DYNORPHIN MODULATES CGRP RELEASE VIA PKC

描述：（申请人摘要） 强啡肽在神经病理学的许多方面起着重要的功能作用， 痛苦本申请旨在进一步探索强啡肽与 神经病理性疼痛通过研究细胞机制的行动， 非阿片肽，脱-Tyr-强啡肽A对脊髓蛋白激酶激活的影响 蛋白激酶C（PKC）和初级传入纤维兴奋性递质的释放。 相当多的证据表明，PKC与下列异常疼痛有关： 神经损伤强啡肽和PKC的激活都被发现增加 辣椒素诱发降钙素基因相关肽（CGRP）释放，特征性 初级传入纤维的激活因此， 强啡肽和PKC是脊髓信号通路的一部分， 兴奋过度由于这些原因，本应用程序将测试假设 （a）强啡肽增强PKC或PKC特定亚型的活性 PKC，通过非阿片机制;和（B）强啡肽促进 辣椒素通过PKC激活引起CGRP释放。为了验证这个假设， 目的1将检查基础和刺激CGRP释放的调制， 强啡肽A（1-17），或其不与 阿片受体这些实验将使用切碎的脊髓 制备以及培养的背根神经节细胞。的 强啡肽及其片段结构-活性关系也将 在这些实验中确定。第二个目标将建立调制 强啡肽A（2-17）对PKC及其同工酶的影响 接近。PKC激活的时间和解剖学相关性 强啡肽在体内将随着痛觉过敏的发展而建立。目标3 将检查阻断PKC或相关亚型是否会阻止 强啡肽A（2-17）增强辣椒素诱发的脊髓CGRP释放 准备工作PKC的特异性抑制剂将与 反义寡脱氧核苷酸（ODN），以“敲低”表达的选择性 同种型。适当的控制措施，如ODN不匹配、与时间相关的行动和 可逆性，并且靶蛋白的定量分析将是 重点介绍最后，将检测相关PKC亚型的敲低 抗强啡肽A（2-17）诱导的疼痛。这些研究将阐明 强啡肽在病理过程中作用的潜在细胞机制 疼痛状态，并可能提供洞察合理的方法来管理这种疼痛。 亚利桑那大学健康科学中心提供了独特的环境 并有机会让候选人扩大他在疼痛领域的背景 在Frank Porreca和Josephine Lai博士的指导下进行药理学研究。这 奖励与候选人的长期职业目标一致， 一个独立的研究者，致力于疼痛的基础研究， 阿片药理学

DESCRIPTION: (Applicant's Abstract) Dynorphin plays an important functional role in many aspects of neuropathic pain. This application aims to further explore the link between dynorphin and neuropathic pain by studying the cellular mechanism for the action of the non-opioid peptide, des-Tyr-dynorphin A on activation of spinal protein kinase C (PKC) and release of excitatory transmitters from primary afferent fibers. Considerable evidence has linked PKC with the abnormal pain seen following nerve injury. Both dynorphin and activation of PKC were found to augment capsaicin-evoked calcitonin gene-related peptide (CGRP) release, characteristic of activation of primary afferent fibers. It is possible therefore that dynorphin and PKC are part of a spinal signaling pathway that promotes sensory hyperexcitability. For these reasons, this application will test the hypothesis that (a) dynorphin potentiates the activity of PKC, or specific isoforms of PKC, via a non-opioid mechanism; and (b) that dynorphin facilitates capsaicin-evoked CGRP release through PKC activation. To test this hypothesis, Aim l will examine the modulation of basal and stimulated-CGRP release by dynorphin A (1-17), or by its des-Tyr fragments which do not interact with opioid receptors. These experiments will be done using a spinal cord minced preparation as well as dorsal root ganglion cells in culture. The structure-activity relationship for dynorphin and its fragments will also be determined in these experiments. The second aim will establish the modulation of PKC and its isozymes by dynorphin A (2-17) using both in vitro and in vivo approaches. Temporal and anatomical correlation between activation of PKC by dynorphin in vivo will be established with development of hyperalgesia. Aim 3 will examine whether blockade of PKC, or relevant isoform(s), will prevent dynorphin A (2-17) enhancement of capsaicin-evoked CGRP release in spinal cord preparations. Specific inhibitors of PKC will be used in conjunction with antisense oligodeoxynucleotides (ODN) to "knock-down" expression of selective isoforms. Appropriate controls such as mismatch ODN, time-related actions and reversibility, and the quantitative analysis of the target proteins will be emphasized. Finally, the knockdown of relevant PKC isoforms will be tested against dynorphin A (2-17)-induced pain in vivo. These studies will elucidate the underlying cellular mechanisms for the role of dynorphin in pathological pain states and may offer insight into rational approaches to manage such pain. The University of Arizona Health Science Center provides a unique environment and opportunity for the candidate to broaden his background in the area of pain pharmacology under the guidance of Drs. Frank Porreca and Josephine Lai. This award is consistent with the long-term career goal of the candidate to become an independent investigator contributing to the basic research of pain and opioid pharmacology.