Autonomic and Antihypertensive Actions of Neuronal CGRP/RAMP1 Receptors

描述（由申请人提供）： 降钙素基因相关肽 (CGRP) 具有自分泌/旁分泌因子、激素和神经调节剂的作用。 CGRP 以其强大的血管舒张活性而闻名，但它也参与许多神经过程，包括伤害感受、神经源性炎症、偏头痛和焦虑。 CGRP 受体必须需要一个称为受体活性修饰蛋白 1 (RAMP1) 的亚基。 RAMP1 增强 CGRP 与受体的结合和受体运输。虽然血管 CGRP 受体的作用已得到充分认识，但神经系统 CGRP 受体在血压调节中的作用仍不清楚。拟议的研究将检验这样的假设：神经系统 CGRP/RAMP1 受体是压力反射敏感性和自主活动的强大调节剂，能够预防或逆转高血压。 本研究的具体目的是：（1）确定神经系统中RAMP1的选择性过度表达是否会改善自主神经调节并减弱血管紧张素II（Ang-II）和去氧肾上腺素诱导的高血压，以及相反，神经系统RAMP1的缺失是否会损害自主神经调节并增强高血压； (2) 鉴定神经系统靶向 RAMP1 转基因和敲除小鼠中负责自主神经和血压表型的 CGRP/RAMP1 受体表达的神经系统位点； (3)检验神经系统CGRP/RAMP1受体的激活通过减少氧化应激消除Ang-II诱导的自主神经功能障碍和高血压的假设。我们的初步数据表明，神经系统中 RAMP1 的过度表达仅 1.5 倍即可改善自主调节并几乎消除 Ang-II 高血压。 我们将使用神经系统特异性巢蛋白 Cre 重组酶来全局靶向神经系统，并局部注射表达 Cre 重组酶的病毒载体来靶向特定的脑区域，包括脑干、穹窿下器官和室旁核，测量雄性和雌性 RAMP1 转基因和敲除小鼠的自主神经和心血管表型。将在植入血压和心电图遥测仪的清醒小鼠中测量血压、心率、运动活动和一系列自主神经指数，无论是在基础条件下还是在通过渗透微型泵输注 Ang-II 和去氧肾上腺素 4 周引起的高血压期间。为了区分压力反射对感觉和中枢部分的影响，将在麻醉小鼠中进行补充实验，包括测量主动脉降压神经的压力感受器传入神经活动以及压力感受器传入分级电刺激期间心率和血压的反射变化。抗氧化剂 tempol 的全身和中央输注以及编码针对 NADPH 氧化酶亚基的 siRNA 的病毒载体的中央注射将用于确定氧化应激在介导自主神经失调和高血压中的作用。将测量相关大脑区域、感觉神经节和外周动脉中 CGRP、CGRP 受体亚基、Ang-II 受体以及促氧化和抗氧化分子的基因表达。 将神经系统 CGRP/RAMP1 受体鉴定为自主神经控制的正调节剂的重要性与其作为治疗高血压和其他病理状态（如心力衰竭和糖尿病）自主神经功能障碍的治疗靶点的潜力有关。通过激活 CGRP/RAMP1 受体来抑制 Ang II 介导的作用对高血压和心力衰竭具有广泛的影响。

DESCRIPTION (provided by applicant): Calcitonin gene-related peptide (CGRP) functions as an autocrine/paracrine factor, hormone and neuro- modulator. While best known for its potent vasodilator activity, CGRP is involved in many neural processes including nociception, neurogenic inflammation, migraine and anxiety. The CGRP receptor has an obligatory requirement for a subunit called Receptor Activity-Modifying Protein 1 (RAMP1). RAMP1 enhances CGRP binding to the receptor and receptor trafficking. While the role of vascular CGRP receptors is well recognized, the role of nervous system CGRP receptors in blood pressure regulation remains unclear. T he proposed studies will test the hypothesis that nervous system CGRP/RAMP1 receptors are powerful modulators of baroreflex sensitivity and autonomic activity capable of preventing or reversing hypertension. The specific aims of the proposed research are to: (1) Determine whether selective overexpression of RAMP1 in the nervous system improves autonomic regulation and attenuates angiotensin II (Ang-II) and phenylephrine-induced hypertension, and conversely if deletion of nervous system RAMP1 impairs auto- nomic regulation and enhances hypertension; (2) Identify nervous system sites of CGRP/RAMP1 receptor expression responsible for autonomic and blood pressure phenotypes in the nervous system-targeted RAMP1 transgenic and knockout mice; and (3) Test the hypothesis that activation of nervous system CGRP/ RAMP1 receptors abrogates Ang-II induced dysautonomia and hypertension by reducing oxidative stress. Our preliminary data indicate that just a 1.5-fold overexpression of RAMP1 in the nervous system improves autonomic regulation and nearly abolishes Ang-II hypertension. We will measure autonomic and cardiovascular phenotypes in male and female RAMP1 transgenic and knockout mice using the nervous system-specific nestin Cre-recombinase to globally target the nervous system and local injections of viral vectors expressing Cre-recombinase to target specific brain regions including brainstem, subfornical organ and paraventricular nucleus. Blood pressure, heart rate, locomotor activity and an array of autonomic indices will be measured in conscious mice implanted with blood pressure and ECG telemeters, both under basal conditions and during hypertension induced by 4-week infusions of Ang-II and phenylephrine delivered by osmotic minipump. To separate effects on sensory vs. central com- ponents of the baroreflex, complementary experiments will be performed in anesthetized mice that include measurements of baroreceptor afferent nerve activity from aortic depressor nerve and reflex changes in heart rate and blood pressure during graded electrical stimulation of baroreceptor afferents. Systemic and central infusions of the antioxidant tempol and central injections of viral vectors encoding siRNAs targeted to NADPH oxidase subunits will be used to determine the role of oxidative stress in mediating autonomic dysregulation and hypertension. Gene expression of CGRP, CGRP receptor subunits, Ang-II receptors, and pro- and anti- oxidant molecules will be measured in the relevant brain regions, sensory ganglia, and peripheral arteries. The significance of identifying nervous system CGRP/RAMP1 receptors as positive modulators of autonomic control relates to their potential as therapeutic targets for treatment of dysautonomia in hyperten- sion, and other pathological states such as heart failure and diabetes. Inhibition of Ang II-mediated effects by activation of CGRP/RAMP1 receptors has widespread implications in hypertension and heart failure.