Agonist selective activation of Mu-opioid receptors

描述（由申请人提供）：对阿片类药物耐受性的发展限制了其治疗疼痛的有效性。各种各样的阿片类药物通常用于临床治疗疼痛和药物成瘾具有显著不同的药理学性质。激动剂之间的一个主要区别是不同的激动剂引起不同数量的阿片受体脱敏和内化的能力。激动剂分为三大类：既能诱导脱敏又能诱导内化的，既能诱导脱敏又不能诱导内化的，以及既能诱导脱敏又不能诱导内化的。脱敏和内化在阿片类药物耐受和依赖发展中的作用一直是一个有争议的主题，已经在各种模型系统中进行了研究。本探索性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mu阿片受体的不同作用机制。这是一种相对较新的方法，它使用光学测量荧光分子在非常小的体积内的迁移率。这些测量是在细胞质膜上进行的，在细胞各部分的细胞内间隔内，包括细胞体、树突、轴突和终末。分子的移动性与分子间的相互作用直接相关，因此可以确定激动剂/受体和受体/效应剂的关联。本研究将在模型系统中使用荧光（aim 1）和荧光标记的mu-阿片受体（aim 2）激动剂，即稳定表达表位标记的mu-阿片受体的HEK293细胞。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导的许多步骤。这一探索性建议将引入一种新的方法来研究阿片样物质激动剂不同药理学特征的机制。在2年的时间里，该方法将使用一种非常有特征的模型HEK293细胞进行开发。这种方法一旦建立，将其应用于初级神经元研究的最终目标将被追求。通过了解受某些激动剂而非其他激动剂选择性影响的过程，可以确定耐受性和依赖性发展的机制，并将其应用于更有效的慢性疼痛和成瘾治疗。

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.