Regulation of opioid receptor function in trigeminal ganglion

描述（由申请人提供）：作用于5阿片受体（MOR）的激动剂（例如吗啡及其类似物）是疼痛管理的主要药物；然而，有严重的副作用（例如依赖）以及社会和法律问题限制了它们的使用。因此，人们对阿片类药物的外周镇痛作用非常感兴趣。然而，尽管阿片受体系统在感觉神经元中表达，但它们在大多数基础条件下功能失活，但当外周组织存在一些刺激（炎症）时，它们会变得功能正常。除了MOR外，4阿片受体（DOR）是一个有吸引力的药物作用靶点，因为与MOR激活相比，DOR的副作用更少。但总体而言，4种激动剂促进镇痛的效果为弱至中等。本研究拟在大鼠三叉神经节神经元原代培养和外周镇痛行为模型中，研究MOR和DOR系统抑制腺苷酸环化酶活性和神经肽释放的功能能力调控机制。我们的具体目标是：1)表征MOR和DOR系统功能能力的诱导和维持动力学。我们将通过给药缓激肽（BK）、花生四烯酸（AA）和纳洛酮来描述诱导能力的时间过程，以及诱导后能力的持久性。2)为了确定MOR和DOR系统在功能能力发展过程中的变化，我们将确定启动诱导阿片受体介导的G蛋白激活增加的机制。3)确定纳洛酮和BK预处理诱导的MOR和DOR受体系统的功能能力是否由于本构脱敏的降低。我们假设MOR/DOR受体系统处于本构脱敏状态，因此对激动剂无反应。用逆激动剂治疗应该减少这种本构性脱敏，从而增强激动剂的反应性。此外，用其他试剂（如BK）启动也可以通过减少本构脱敏来诱导能力。4)探讨MOR和DOR在镇痛行为分析中的功能能力。这一目标提供了目标1-3基础工作的翻译延伸。利用新建立的口腔面部疼痛模型，我们将1)确定BK、AA和纳洛酮是否能诱导MOR和DOR系统的功能能力，2)研究能力的时间过程，3)描述PKC5在诱导BK、AA和纳洛酮诱导的功能能力中的作用，4)确定BK受体和PKC在炎症性疼痛模型中介导功能能力的作用。这些实验将增加我们对5和4阿片受体信号在初级感觉神经元中的调节的理解，并可能导致新的治疗疼痛的方法。公共卫生相关性：在过去几年中，在秀丽隐杆线虫、黑胃线虫和小鼠中分离出许多不同基因的突变，这些突变在囊泡融合和突触可塑性中起作用。当我们了解不同的保守信号机制如何控制这一过程时，对人类突触可塑性过程和调节的基本见解将会出现。果蝇的神经分泌研究作为一个模型系统，为使用与哺乳动物突触释放和可塑性直接相关的基因操作和基因组学的不同方法提供了非凡的机会。

Description (provided by applicant): Agonists acting at the 5 opioid receptor (MOR) (e.g. morphine and its analogs) are the mainstay of pain management; however there are serious adverse effects (e.g. dependence) and social and legal issues which limit their use. Consequently there has been considerable interest in the peripheral analgesic effects of opioids. However, although opioid receptor systems are expressed in sensory neurons, they are functionally inactive under most basal conditions, but become functionally competent when some stimulus (inflammation) is present in the peripheral tissue. In addition to MOR, the 4 opioid receptor (DOR) is an attractive target for drug action since there are fewer adverse effects than with MOR activation. However, in general, the efficacy of 4 agonists to promote analgesia is weak to moderate. Here we propose to study mechanisms involved in the regulation of the functional competence of the MOR and DOR systems to inhibit adenylyl cyclase activity and neuropeptide release in primary cultures of rat trigeminal ganglion neurons and in behavioral models of peripheral analgesia. Our specific aims are: 1) To characterize the kinetics of induction and maintenance of functional competence of MOR and DOR systems. We will delineate the time course for induction of competence, and the persistence of competence after induction, by administration of bradykinin (BK), arachidonic acid (AA) and naloxone. 2) To determine the changes in the MOR and DOR systems which underlie the development of functional competence In this aim, we will determine the mechanism for the priming-induced increase in opioid receptor-mediated G protein activation. 3) To determine if functional competence of MOR and DOR receptor systems induced by naloxone and BK pre-treatment is due to reduced constitutive desensitization.. We hypothesize that the MOR/DOR receptor systems exist in a constitutively desensitized state thus are non-responsive to agonist. Treatment with inverse agonists should reduce this constitutive desensitization leading to enhanced agonist responsiveness. Also priming with other agents (e.g. BK) may induce competence by reducing constitutive desensitization. 4) To investigate the functional competence of MOR and DOR in behavioral assays of analgesia. This aim provides a translational extension of the foundation work of Aims 1-3. Using a newly developed model of orofacial pain, we will 1) determine if functional competence of the MOR and DOR systems can be induced by BK, AA, and naloxone, 2) investigate the time course of competence, 3) delineate the role of PKC5 in induction of functional competence induced by BK, AA and naloxone and 4) determine the role of BK receptors and PKC in mediating functional competence in an inflammatory model of pain. These experiments will increase our understanding of the regulation of 5 and 4 opioid receptor signaling in primary sensory neurons and may lead to novel therapeutic approaches for the treatment of pain. PUBLIC HEALTH RELEVANCE: In the last few years many mutations in different genes have been isolated in C. elegans, D. Melanogaster and mice which plays role in vesicle fusion and synaptic plasticity. Fundamental insights into the processes and regulation of human synaptic plasticity will occur when we understand how different conserved signaling mechanism controls this process. The neurosecretion study of Drosophila as a model system provides extraordinary opportunities to use different methods of gene manipulation and of genomics with direct relevance to mammalian synaptic release and plasticity.