DYNORPHIN MODULATES CGRP RELEASE VIA PKC

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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.