AT2受体调控主动脉压力反射的新靶点-Nav1.9通道

AT2R is the receptor that highly expresses in heart and kidney of females and plays a potential role in heart and blood vessle protection to counteract to AT1 receptor compared with male. Our recent study showed a gender-specific subpopulation of myelianted Ah-type baroreceptor neuron (Ah-BRN) distributed in nodose ganglion of female rats, which contributes greatly to baroreceptor reflex because of its electrophysilogical properties, i.e. lower threshold in action potential firings and higher frequency in repetitive discharges responding to afferent signal of blood presure changes rasing from baroreceptor endings located in the aortic wall. As an important modulator of neuronal and humoral regulation system, whether and how AT2R modulate blood pressure through the baroreceptor reflex in female was largely unknown. Our prelimitory data demonstrated a higher AT2R expression in nodose neurons of female rats and this result was strongly supported by the notion of real time RT-PCR from single identified Ah-BRN of female rats, suggesting AT2R may play a key role in baroreflex to make female blood pressure significantly lower than that of males. Our further study attempts to investigate if AT2 receptor in female could modulate baroreceptor reflex through Nav1.9 channel using patch clamp technique, cell transfection, and molecular methods. Our hypothethsis is that AT2R could increase Nav1.9 channel expression and elevate Ah-BRN excitibility which was related with activation of MAPK signaling pathway and down-regulation of miR-145/miR-485 expression. Our project will verify the physiological and clinical significance of AT2R on modulation of blood pressure in female and provide a valuable information for publics and clinicians of how to effiicently menagement of cardiovascular disease and also to guide the development of new drug.

AT2受体（AT2R）在女性组织中高表达，是女性一种心脏和血管潜在的保护型受体。最新研究表明结状神经节内髓鞘化Ah-型压力感受器神经元（Ah-BRN）是仅存在于雌性大鼠的神经元亚型，其以独特的生理特性参与主动脉压力反射。申请人初步证实相比雄性，雌性大鼠结状神经节高度表达AT2R，单细胞RT-PCR结果显示其主要表达于Ah-BRN。本课题将深入研究AT2受体如何调节主动脉压力反射及其分子机制。实验拟用细胞转染，膜片钳及分子生物学等方法证实AT2R激活可通过增加Nav1.9通道表达提高雌鼠压力感受器敏感性而增强主动脉压力反射，使其血压维持在较低水平。并研究MAPK信号通路和miR-145/miR-485参与AT2受体调控Nav1.9通道的调节机制。本研究结果将从传入神经调控机制入手揭示血压调节的性别差异，对心血管疾病合理地诊断、治疗和预后评估有重大科学意义。

本项目组通过系列电生理学的研究发现，成年雌性大鼠结状神经节内除髓鞘化A型和非髓鞘化C型神经元外，尚存在一种性别特异性分布的髓鞘化Ah-型压力感受器神经元（Ah-BRN），具有低兴奋阈值和高放电频率等电生理学特征，即Ah-BRN末梢对较低血压变化就能产生兴奋，通过主动脉压力反射使中枢副交感传出的活性增强，交感传出的活性相对减弱，致使血压下调，并保持血压在较低的水平。Ah-BRN兴奋性在去除卵巢（OVX）雌性大鼠显著下降，雌激素可完全恢复下调的神经元兴奋性。.本项目研究发现SD大鼠血管紧张素II（Ang II）的AT1 和AT2受体表达存在性别差异，RT-PCR、Western blot和共聚焦实验结果都表明去卵巢后AT1受体表达量没有明显的变化即其无性别差异，而去卵巢大鼠AT2受体表达明显下降接近于雄性的表达量，即AT2受体表达量存在明显的性别差异。Ang II 50 ng/kg/day连续灌流15天，去卵巢雌鼠动脉压力感受器敏感性下降，与以往雄鼠的报道一致；但不同的是雌鼠压力感受器敏感性增强。电生理结果显示各组A和C-BRN兴奋性不变或降低；雌性组Ah-BRN兴奋性增高；利用Single cell PCR方法检测单个神经元细胞的AT2受体的表达，结果显示AT2受体在Ah神经元中表达量最高，雌性结状神经节Nav1.9通道表达增加。全细胞膜片钳结果显示Ang II 100nM对三种结状神经节神经元的内向电流和放电频率作用不同，Ang II明显增加Ah神经元的放电频率，并产生了较强的内向电流。因此，得出结论Ang II可通过激活AT2受体增加Nav1.9表达而提高Ah-BRN兴奋性，使雌鼠主动脉减压反射活动增强。.应用Targetscan软件预测和手工序列比对发现，Nav1.9通道是miR-145/miR-485的潜在作用靶点。荧光素酶活性实验证明miR-145/miR-485对SCN11a基因直接调控作用。即miRNA可影响Nav1.9通道表达而改变神经元细胞的兴奋阈值，提高神经元的兴奋性。.本项目首次证明AngII对Ah-型神经元的调节作用，从离子通道和分子生物学水平揭示AngII调节血压性别差异的新机制，为寻找治疗高血压的新靶点提供理论依据。