FLUORESCENT LIGANDS FOR OPIOID RECEPTORS

申请人摘要：这项提案的主要目标是发展 检测阿片类药物的高灵敏荧光方法 免疫系统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 基于藻红蛋白的扩增程序，特异性结合 小鼠胸腺瘤R1.1细胞表面kappa受体的荧光阿片受体 用荧光显微镜可以检测到品系和小鼠的胸腺细胞和 通过荧光激活的细胞分选(FACS)。此应用程序将 将这些初步观察推广到合成和 新型荧光素标记的特异性阿片类药物的表征 用于Mu、Delta和kappa受体。Mu，Delta， 既是激动剂又是拮抗剂的kappa受体将是 通过在分子中掺入荧光素基团合成 莱兰德。由于许多选择性配体是小分子多肽， 荧光素将与适当的体内氨基酸偶联。两者都有 将准备激动剂和拮抗剂，因为有可能 激动剂与免疫细胞的结合将会减弱，因为 实验将在生理盐水中进行。另一方面， 不应因此而削弱拮抗剂的结合能力。 中号的。荧光素标记的化合物将被测试其 豚鼠脑内阿片受体的亲和力和选择性 膜。这三种配体的KI值将被确定 阿片受体的主要类型。神经母细胞瘤和淋巴瘤细胞系， 表达Mu、Delta或kappa受体的人将用特定的 荧光标记化合物。标记的单元格将由这两个人查看 荧光显微镜和流式细胞仪。对方法进行优化后 荧光配体将用于分离的细胞系 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Mu、Delta和kappa受体 淋巴细胞亚群。该方法也将应用于 原代培养神经元细胞以确定神经元是否表达 可以检测到一种特定类型的阿片受体。最后一种方法是 将被应用于脑片以确定组织化学 Mu、Delta和kappa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.