Regulation of opioid receptor function in trigeminal ganglion

说明(申请人提供)：作用于5阿片受体(MOR)的激动剂(如吗啡及其类似物)是止痛的主要药物；但有严重的不良反应(如依赖性)以及限制其使用的社会和法律问题。因此，阿片类药物的外周镇痛作用引起了人们的极大兴趣。然而，尽管阿片受体系统在感觉神经元中表达，但在大多数基础条件下，它们在功能上不活跃，但当周围组织中存在一些刺激(炎症)时，它们变得有功能。除了MOR，4阿片受体(DOR)也是一个有吸引力的药物作用靶点，因为它的副作用比MOR激活时要少。但总体而言，4种激动剂促进镇痛的效果为弱至中度。在此，我们建议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系统抑制腺酰环化酶活性和神经肽释放的功能调节机制。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的动力学。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进程和诱导后能力的持久性。2)为了确定作为功能能力发展基础的MOR和DOR系统的变化，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)研究纳洛酮和BK预处理对MOR和DOR受体系统功能的影响是否与结构性脱敏作用减弱有关。我们假设MOR/DOR受体系统处于结构性脱敏状态，因此对激动剂无反应。反向激动剂的治疗应减少这种结构性脱敏，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结构性脱敏来诱导能力。4)探讨MOR和DOR在镇痛行为检测中的功能。这一目标提供了目标1-3的基础工作的翻译延伸。我们将利用新发展的口面部疼痛模型，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)研究功能的时间进程，3)描述PKC5在BK、AA和纳洛酮诱导的功能能力诱导中的作用，4)在疼痛炎症模型中确定BK受体和PKC在介导功能能力中的作用。这些实验将增加我们对初级感觉神经元中5和4阿片受体信号调节的理解，并可能导致治疗疼痛的新方法。公共卫生相关性：在过去的几年中，在线虫、黑腹线虫和小鼠中分离到了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发挥作用。当我们了解不同的保守信号机制是如何控制这一过程时，就会对人类突触可塑性的过程和调控有基本的了解。以果蝇为模型系统的神经分泌研究为使用与哺乳动物突触释放和可塑性直接相关的不同的基因操作和基因组学方法提供了难得的机会。

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.