DYNORPHIN MODULATES CGRP RELEASE VIA PKC

描述：(申请人摘要) 强啡肽在神经病的许多方面起着重要的功能作用 疼痛。这项申请旨在进一步探索强啡肽和 神经病理性疼痛的细胞机制研究 非阿片肽Des-Tyr-强啡肽A对脊髓蛋白激酶活性的影响 C(PKC)和初级传入纤维释放兴奋性递质。 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的激活都被发现增强 辣椒素诱导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 强啡肽和蛋白激酶C是促进感觉的脊髓信号通路的一部分。 过度兴奋。出于这些原因，这个应用程序将检验该假设 (A)强啡肽增强PKC的活性，或特定的亚型 PKC，通过非阿片机制；和(B)强啡肽促进 辣椒素通过激活PKC诱导CGRP的释放。为了检验这一假设， 目的L研究降钙素基因相关肽对基础和刺激释放的调节作用。 强啡肽A(1-17)或其不与之相互作用的Des-Tyr片段 阿片受体。这些实验将使用切碎的脊髓进行 制备以及培养背根神经节细胞。这个 强啡肽及其片段的构效关系也将是 在这些实验中确定的。第二个目标将建立调制 强啡肽A(2-17)对PKC及其同工酶的体内外研究 接近了。蛋白激酶C激活的时间和解剖学相关性 随着痛觉过敏的发展，强啡肽将在体内建立。目标3 将研究是否封锁PKC或相关的亚型(S)将阻止 强啡肽A(2-17)促进辣椒素诱导的脊髓降钙素基因相关肽释放 准备工作。PKC的特定抑制剂将与 反义寡核苷酸(ODN)选择性“敲除”表达 异构体。适当的控制，如不匹配的ODN、与时间相关的操作和 可逆性，并将对目标蛋白进行定量分析 强调。最后，将测试相关的PKC亚型的敲除。 在体内对抗强啡肽A(2-17)引起的疼痛。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，并可能提供对合理的方法来管理这种疼痛的洞察。 亚利桑那大学健康科学中心提供了一个独特的环境 并为候选人提供了拓宽其痛苦领域背景的机会 由Frank Porreca博士和Josephine Lai博士指导的药理学。这 奖项与候选人的长期职业目标是一致的 为疼痛和疼痛的基础研究做出贡献的独立研究员 阿片类药理学。

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.