Agonist selective activation of Mu-opioid receptors

描述(申请人提供)：对阿片类药物的耐受性的形成限制了它们治疗疼痛的有效性。临床上常用于治疗疼痛和药物成瘾的阿片类药物具有截然不同的药理特性。激动剂之间的一个主要区别是不同的激动剂导致不同量的u阿片受体脱敏和内化的能力。激动剂分为三大类，既能诱导脱敏又能内化的，能诱导脱敏但不能内化的，以及对两者都无效的。脱敏和内化在阿片类药物耐受和依赖的形成中所起的作用一直是一个有争议的课题，已经在各种模型系统中进行了研究。这一探索性的建议将发展荧光相关光谱的方法来研究不同阿片激动剂对u阿片受体不同作用的机制。这是一种相对较新的方法，它使用光学方法在非常小的体积内测量荧光分子的迁移率。这些测量是在细胞膜上进行的，在细胞不同部分的细胞内隔室内进行，包括细胞体、树突、轴突和终末。分子的流动性与分子间的相互作用直接相关，从而识别激动剂/受体和受体/效应器之间的相互作用。这项研究将在一个模型系统中使用荧光(Aim 1)和荧光中心标记的u-阿片受体(Aim 2)的激动剂，HEK293细胞稳定表达表位标记的u-阿片受体。电生理、生化和成像方法已被用于表征阿片受体依赖信号传递的许多步骤。这一探索性建议将引入一种新的方法来研究阿片类激动剂不同药理特征背后的机制。在这项提议提出的两年内，这种方法将使用一个非常有特点的模型-HEK293细胞来开发。一旦建立了这种方法，就会追求将其应用于初级神经元研究的最终目标。通过了解某些激动剂而不是其他激动剂选择性影响的过程，可以确定耐受和依赖发展的潜在机制，并将其应用于更有效地治疗慢性疼痛和成瘾。

DESCRIPTION (provided by applicant): The development of tolerance to opioids limits their effectiveness for the treatment of pain. A wide variety of opioids that are commonly used in the clinic for the treatment of pain and drug addiction have dramatically different pharmacological properties. One primary difference between agonists is the ability of different agonist to cause varying amounts of desensitization and internalization of mu opioid receptors. Agonists fall into three major groups, those that induce both desensitization and internalization, those that induce desensitization but not internalization and those that are ineffective at both. The role that desensitization and internalization have in the development to tolerance and dependence to opioids has been a controversial subject that has been studied in a variety of model systems. This exploratory proposal will develop the method of Flurescence Correlation Spectroscopy to investigate the mechanism that accounts for the varying actions of different opioid agonists on the mu opioid receptor. This is a relatively new method that uses optical measurements of the mobility of fluorescent molecules within a very small volume. These measurements are made at the plasma membrane, within intracellular compartments in various parts of the cell, including the cell body, dendrites, axons and terminals. The mobility of molecular is directly related to the molecular interactions such that agonist/receptor and receptor/effector associations will be identified. This study will use agonists that are fluorescent (aim 1) and fluorencescently tagged mu-opioid receptors (aim 2) in a model system, HEK293 cells that stably express epitope-tagged mu-opioid receptors. Electrophysiological, biochemical and imaging methods have been used to characterize many of the steps in opioid-receptor dependent signaling. This exploratory proposal will introduce a new way to investigate the mechanisms that underlie the different pharmacological profiles of opioid agonists. In the 2 years afforded this proposal, this method will be developed using a very well characterized model, the HEK293 cells. Once this method is established, the ultimate goal of applying it to study of primary neurons will be pursued. By gaining knowledge of the processes that are selectively affected by some agonists and not others, the mechanisms underlying the development of tolerance and dependence may be identified and applied to more effective treatment of chronic pain and addiction.