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）调节三叉神经元降钙素基因相关肽（CGRP）基因表达的机制。CGRP的血清水平在包括偏头痛在内的血管性头痛中升高。已知神经肽CGRP在偏头痛的潜在病理学中起关键作用，这是由于其调节脑血流、介导神经源性炎症和将伤害性信息传递至CNS的能力。与偏头痛病理学有关的另一种药剂是一氧化氮（NO）。甘油三硝酸酯，一种外源性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。NO的脑血管效应被认为是通过三叉神经元局部释放神经肽介导的。在这个建议中，我将测试的假设，NO直接刺激CGRP基因的表达，并确定是否能抗偏头痛药物可以抑制NO的影响。研究提出的第一个具体目标将确定NO单独或与其他炎症介质结合对三叉神经元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种影响。第二个目标将集中在确定CGRP启动子的基础和NO反应的调节位点。原代三叉神经节培养物将用CGRP-荧光素酶报告基因DNA瞬时转染，并测量报告基因活性。将测定舒马曲坦对基础和NO刺激的CGRP启动子活性的影响。第三个目标是阐明三叉神经元中NO信号通路。最初，将使用特异性环化酶和激酶抑制剂和激活剂来鉴定参与调节CGRP合成和释放的主要途径。进一步研究个别途径将利用磷酸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元中CGRP基因表达的基础和NO调节，这可能导致开发用于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.