Desensitization and Internalization of Mu-Opioid Receptors in the Locus Coeruleus

说明(申请人提供)：阿片类药物是广泛使用的镇痛剂；然而，长期使用会导致耐受性，对药物的反应性逐渐下降。U-阿片受体(MOR)的脱敏和内化在阿片类药物的作用中很重要，并且似乎有助于耐受的发展。脱敏和内化在幼稚细胞中已经有了很好的特征，但在慢性治疗期间这些过程是如何在神经元中调节的还不清楚。了解慢性激动剂暴露后细胞的适应情况将有助于我们更好地理解导致耐受的机制。这项建议的目的是研究在慢性吗啡治疗过程中，u-阿片受体脱敏和内吞的调节是如何改变的。急性脱敏是指在持续应用高浓度激动剂时，受体从G蛋白激活中解偶联的现象。激动剂结合的受体也会发生内吞作用。我假设在慢性吗啡治疗的动物中，激动剂诱导的内化上调，导致从脱敏中恢复的减少。这一假说是基于对阿片类药物在慢性吗啡治疗后大鼠蓝斑神经元中作用变化的研究。慢性吗啡处理大鼠LC神经元的激动剂效应降低，脱敏作用增强，恢复不完全。为了验证我的假设，我将记录表达标志标记的U-阿片受体(FLAG-MOR)的小鼠蓝斑(LC)神经元的莫尔偶联与钾通道的激活。这将使我们能够在活体脑片上同时研究MOR偶联和贩运。这个项目的第一个目标是利用电生理学和成像相结合的方法来表征NATve细胞中FLAG-MOR对[Met]5-脑啡肽的激活、脱敏和内化。第二个目标是用吗啡慢性治疗小鼠，并确定内化在所观察到的脱敏恢复减慢中所起的作用。了解吗啡慢性暴露过程中MOR调节的细胞机制将有助于深入了解导致阿片类药物耐受形成的过程。这一理解可能允许开发不产生耐受性的有效止痛剂。

DESCRIPTION (provided by applicant): Opioids are widely used analgesics; however prolonged use results in tolerance, a progressively decreasing responsiveness to the drug. Desensitization and internalization of u-opioid receptors (MOR) are important in the actions of opioids and appear to contribute to the development of tolerance. Desensitization and internalization have been well characterized in naive cells, but how these processes are regulated in neurons during chronic treatment is unclear. Knowing what cellular adaptations occur in response to chronic agonist exposure will better our understanding of the mechanisms contributing to tolerance. The purpose of this proposal is to investigate how regulation of u-opioid receptor desensitization and endocytosis are altered during chronic morphine treatment. Acute desensitization is an uncoupling of the receptor from G-protein activation that occurs during a sustained application of a high concentration of agonist. Agonist-bound receptors also undergo endocytosis. I hypothesize that an upregulation of agonist-induced internalization occurs in chronic-morphine treated animals, resulting in reduced recovery from desensitization. This hypothesis is based on studies examining alterations in the actions of opioids in rat locus coeruleus neurons following chronic-morphine treatment. Agonist efficacy was decreased, desensitization was facilitated, and recovery was incomplete in LC neurons from chronic-morphine-treated rats. To test my hypothesis, I will record MOR coupling to activation of potassium channels in locus coeruleus (LC) neurons from mice expressing a FLAG-tagged u-opioid receptor (FLAG-MOR). This will allow us to study MOR coupling and trafficking simultaneously in a live brain slice. The first goal of this project is to characterize activation, desensitization, and internalization of FLAG-MOR to [Met]5-enkephalin in naTve cells using a combination of electrophysiology and imaging. The second goal is to treat mice chronically with morphine and determine what role internalization plays in the reduced recovery from desensitization that is observed. Understanding the cellular mechanisms of MOR regulation during chronic exposure to morphine will provide insight into the processes that lead to the development of tolerance to opioids. This understanding may allow for the development of effective analgesics which do not produce tolerance.