BIOCHEMICAL CHARACTERIZATION OF OPIOID BINDING SITES

阿片类药物结合位点的生化特征

• 批准号: 2116613
• 负责人: GAVRIL W PASTERNAK
• 金额: $ 29.39万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2116613
• 关键词: affinity labeling; analgesics; autoradiography; brain mapping; chemical structure function; cyclic AMP; drug addiction antagonist; drug metabolism; drug tolerance; forskolin; genetic strain; hydrazones; laboratory mouse; laboratory rat; monoclonal antibody; morphine; nalorphine; naloxone; neuroblastoma; opiate alkaloid; opioid receptor; radiotracer; receptor binding; stimulant /agonist; tissue /cell culture

The concept of multiple opioid receptors, first proposed almost 25 years ago, has become increasingly important. Evidence now suggests subtypes of both mu and kappa receptors. Pharmacological and binding approaches suggest that existence of mu1 and mu2 receptors. The mu2 site corresponds to the classical morphine-selective mu receptor identified in the guinea pig ileum bioassay. In addition to differences in their binding profiles, mu1 and mu2 actions are easily differentiated pharmacologically. Although both subtypes elicit analgesia, mu1 receptors act supraspinally while mu2 receptors act at the level of spinal cord. Furthermore, mu2 receptors mediate morphine's respiratory depression and inhibition of gastrointestinal transit. More recent work has suggested multiple kappa subtypes as well. The kappa1 subtype is defined by its selectivity for the agonists U50,488H and U69,593 and the antagonist nor-binaltorphimine. Studies from our laboratory with naloxone benzoylhydrazone (NalBzoH) identified a novel site (kappa3) with a binding profile unlike any previously described receptor. With a density in calf, rat and mouse brain 2-fold higher than either mu or delta receptors, kappa3 receptors are the predominant opioid receptor in the CNS. Pharmacologically, kappa3 receptors elicit analgesia through a supraspinal mechanism which is easily separated from mu, delta and kappa1 receptor mechanisms. Additional studies now suggest that kappa3 receptors correspond to the "N", or nalorphine, receptor first proposed by Martin in his concept of "Receptor Dualism". A human neuroblastoma cell line expressing functionally active mu and kappa3 receptors has now been identified. Morphine (IC50 0.2 muM) and NalBzoH (IC50 2 muM) both inhibited forskolin-stimulated cyclase by 80% through discrete mu and kappa3 receptor mechanisms, respectively. In the present application, we propose to continue our studies of mu1, mu2 and kappa3 subtypes in this cell line. Studies will characterize the binding of the subtypes and their actions in function assays, specifically forskolin-stimulated cyclase. Experiments will investigate the effects of chronic agonist and antagonist exposure on function and receptor turnover. Our second aim is to utilize the selective mu1, mu2 and kappa3 assays developed in our laboratory to examine opioid binding in brain. Binding levels and selectivities will be examined in various species. The developmental appearance of kappa3 site will be compared to the other subtypes and its regional distributions will be examined autoradiographically. Finally, we will extend our studies utilizing affinity labels synthesized in our laboratory to investigate binding heterogeneity at the molecular level. Through an understanding of opioid receptor multiplicity we hope to better use the analgesics currently available and perhaps to develop new approaches to the design of novel, innovative drugs lacking the problematic side-effects of morphine and its analogs.

多种阿片受体的概念，首次提出近25年 现在，它变得越来越重要。 现在有证据表明 μ和κ受体。 药理学和结合方法 表明存在μ 1和μ 2受体。 mu2位点对应于 与在几内亚发现的经典吗啡选择性μ受体 猪回肠生物测定。 除了它们结合特征的差异外， mu1和mu2动作很容易区分。 虽然 这两种亚型都能引起镇痛作用，mu1受体作用于脊髓上，而mu2 受体在脊髓水平起作用。 此外，μ 2受体 介导吗啡的呼吸抑制和 胃肠转运 最近的研究表明， 子类型也是。 kappa1亚型是由其对 激动剂U50，488H和U69，593和拮抗剂nor-binaltorphimine。 来自我们实验室的纳洛酮苯甲酰腙（NalBzoH）研究 确定了一个新的网站（kappa3）的结合概况不同于任何 先前描述的受体。 在小牛、大鼠和小鼠的大脑中有密度 2-kappa3受体比mu或delta受体高1倍， 中枢神经系统中的主要阿片受体。 药理学，kappa3 受体通过脊髓上机制引起镇痛， 与μ、δ和κ 1受体机制分开。 额外 现在的研究表明，kappa3受体对应于"N"，或 纳洛啡，由Martin在他的"受体"概念中首次提出 二元论"。 表达功能活性的人神经母细胞瘤细胞系 现在已经鉴定了μ和κ 3受体。 吗啡（IC50 0.2 μ M） 和NalBzoH（IC 50 2 μ M）均抑制毛喉素刺激的环化酶80% 分别通过离散的μ和κ 3受体机制。 在 在本申请中，我们建议继续我们对μ 1，μ 2和μ 3的研究。 kappa3亚型在该细胞系中。 研究将描述 亚型及其在功能测定中的作用，特别是 毛喉素刺激的环化酶。 实验将调查的影响， 慢性激动剂和拮抗剂暴露对功能和受体转换的影响。 我们的第二个目标是利用选择性mu1、mu2和kappa3测定法 在我们的实验室开发，以检查阿片类药物在大脑中的结合。 结合 水平和选择性将在各种物种中进行检查。 的 将kappa3位点的发育外观与其他位点进行比较 亚型及其区域分布将被检查 放射自显影。 最后，我们将扩大我们的研究利用 在我们的实验室合成的亲和标记，以研究结合 分子水平上的异质性。 通过了解阿片类药物 受体多样性，我们希望更好地利用目前的镇痛药， 也许可以开发新的方法来设计新颖的， 创新药物没有吗啡的问题副作用， 类似物