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

描述(申请人提供)：慢性心力衰竭(CHF)是美国发病率和死亡率的主要原因，其特征是交感神经过度活跃和肾素-血管紧张素系统的激活。这些都是这种综合征的主要治疗靶点。在这个项目中，我们提出了三个目标，以探索血管紧张素2型受体(AT2R)在充血性心力衰竭大鼠延髓头端腹外侧区(RVLM)过度表达的潜在益处。此外，我们还将确定AT2R在RVLM中过度表达所涉及的交感抑制效应的潜在机制。已经确定，与AT1R的影响相反，AT2R促进神经元钾通道和电流，从而使膜电位超极化，抑制神经元的兴奋性。另一方面，我们的初步实验表明，CHF大鼠RVLM中AT2R蛋白的表达下调。这些现象使我们推测，RVLM中AT2R信号的减少通过提高交感前神经元的兴奋性而参与了这种综合征的交感兴奋。我们在这个方案中的总体假设是，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，从而有利于该综合征。在初步实验中，我们成功地建立了一种大鼠模型，在该模型中，通过将AT2R病毒载体直接输送到该区域，AT2R在RVLM中选择性地过表达。利用这个动物模型，我们将通过追求以下三个具体目标来验证我们的假设。目的：研究血管紧张素Ⅱ受体(AT2R)在正常和充血性心力衰竭(CHF)大鼠的血流动力学、心功能和交感神经流出情况。这一目标包括两个组成部分。首先，我们将确定AT2R在RVLM中过度表达对清醒正常和CHF大鼠的动脉血压(AP)、心率(HR)、心功能和去甲肾上腺素排泄的慢性影响。此外，还将测量水分摄入量、尿液排泄量和体重。其次，我们将观察AT1R和AT2R激动剂和拮抗剂微量注射到AT2R高表达的RVLM对AP、HR和肾交感神经活动(RSNA)的急性影响。我们还将探索细胞内AR2R信号交感抑制的参与。这包括NO/cGMP和PLA2/AA/12-LO/PP2A通路。目的：研究血管紧张素Ⅱ受体AT2R过度表达对麻醉大鼠RVLM单个交感前神经元活动的影响。我们将直接记录AT2R过表达后RVLM前交感神经元的细胞外单位放电。目的：研究血管紧张素Ⅱ受体(AT2R)过表达对RVLM交感前神经元钾电流的影响。利用膜片钳和脑干切片技术，我们将直接记录AT2R过表达后的交感前神经元钾电流。这些研究将有助于更好地理解CHF时交感前神经元中的血管紧张素信号。他们将强调AT1和AT2受体信号之间的平衡在设定交感神经兴奋水平方面的重要性，并确定可能的治疗CHF的新靶点。公共卫生相关性：拟议的研究将为慢性心力衰竭大鼠特定脑区血管紧张素2型受体过度表达的交感抑制效应提供见解。该项目的结果将为交感神经兴奋性疾病的治疗提供潜在的策略，如心力衰竭、高血压和糖尿病。

DESCRIPTION (provided by applicant): 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 overexpressed 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. PUBLIC HEALTH RELEVANCE: The proposed research will provide insights into the sympatho-inhibitory effects of over expression of Angiotensin type 2 receptors in a specific brain area in rats with chronic heart failure. The results from this project will provide a potential strategy for the treatment of sympatho-excitatory diseases such as heart failure, hypertension, and diabetes.