Nitric Oxide Control of CGRP in Trigeminal Neurons

描述(申请人提供)：拟议研究的目的是了解一氧化氮(NO)调节三叉神经细胞降钙素基因相关肽(CGRP)基因表达的机制。包括偏头痛在内的ALT类型的血管性头痛患者的血清CGRP水平升高。已知神经肽CGRP在偏头痛的潜在病理机制中发挥关键作用，因为它能够调节脑血流，介导神经源性炎症，并将伤害性信息传递到中枢神经系统。另一种与偏头痛病理有关的因素是一氧化氮(NO)。硝酸甘油是一种外源性NO供体，可触发偏头痛发作，而阻断NO合成可中止急性偏头痛发作。NO对脑血管的影响被认为是通过三叉神经细胞局部释放神经肽来实现的。在这个提案中，我将检验NO直接刺激CGRP基因表达的假设，并确定5-羟色胺能抗偏头痛药物是否可以抑制NO的作用。在第一个特定目标中提出的研究将确定NO单独或与其他炎症介质联合对三叉神经细胞释放CGRP的影响，以及抗偏头痛药物舒马曲坦是否可以抑制这一作用。第二个目标将集中于确定CGRP启动子中的基础调控位点和无反应调控位点。将CGRP-荧光素酶报告DNA瞬时导入原代培养的三叉神经节细胞，并测定其活性。苏马曲坦对基础和NO刺激的CGRP启动子活性的影响将被确定。第三个目标将阐明三叉神经细胞中参与NO信号传递的途径。最初，我们将使用特定的循环酶和激酶抑制剂和激活剂来确定参与调节CGRP合成和释放的主要途径(S)。对单个通路的进一步研究将利用磷酸特异性抗体和信号通路检测试剂盒。舒马曲坦对非激活通路的影响将被确定。这些研究的总体目标是深入了解三叉神经细胞CGRP基因表达的基础和无调控，这可能导致开发偏头痛和其他涉及神经源性炎症的疾病的新治疗策略。

DESCRIPTION (provided by applicant): The objective of the proposed research is to understand the mechanisms by which nitric oxide (NO) regulates calcitonin gene-related peptide (CGRP) gene expression in trigeminal neurons. Serum levels of CGRP are elevated in alt forms of vascular headaches, including migraine. The neuropeptide CGRP is known to play a critical role in the underlying pathology of migraine due to its ability to regulate cerebral blood flow, mediate neurogenic inflammation, and relay nociceptive information to the CNS. Another agent implicated in migraine pathology is nitric oxide (NO). Glyceryl trinitrate, an exogenous NO donor, triggers migraine attacks, while blockade of NO synthesis aborts acute migraine attacks. The cerebrovascular affect of NO is thought to be mediated by the local release of neuropeptides from trigeminal neurons. In this proposal, I will test the hypothesis that NO directly stimulates CGRP gene expression and determine whether serotonergic anti-migraine drugs can repress the effect of NO. Studies proposed in the first specific aim will determine the effect of NO alone or in combination with other inflammatory mediators on CGRP release from trigeminal neurons and whether the anti-migraine drug sumatriptan can repress this effect. The second aim will focus on identifying the basal and NO-responsive regulatory sites in the CGRP promoter. Primary trigeminal ganglia cultures will be transiently transfected with CGRP-luciferase reporter DNA and reporter activity measured. The effect of sumatriptan on basal and NO-stimulated CGRP promoter activity will be determined. The third aim will elucidate the pathways involved in NO signaling in trigeminal neurons. Initially, specific cyclase and kinase inhibitors and activators will be used to identify the major pathway(s) involved in regulating the synthesis and release of CGRP. Further studies of individual pathways will utilize phosphospecific antibodies and signaling pathway detection kits. The effect of sumatriptan on NO-activated pathways will be determined. The overall goal of these studies is to gain insight into basal and NO regulation of CGRP gene expression in trigeminal neurons that may lead to the development of novel therapeutic strategies for migraine and other diseases involving neurogenic inflammation.