Nitric Oxide Control of CGRP in Trigeminal Neurons

描述（由申请人提供）：拟议研究的目的是了解一氧化氮（NO）调节三叉神经细胞中降钙素基因相关肽（CGRP）基因表达的机制。血清CGRP水平升高在所有形式的血管性头痛，包括偏头痛。神经肽CGRP由于其调节脑血流、介导神经源性炎症和向中枢神经系统传递伤害性信息的能力，在偏头痛的潜在病理中起着关键作用。另一种与偏头痛病理有关的物质是一氧化氮（NO）。三硝酸甘油，外源性NO供体，触发偏头痛发作，而阻断NO合成中止急性偏头痛发作。一氧化氮对脑血管的影响被认为是由三叉神经神经元局部释放神经肽介导的。在本提案中，我将检验NO直接刺激CGRP基因表达的假设，并确定5 -羟色胺类抗偏头痛药物是否可以抑制NO的作用。第一个具体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CGRP释放的影响，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启动子中的基础和no反应性调控位点。将cgrp -荧光素酶报告基因瞬时转染三叉神经节培养物并测量报告基因活性。舒马匹坦对基础和no刺激CGRP启动子活性的影响将被确定。第三个目的是阐明三叉神经中NO信号通路。最初，将使用特定的环化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。个体途径的进一步研究将利用磷特异性抗体和信号通路检测试剂盒。将确定舒马曲坦对no激活途径的影响。这些研究的总体目标是深入了解三叉神经细胞中CGRP基因表达的基础和NO调节，这可能导致偏头痛和其他涉及神经源性炎症的疾病的新治疗策略的发展。

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.