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（Calcitonin gene-related peptide，CGRP）是一种自分泌/旁分泌因子、激素和神经调节剂。虽然以其有效的血管扩张活性而闻名，但CGRP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受体对称为受体活性修饰蛋白1（RAMP 1）的亚基具有强制性要求。RAMP 1增强CGRP与受体的结合和受体运输。虽然血管CGRP受体的作用是公认的，但神经系统CGRP受体在血压调节中的作用仍不清楚。这项研究将验证神经系统CGRP/RAMP 1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的假设。 本研究的具体目的是：（1）确定RAMP 1在神经系统中的选择性过表达是否改善自主调节并减轻血管紧张素II（Ang-II）和苯肾上腺素诱导的高血压，以及相反，是否神经系统RAMP 1的缺失损害自主调节并增强高血压;（2）在神经系统靶向的RAMP 1转基因和敲除小鼠中鉴定负责自主和血压表型的CGRP/RAMP 1受体表达的神经系统位点;和（3）检验神经系统CGRP/RAMP 1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，RAMP 1在神经系统中仅过表达1.5倍就能改善自主调节，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重组酶来全面靶向神经系统，并局部注射表达Cre重组酶的病毒载体来靶向特定的大脑区域，包括脑干、穹窿下器官和室旁核，来测量雄性和雌性RAMP 1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ECG遥测仪的清醒小鼠中测量血压、心率、自主活动和一系列自主指数，包括在基础条件下和在通过渗透性微型泵递送的Ang-II和苯肾上腺素的4周输注诱导的高血压期间。为了分离对压力感受器反射的感觉与中枢成分的影响，将在麻醉小鼠中进行补充实验，包括测量来自主动脉减压神经的压力感受器传入神经活动以及在压力感受器传入神经的分级电刺激期间心率和血压的反射变化。抗氧化剂tempol的全身和中枢输注以及编码靶向NADPH氧化酶亚基的siRNA的病毒载体的中枢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。将在相关脑区、感觉神经节和外周动脉中测量CGRP、CGRP受体亚单位、Ang-II受体以及促氧化剂和抗氧化剂分子的基因表达。 鉴定神经系统CGRP/RAMP 1受体作为自主控制的正调节剂的意义涉及它们作为治疗高血压和其他病理状态如心力衰竭和糖尿病中的自主神经功能障碍的治疗靶点的潜力。通过激活CGRP/RAMP 1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.