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

描述（由申请人提供）：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-血管紧张素系统激活。这些是该综合征的主要治疗靶点。在这个项目中，我们提出了三个目的来探讨在CHF大鼠的吻侧腹外侧髓质（RVLM）中过度表达血管紧张素2型受体（AT2R）的潜在益处。此外，我们还将确定RVLM中AT2R过表达的交感神经抑制作用的潜在机制。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并抑制神经元的兴奋性。另一方面，我们的初步实验显示CHF大鼠RVLM中AT2R蛋白表达下调。这些现象使我们假设RVLM中AT2R信号的减少通过提高前交感神经元的兴奋性来促进该综合征的交感神经兴奋。我们在本提案中的总体假设是，通过基因转移，RVLM中AT2R的过表达将减少或正常化CHF的交感神经激活，从而使该综合征受益。在初步实验中，我们成功地建立了一个大鼠模型，通过将AT2R病毒载体直接递送到RVLM区域，AT2R在RVLM中有选择性地过表达。利用这个动物模型，我们将通过追求以下3个具体目标来检验我们的假设。目的1：测定RVLM中AT2R过表达的正常大鼠和CHF大鼠的血流动力学、心功能和交感神经流出。这一目标包括两个部分。首先，我们将确定RVLM中过度表达AT2R对有意识的正常大鼠和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，还将测量饮水量、排尿量和体重。其次，我们将观察微注射AT1R和AT2R激动剂和拮抗剂到RVLM的急性效应，并观察AT2R对AP、HR和肾交感神经活性（RSNA）的过表达。我们还将探讨细胞内AR2R信号的交感神经抑制作用。这包括NO/cGMP和PLA2/AA/12-LO/PP2A途径。目的2：探讨AT2R过表达对麻醉大鼠RVLM单个前交感神经元活动的影响。我们将在AT2R过表达后直接记录RVLM前交感神经元的细胞外单单位放电。目的3：探讨过表达AT2R对RVLM前交感神经元钾电流的影响。通过膜片钳和脑干切片制备，我们将直接记录AT2R过表达后交感前神经元的钾电流。这些研究将有助于加深对CHF前交感神经元中血管紧张素信号的理解。他们将强调AT1和AT2受体信号平衡在设定交感神经兴奋水平中的重要性，并确定CHF治疗的可能新靶点。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神经抑制作用提供见解。该项目的结果将为治疗交感神经兴奋性疾病（如心力衰竭、高血压和糖尿病）提供潜在的策略。

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.