TRANSDUCTION MECHANISMS OF OPIOID AGONIST EFFICACY

阿片类激动剂功效的转导机制

• 批准号: 6174848
• 负责人: DANA E SELLEY
• 金额: $ 10.34万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-05 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6174848
• 关键词: G protein; biological signal transduction; drug screening /evaluation; guanine nucleotide exchange factors; neoplastic cell culture for noncancer research; northern blottings; opioid receptor; pertussis toxin; receptor binding; receptor sensitivity; stimulant /agonist; tissue /cell culture; transfection

DESCRIPTION: (Applicant's Abstract) Opioid analgesic drugs, including heroin and morphine, form a major class of drugs with potential for abuse. Different opioid agonists exhibit different efficacies in various biological systems, and agonist efficacy may not only determine the response produced by the drugs, but also contribute to the development of tolerance to and dependence upon the drug. The proposed project will examine the signal transduction mechanisms underlying agonist efficacy and intrinsic efficacy at mu and delta opioid receptors in two well-established cell culture model systems: mMOR-CHO cells that have been transfected with the mouse mu opioid receptor, SK-N-SH neuroblastoma cells, which contain primarily, mu receptors, and NG108-15 neuroblastoma x glioma cells which contain only delta opioid receptors. Agonist-stimulated [35S]-GTPgS binding, and agonist displacement of [3H]antagonist binding, will be used to measure agonist efficacy and intrinsic efficacy. Efficacy will be defined as maximal stimulation of [35S]GTPgS binding and intrinsic efficacy as both the maximal stimulation and the ratio of Ki in competition binding assays to ED50 in [35S]GTPgS assays. Scatchard analysis of agonist-stimulated [35S]GTPgS binding will be determined whether agonists of different efficacies change the affinity or number of activated G-proteins. Catalytic amplification factors will be calculated by comparing the receptor Bmax to the agonist-stimulated GTPgS binding Bmax, and the role of GDP and sodium in regulating agonist efficacy will be examined. The second phase of the project will examine the relationship between drug efficacy and receptor desensitization and downregulation after chronic drug treatment of cells. In these experiments, cells will be chronically treated with agonists of different efficacies to determine how much the response is decreased, to what extent cross-desensitization to the response produced by drugs of different efficacies develops, and the signal transduction mechanisms underlying the loss in agonist efficacy after chronic drug treatment. In the last phase of the proposed project, the contribution of receptor:transducer ratio and specific transducer reserve to agonist efficacy will be addressed using irreversible antagonists, pertussis toxin, and molecular transfection. A cDNA encoding a cloned Gia2 subunit will be transfected into cells to determine the contribution of specific transducer reserve to opioid agonist efficacy. Thus, the studies in this project are aimed at a mechanistic understanding of how opioid agonist efficacy is produced, and how desensitization effects agonist efficacy, at the level of the receptor-transducer interaction.

描述：（申请人摘要） 阿片类镇痛药物，包括海洛因和吗啡，形成了一个主要的类别， 有可能被滥用的药物。 不同的阿片激动剂表现出不同的 在各种生物系统中的功效，并且激动剂功效不仅可以 决定药物产生的反应，但也有助于 对药物的耐受性和依赖性的发展。 拟议 该项目将研究激动剂的信号转导机制 在两种情况下，对μ和δ阿片样物质受体的功效和内在功效 完善的细胞培养模型系统：已被 转染小鼠μ阿片受体，SK-N-SH神经母细胞瘤细胞， 主要含有μ受体，NG 108 -15神经母细胞瘤×神经胶质瘤 仅含有δ阿片受体的细胞。 激动剂刺激的 [35 S]-GTPgS结合和[3 H]拮抗剂结合的激动剂置换， 将用于测量激动剂功效和内在功效。 疗效 将被定义为[35 S]GTPgS结合的最大刺激和内源性 最大刺激和竞争中Ki的比值 在[35 S]GTPgS测定中结合测定ED 50。 Scatchard分析 激动剂刺激的[35 S]GTPgS结合将确定激动剂刺激的[35 S]GTPgS结合是否为 不同的功效改变了活化的G蛋白的亲和力或数量。 催化放大因子将通过比较受体 Bmax与激动剂刺激的GTPgS结合Bmax的关系，以及GDP和 将检查钠在调节激动剂功效中的作用。 第二阶段 该项目将研究药物疗效与受体之间的关系， 在慢性药物处理细胞后的脱敏和下调。 在这些实验中，细胞将用以下激动剂长期处理： 不同的功效，以确定反应降低了多少， 对药物产生的反应的交叉脱敏程度 产生不同的功效，信号传导机制 这是慢性药物治疗后激动剂功效丧失的根本原因。 在 项目的最后一个阶段， 受体：换能器比和对激动剂的特异性换能器储备 使用不可逆拮抗剂，百日咳毒素， 和分子转染。 编码克隆的Gia 2亚基的cDNA将被克隆。 转染到细胞中，以确定特异性换能器的贡献 保留阿片类激动剂功效。 因此，本项目的研究是 旨在从机理上理解阿片类激动剂的疗效如何 产生的，以及脱敏如何影响激动剂的功效，在水平 受体-传感器相互作用。