DYNORPHIN MODULATES CGRP RELEASE VIA PKC

强啡肽通过 PKC 调节 CGRP 释放

• 批准号: 6557991
• 负责人: Zaijie Jim Wang
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6557991
• 关键词: antisense nucleic acid; calcitonin gene related peptide; capsaicin; dynorphins; enzyme activity; enzyme inhibitors; gene expression; histochemistry /cytochemistry; hyperalgesia; immunologic assay /test; isozymes; laboratory rat; naloxone; neuropathology; neurotransmitters; opioid receptor; pain; pain threshold; posttranslational modifications; protein kinase C; protein protein interaction; protein structure function; spinal ganglion; tissue /cell culture; tissue /cell preparation; western blottings

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.

描述：（申请人摘要） 强啡肽在神经病的许多方面发挥着重要的功能作用 疼痛。该应用旨在进一步探讨强啡肽和强啡肽之间的联系 通过研究神经病理性疼痛的细胞机制 非阿片肽、脱酪氨酸强啡肽 A 对脊髓蛋白激酶激活的影响 C (PKC) 和初级传入纤维兴奋性递质的释放。 大量证据表明 PKC 与以下异常疼痛有关 神经损伤。强啡肽和 PKC 的激活都被发现可以增强 辣椒素诱发的降钙素基因相关肽 (CGRP) 释放，特征 初级传入纤维的激活。因此有可能 强啡肽和 PKC 是促进感觉的脊髓信号传导通路的一部分 过度兴奋。由于这些原因，该应用程序将检验假设 (a) 强啡肽增强 PKC 或特定亚型的活性 PKC，通过非阿片类药物机制； (b) 强啡肽促进 辣椒素通过 PKC 激活诱发 CGRP 释放。为了检验这个假设， 目标 l 将通过以下方式检查基础和刺激 CGRP 释放的调节 强啡肽 A (1-17)，或其 des-Tyr 片段，不与 阿片受体。这些实验将使用切碎的脊髓进行 制备以及培养中的背根神经节细胞。这 强啡肽及其片段的构效关系也将是 在这些实验中确定。第二个目标将建立调制 强啡肽 A (2-17) 在体外和体内对 PKC 及其同工酶的影响 接近。 PKC 激活之间的时间和解剖学相关性 体内强啡肽将随着痛觉过敏的发展而建立。目标 3 将检查 PKC 或相关亚型的封锁是否会阻止 强啡肽 A (2-17) 增强脊髓中辣椒素诱发的 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）“敲低”选择性表达 同工型。适当的控制，例如不匹配 ODN、与时间相关的操作和 可逆性，目标蛋白的定量分析将是 强调。最后，将测试相关 PKC 同工型的敲低 对抗强啡肽 A (2-17) 引起的体内疼痛。这些研究将阐明 强啡肽在病理学中作用的潜在细胞机制 疼痛状态并可能提供对管理此类疼痛的合理方法的见解。 亚利桑那大学健康科学中心提供独特的环境 候选人有机会扩大其在疼痛领域的背景 在博士的指导下药理学。弗兰克·波雷卡和约瑟芬·赖。这 奖项与候选人的长期职业目标一致 一位致力于疼痛和基础研究的独立研究者 阿片类药理学。