OPIOID MODULATION OF IMMUNOCOMPETENCE

这项为期3年的竞争性续签提案的一个主要目标是 分离表达阿片类药物的小鼠淋巴细胞亚群 受体。这个实验室之前的研究表明， 小鼠胸腺瘤细胞株R1.1表达kappa1阿片受体 通过百日咳毒素与腺苷环化酶负偶联- 敏感的G蛋白，表明免疫系统的某些细胞 可以表达阿片受体。将使用两种方法来分离 表达阿片受体的小鼠淋巴细胞亚群。一 该方法包括将14β-溴乙酰胺衍生物偶联 纳曲酮到磁珠，含有硫醇基团。小白鼠 胸腺细胞、脾细胞和浓缩的T细胞群将 与阿片结合的珠子孵育，然后洗涤。细胞， 含有阿片受体，会结合到珠子上，并随使用 磁铁，这些细胞将从缺乏阿片类药物的细胞中分离出来 感受器。亲和配体识别我、Delta和kappa阿片类药物 受体，在分离包含任何类型的细胞时应该是有用的 阿片受体。第二种方法涉及使用高 亲和kappa选择性荧光阿片类药物标记细胞， 通过使用藻红蛋白的扩增程序。这两个都是 已经显示了选择性地为R1.1贴标签的程序。和派生的 胸腺瘤细胞株，表明这些方法将是成功的 在分离表达阿片类药物的小鼠淋巴细胞亚群中 感受器。表型标记将被用来表征纯化的 细胞和受体的性质将通过结合和 第二信使分析。 来自R1.1胸腺瘤的两个商业上可用的细胞系， 比亲本R1.1表达更多数量的kappa阿片受体 细胞。-GTP抑制激动剂结合的效力及其最大值 腺酰环化酶活性的抑制在三种细胞中是不同的 台词。使用特定的抗体，偶联到 将测定三种细胞系中的Kappa阿片受体。 低KM GTP酶活性的阿片类刺激将被用作一种措施 Kappa阿片受体与G蛋白偶联。研究还将 确定备用受体的数量与 Kappa阿片受体的脱敏/下调。 通过确定表达阿片受体的免疫细胞的类型 以及影响淋巴细胞kappa阿片偶联的因素 腺酰环化酶的受体，拟议的研究将导致 更好地了解参与改变的机制 麻醉剂的免疫功能。

A major objective of this 3-year competitive renewal proposal is to isolate a subpopulation of mouse lymphocytes that express an opioid receptor. Previous studies from this laboratory have shown that the mouse R1.1 thymoma cell line expresses a kappa1 opioid receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin- sensitive G protein, suggesting that certain cells of the immune system can express opioid receptors. Two approaches will be used to isolate a subpopulation of mouse lymphocytes that express opioid receptors. One approach involves conjugating a 14beta-bromoacetamido derivative of naltrexone to magnetic beads, containing a thiol group. Mouse thymocytes, splenocytes, and enriched T-cell populations will be incubated with the opioid-conjugated beads, followed by washing. Cells, containing an opioid receptor, will bind to the beads, and with the use of magnet, these cells will be separated from cells that lack opioid receptors. The affinity ligand recognizes me, delta, and kappa opioid receptors, and should be useful in isolating cells that contain any type of opioid receptor. The second approach involves the use of a high affinity kappa-selective fluorescent opioid to label the cells, followed by an amplification procedure using phycoerythrin. Both of these procedures have been shown to selectively label the R1.1. and derivative thymoma cell lines, suggesting that these approaches will be successful in isolating a murine lymphocyte subpopulation that expresses opioid receptors. Phenotypic markers will be used to characterize the purified cells and the receptors properties will be determined by binding and second messenger assays. Two commercially available cell lines, derived from the R1.1 thymoma, express a larger number of kappa opioid receptors than the parent R1.1 cells. The potency of -GTP to inhibition agonist binding and the maximal inhibition of adenylyl cyclase activity varies among the three cell lines. Using specific antibodies, the G proteins that are coupled to the kappa opioid receptor in the three cell lines will b e determined. Opioid stimulation of low Km GTPase activity will b e used as a measure of kappa opioid receptor coupling to G proteins. Studies will also determine the correlation between the number of spare receptors and desensitization/downregulation of the kappa opioid receptor. By determining the types of immune cells that express opioid receptors and the factors involved in the coupling of the lymphocytic kappa opioid receptor to adenylyl cyclase, the proposed studies will result in a better understanding of the mechanisms involved in the alteration of immune function by narcotics.