FLUORESCENT LIGANDS FOR OPIOID RECEPTORS

申请人摘要：本提案的主要目标是开发 用于检测阿片样物质的高灵敏度荧光方法 免疫系统的神经元和细胞上的受体。初步研究 已经表明，通过使用荧光素标记的阿片类药物， 基于藻蓝蛋白的扩增程序， 小鼠胸腺瘤R1.1细胞上荧光阿片受体的研究 系和小鼠胸腺细胞可通过荧光显微镜检测， 通过荧光激活细胞分选（FACS）。此应用程序将 将这些初步意见扩展到综合报告， 特异性的新荧光素标记的阿片样物质的表征 μ δ和κ受体。μ，δ， 激动剂和拮抗剂的κ受体将被 通过将荧光素基团引入到荧光素的分子中而合成。 配体。由于许多选择性配体是小多肽， 荧光素将与适当的内部氨基酸偶联。两 激动剂和拮抗剂将被制备， 激动剂与免疫细胞的结合将减少， 实验将在盐水溶液中进行。另一方面，在一项研究中， 拮抗剂的结合能力不应降低 一个灵媒。将测试黄绿素标记的化合物的 豚鼠脑阿片受体的亲和力和选择性 膜。将测定这三种配体的Ki值 阿片受体的主要类型。神经母细胞瘤和淋巴瘤细胞系， 表达μ、δ或κ受体的细胞将用特异性抗体进行测试。 荧光标记化合物。标记的细胞将由两个 荧光显微镜和流式细胞仪。一旦方法被优化， 细胞系荧光配体将与分离自 免疫系统，如胸腺细胞和脾细胞。这些研究 将显示是否可以检测到μ，δ和κ受体， 淋巴细胞亚群。该方法还将应用于 原代神经元细胞培养，以确定表达 可以检测特定类型的阿片受体。最后，方法 将应用于脑切片，以确定组织化学 μ、δ和κ受体的定位。

APPLICANT'S ABSTRACT: The major objective of this proposal is to develop highly sensitive fluorescent methods for the detection of opioid receptors on neurons and cells of the immune system. Preliminary studies have shown that by using a fluorescein-labelled opioid followed by a phycoerythrin-based amplification procedure, specific binding of the fluorescent opioid to kappa receptors on the mouse thymoma R1.1 cell line and mouse thymocytes can be detected by fluorescent microscopy and by fluorescent-activated cell sorting (FACS). This application will extend these preliminary observations to the synthesis and characterization of new fluorescein-labelled opioids that are specific for mu, delta, and kappa receptors. Selective ligands for mu, delta, and kappa receptors that are both agonists and antagonists will be synthesized by incorporating a fluorescein group in the molecule of the ligand. Since many of the selective ligands are small polypeptides, the fluorescein will be coupled to an appropriate internal amino acid. Both agonists and antagonists will be prepared since it is possible that the binding of the agonists to immune cells will be diminished since the experiments will be performed in a saline solution. On the other hand, the binding capacity of the antagonists should not be diminished in such a medium. Fluorescein labelled compounds will be tested for their affinity and selectivity for the opioid receptors using guinea pig brain membranes. Ki values for the ligands will be determined for these three major types of opioid receptors. Neuroblastoma and lymphoma cell lines, expressing mu, delta, or kappa receptors will be tested with specific fluorescent-labelled compounds. Labelled cells will be viewed by both fluorescence microscopy and FACS. Once methods have been optimized with cell lines the fluorescent ligands will be used with cells isolated from the immune system, such as thymocytes and splenocytes. These studies will show whether mu, delta, and kappa receptors can be detected on subpopulations of lymphocytes. The method will also be applied to primary neuronal cell cultures to determine whether neurons that express a specific type of opioid receptor can be detected. Finally the method will be applied to brain slices to determine histochemically the localization of mu, delta, and kappa receptors.