TRANSDUCTION MECHANISMS OF OPIOID AGONIST EFFICACY

描述：(申请人摘要) 阿片类止痛药，包括海洛因和吗啡，构成了一大类 有可能被滥用的药物。不同的阿片类激动剂表现不同 在各种生物系统中的疗效，以及激动剂的疗效不仅可以 确定药物产生的反应，也有助于 对药物的耐受性和依赖性的发展。建议数 该项目将研究激动剂潜在的信号转导机制 Mu和Delta阿片受体的疗效和内在疗效 成熟的细胞培养模型系统：mmor-CHO细胞 转染鼠MU阿片受体的SK-N-SH神经母细胞瘤细胞， 主要含有Mu受体和NG108-15神经母细胞瘤x神经胶质瘤 只含有阿片受体的细胞。激动剂刺激性 [35S]-GTPgS结合和[~3H]拮抗剂结合的激动剂置换， 将被用来衡量激动剂的疗效和内在疗效。功效 将被定义为最大限度地刺激[35S]GTPgS结合和本征 效率既是最大的刺激，也是竞争中的KI比率 [35S]GTPgS分析中与ED50结合的分析。Scatchard分析 激动剂刺激的[35S]GTPgS结合将确定激动剂是否 不同的功效会改变激活的G蛋白的亲和力或数量。 催化放大系数将通过比较受体来计算 Bmax到激动剂刺激的GTPgS结合Bmax，以及GDP和Bmax的作用 将考察钠在调节激动剂中的作用效果。第二阶段 该项目将研究药物疗效和受体之间的关系。 细胞慢性药物治疗后的脱敏和下调。 在这些实验中，细胞将被长期使用激动剂处理 不同的效率来确定反应减少了多少， 对药物的反应有多大程度的交叉脱敏 不同的药效发展和信号转导机制 慢性药物治疗后激动剂疗效丧失的根本原因。在……里面 拟议项目的最后阶段， 受体：转导比和特异性转导对激动剂的储备 疗效将使用不可逆转的拮抗剂百日咳毒素， 分子转染法。编码克隆的Gia2亚单位的cDNA会是 转染细胞以确定特异性转导分子的作用 保留阿片类激动剂的疗效。因此，本项目的研究内容包括 旨在从机制上理解阿片类激动剂的疗效 产生，以及脱敏如何影响激动剂的疗效，在 受体-传导器的相互作用。

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.