动力学特性，纯化的GTP- 调节蛋白将通过膜融合融合，及其 通过百日咳毒素和碱性处理调节的浓度。 在转移之后，还将评估受体效应器耦合 从膜到原发性的受体 - 脱节星形胶质细胞的阿片类药物受体 文化。 Mu-，delta-和kappa受体的荧光标记 将进行，并用于评估横向的标记受体 膜移动性是其碰撞耦合到与 效应子G蛋白和腺苷酸环化酶。 描述 配体 - 受体效应器相互作用对化学物质的依赖性 组成和/或物理化学特性（流动性和 膜的疏水性），分离的脂质转移蛋白将是 用于系统地改变神经元细胞膜的组成。 通过诱导受体和融合的荧光能量转移 磷脂，功能意义的脂质边界的结构 将表征Mu-，delta-和Kappa受体周围的层。 在神经元细胞和神经元间共培养中，长期修饰 特定的脂质掺入，受体占用率和耦合到g- 将评估蛋白质/腺苷酸环化酶的重点 细胞耐受性和依赖性条件下的膜调节 去鸦片。