BIOCHEMICAL CHARACTERIZATION OF OPIOID BINDING SITES

多个阿片受体的概念，首次提出近25年 以前，已经变得越来越重要。现在有证据表明， Mu和kappa受体都有。药理学和约束性方法 提示MU1和MU2受体的存在。MU2站点对应 与豚鼠中发现的经典的吗啡选择性Mu受体结合 猪回肠生物测定。除了它们结合特性的不同之外， MU1和MU2的作用在药理学上很容易区分。虽然 这两种亚型都有镇痛作用，MU1受体起作用于冈上，而MU2受体起作用 受体在脊髓水平起作用。此外，MU2受体 介导吗啡的呼吸抑制及对其的抑制 胃肠道转运。最近的研究表明，多个卡帕 子类型也一样。Kappa1子类型由其对 激动剂U50,488H和U69,593以及拮抗剂Nor-binaltorphimine。 纳洛酮苯甲酰肼(NalBzoH)的实验室研究 鉴定了一个新的位点(Kappa3)，其结合谱不同于任何 先前描述的受体。在小牛、大鼠和小鼠脑中的密度 比Mu或Delta受体高2倍，kappa3受体是 中枢神经系统中占优势的阿片受体。药理上，kappa3 受体通过一种脊髓上机制引起镇痛，这种机制很容易实现 分离出Mu、Delta和kappa1受体机制。其他内容 现在的研究表明kappa3受体对应于“N”，或 纳洛啡，受体最先由马丁在他的“受体”概念中提出 表达功能活性的人神经母细胞瘤细胞系 现在已经确定了Mu和kappa3受体。吗啡(IC50 0.2微米) 和NalBzoH(IC_(50)2微米)均抑制Forsklin刺激的环化酶80% 分别通过不同的Mu和kappa3受体机制。在 目前，我们建议继续我们对MU1、MU2和MU2的研究 该细胞系中的kappa3亚型。研究将描述这种结合的特征 亚型及其在功能分析中的作用，特别是 Forsklin刺激的环化酶。我们将通过实验来研究 慢性激动剂和拮抗剂暴露对功能和受体周转的影响。 我们的第二个目标是利用选择性的MU1、MU2和kappa3检测 我们实验室开发的用于检测大脑中阿片类药物结合的药物。装订 水平和选择性将在不同的物种中进行检查。这个 Kappa3站点的发育外观将与其他站点进行比较 我们将研究亚型及其地区分布。 放射自显影。最后，我们将扩大我们的研究范围，利用 我们实验室合成的亲和标记来研究结合 分子水平上的异质性。通过对阿片类药物的理解 受体的多样性我们希望更好地使用目前的止痛药 可以利用，也许可以用来开发新的小说设计方法， 没有吗啡及其副作用的创新药物 类比。

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.