Agonist selective activation of Mu-opioid receptors

描述（由申请人提供）：阿片类药物耐受性的发展限制了其治疗疼痛的有效性。 临床上常用于治疗疼痛和药物成瘾的各种阿片类药物具有显著不同的药理学特性。激动剂之间的一个主要差异是不同激动剂引起μ阿片受体的不同量的脱敏和内化的能力。 激动剂分为三大类：既诱导脱敏又诱导内化的激动剂，诱导脱敏但不诱导内化的激动剂，以及对脱敏和内化均无效的激动剂。脱敏和内化在阿片类药物耐受性和依赖性发展中的作用一直是一个有争议的主题，已在各种模型系统中进行了研究。 该探索性建议将开发荧光相关光谱法，以研究不同阿片激动剂对μ阿片受体的不同作用的机制。这是一种相对较新的方法，它使用非常小体积内荧光分子迁移率的光学测量。这些测量是在质膜上进行的，在细胞的各个部分的细胞内区室中，包括细胞体、树突、轴突和终末。 分子的迁移率与分子相互作用直接相关，因此将鉴定激动剂/受体和受体/效应物缔合。 本研究将在模型系统中使用荧光（aim 1）和荧光标记的μ阿片受体（aim 2）激动剂，HEK 293细胞稳定表达表位标记的μ阿片受体。 电生理学、生物化学和成像方法已被用于表征阿片受体依赖性信号传导中的许多步骤。 这一探索性的建议将引入一种新的方法来研究阿片类激动剂不同药理学特征的机制。 在提供该提案的2年内，将使用非常好表征的模型HEK 293细胞开发该方法。 一旦这种方法建立起来，将其应用于初级神经元研究的最终目标将是追求。通过了解某些激动剂选择性影响而非其他激动剂影响的过程，可以确定耐受性和依赖性发展的潜在机制，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.