AT2 Receptors in the RVLM: Regulation of Sympathetic Outflow in Heart Failure

摘要 慢性心力衰竭(CHF)是美国发病率和死亡率的主要原因， 交感神经过度活动和肾素-血管紧张素系统激活的特点。这些是 这种综合征的主要治疗目标。在这个项目中，我们提出了三个开发潜力的目标 延髓吻侧腹外侧区血管紧张素2型受体(AT2R)过度表达的益处 (RVLM)对CHF大鼠的影响。此外，我们还将确定 RVLM中AT2R过度表达的交感神经抑制作用已经确凿地证明，在 与AT1R的影响相比，AT2R促进了神经元的钾通道和电流， 它超极化膜电位并抑制神经元的兴奋性。另一方面，我们的 初步实验表明，心力衰竭大鼠RVLM中AT2R蛋白表达下调。 这些现象使我们假设RVLM中AT2R信号的减少有助于 交感神经兴奋通过提高交感前神经元的兴奋性而引起的这种综合征。我们的全球 该方案中的假设是，通过基因转移在RVLM中过度表达AT2R将 减少或正常化CHF的交感神经激活，从而有益于这一综合征。在……里面 初步实验，我们已经成功地建立了一种AT2R选择性终止的大鼠模型 通过将AT2R病毒载体直接输送到该区域，在RVLM中表达。利用这种动物模型， 我们将通过追求以下三个具体目标来检验我们的假设。目标1：确定 正常和慢性心力衰竭大鼠的血流动力学、心功能和交感神经流出 AT2R在RVLM中的过度表达。这一目标包括两个组成部分。首先，我们将确定 血管紧张素Ⅱ受体AT2R在RVLM中过度表达对动脉血压(AP)、心率(HR)、 清醒的正常大鼠和心力衰竭大鼠的心功能和去甲肾上腺素排泄。此外，水的摄入， 尿液排泄，并测量体重。第二，我们将观察其急性影响 RVLM内微量注射AT1R和AT2R激动剂和拮抗剂AT2R高表达 AP、HR和肾交感神经活动(RSNA)。我们还将探索细胞内的参与 AR2R信号转导交感抑制。这包括NO/cGMP和PLA2/AA/12-LO/PP2A通路。目标 2.研究AT2R过表达对大鼠单个交感神经元电活动的影响 麻醉大鼠的RVLm。我们将直接记录RVLM的细胞外单单位放电 AT2R过表达后的交感前神经元。目标3：确定 AT2R在RVLM交感前神经元钾电流上的过度表达。用人 膜片钳和脑干切片准备，我们将直接记录交感神经前膜的钾电流 AT2R过表达后的神经元。这些研究将有助于加深对 慢性心力衰竭时交感前神经元的血管紧张素信号转导。他们将强调 AT1和AT2受体信号在设定交感兴奋水平和识别中的平衡 可能成为CHF治疗的新靶点。

ABSTRACT Chronic heart failure (CHF) is a leading cause of morbidity and mortality in the United States with the characteristics of sympathetic overactivity and activation of the renin-angiotensin system. These are the primary therapeutic targets for this syndrome. In this project, we propose three aims to explore the potential benefit of over expressing angiotensin type 2 receptor (AT2R) expression in the rostral ventrolateral medulla (RVLM) in rats with CHF. Moreover we will also determine the underlying mechanisms involved in the sympatho-inhibitory effects of AT2R overexpression in the RVLM. It has been firmly established that, in contrast to the influences of the AT1R, the AT2R facilitates the neuronal potassium channel and current, which hyperpolarizes membrane potential and suppresses neuronal excitability. On the other hand, our preliminary experiments show a down regulation of AT2R protein expression in the RVLM of rats with CHF. These phenomena lead us to postulate that a decrease in AT2R signaling in the RVLM contributes to sympatho-excitation of this syndrome by elevating the excitability of presympathetic neurons. Our global hypothesis in this proposal is that over expression of the AT2R in the RVLM by gene transfer will reduce or normalize sympathetic activation in CHF, and therefore benefit this syndrome. In preliminary experiments, we have successfully produced a rat model in which the AT2R is selectively over expressed in the RVLM by direct delivery of AT2R viral vectors into this area. Employing this animal model, we will test our hypothesis by pursuing the following 3 Specific Aims. AIM 1: To determine the hemodynamic, cardiac function, and sympathetic outflow in normal and CHF rats with overexpression of AT2R in the RVLM. This Aim includes two components. First, we will determine the chronic effects of over expressing AT2R in the RVLM on arterial blood pressure (AP), heart rate (HR), cardiac function, and norepinephrine excretion in conscious normal and CHF rats. In addition, water intake, urine excretion, and body weight will be measured. Second, we will observe the acute effects of microinjecting agonists and antagonists of AT1R and AT2R into the RVLM with AT2R over expression on AP, HR, and renal sympathetic nerve activity (RSNA). We will also explore the involvement of intracellular AR2R signaling sympatho-inhibition. This includes the NO/cGMP and PLA2/AA/12-LO/PP2A pathways. AIM 2: To determine the effects of overexpressing AT2R on single presympathetic neuronal activity in the RVLM of anesthetized rats. We will directly record extracellular single unit firing of RVLM presympathetic neurons following overexpression of AT2R. AIM 3: To determine the effects of overexpressing AT2R on potassium current of presympathetic neurons in the RVLM. Employing patch clamp and brainstem slice preparations, we will directly record potassium currents of presympathetic neurons following overexpression of AT2R. These studies will lead to an enhanced understanding of angiotensin signaling in presympathetic neurons in the setting of CHF. They will highlight the importance of a balance between AT1 and AT2 receptor signaling in setting the level of sympatho-excitation and identify possible new targets for therapy in CHF.