Role of nonnuclear estrogen receptor GPR30 in preganglionic vagal neurons

描述(申请人提供)：心血管疾病是导致发病率和死亡率的主要原因。雌激素被认为具有保护作用。除了与两个经典的核受体相互作用外，雌激素还激活一个非核受体GPR30。我们的长期目标是了解GPR30在雌激素介导的神经元效应中的作用。该应用的目的是确定GPR30在心血管调节中的作用。我们的中心假设是，雌激素作用于位于疑核神经元上的GPR30，增加了迷走神经对心脏的活动，这可能具有心脏保护作用。在这种背景下，GPR30是开发新型心血管治疗药物的潜在靶点。我们将使用多学科方法，包括免疫组织化学、共聚焦成像、血压和心率的活体监测、胞浆钙的荧光测量和全细胞膜片钳记录来实现以下目标。第一，GPR30在心脏疑核节前神经元的定位。我们的初步结果表明GPR30存在于疑核的胆碱能神经元中。我们将使用逆行标记、单免疫组织化学和双重免疫组织化学染色来确定GPR30在疑核心脏投射神经元中的存在及其与芳香化酶的共存。第二，在体内测量GPR30激动剂的心率和血压。我们的初步结果表明，向疑核微量注射GPR30特异性激动剂G-1或17-雌二醇(E_2)会产生心动过缓；这种作用可被预先给予GPR30拮抗剂G-36所消除。此外，初步实验表明，静脉注射G-1和E_2可降低乌拉坦麻醉大鼠的平均动脉压和心率。我们将确定静脉注射GPR30激动剂对血压、心率的影响以及迷走神经参与这些反应的情况。GPR30在体外激活单个疑核神经元的电生理反应。我们的初步结果表明，G-1兴奋疑核神经元。我们将利用脑干切片的全细胞膜片钳记录来确定GPR30激动剂对疑核神经元膜特性、诱发和自发突触电流的影响。第四，明确参与GPR30介导的神经元反应的钙通路。我们的初步结果表明，在培养的大鼠下丘脑神经元中，GPR30的激活产生来自外部和内部的钙动员；这一作用可被G-36预处理所消除。我们将确定参与运动前心脏迷走神经GPR30信号转导的钙池。这项研究的结果将扩大我们对雌激素对参与心血管调节的神经元的作用机制的理解，对开发有效的心血管疾病治疗方法具有重要意义。公共卫生相关性：我们将研究一种新的雌激素受体在心血管功能中枢控制中的作用。更好地了解雌激素的作用机制对于开发心血管疾病的新疗法至关重要。

DESCRIPTION (provided by applicant): Cardiovascular disease is a leading cause of morbidity and mortality. Estrogen has been proposed to have a protective effect. In addition to the interaction with two classical nuclear receptors, estrogen activates a non nuclear receptor, GPR30. Our long-term goal is to understand the role of GPR30 in estrogen-mediated neuronal effects. The objective of this application is to determine the role of GPR30 in cardiovascular regulation. Our central hypothesis is that estrogen acting on GPR30 located on neurons of the nucleus ambiguus increases vagal activity to the heart, which may be cardioprotective. Viewed in this context, GPR30 is a potential target for the development of novel cardiovascular therapeutic agents. We will use a multidisciplinary approach including immunohistochemistry, confocal imaging, in vivo monitoring of blood pressure and heart rate, fluorimetric measurement of cytosolic calcium and whole-cell patch-clamp recording to address the following aims. First, localization of GPR30 to cardiac preganglionic neurons of the nucleus ambiguus. Our preliminary results indicate that GPR30 is present in cholinergic neurons of the nucleus ambiguus. We will use retrograde labeling, single and double immunohistochemical staining to identify the presence of GPR30 in cardiac projecting neurons of the nucleus ambiguus and its colocalization with aromatase. Second, measurement of heart rate and blood pressure in response to GPR30 agonists in vivo. Our preliminary results indicate that microinjection of G-1, a specific GPR30 agonist, or 17-estradiol (E2) into the nucleus ambiguus produces bradycardia; the effect was abolished by prior administration of G-36, a GPR30 antagonist. In addition, pilot experiments indicate that intravenous administration of G-1 and E2 decreased mean arterial pressure and heart rate in urethane-anesthetized rats. We will determine the effect of intravenous administration of GPR30 agonists on blood pressure, heart rate and the participation of the vagus nerve in these responses. Third, electrophysiological response of GPR30 activation in single nucleus ambiguus neurons in vitro. Our preliminary results indicate that G-1 excites neurons from nucleus ambiguus. We will determine the effect of GPR30 agonists on membrane properties, evoked and spontaneous synaptic currents from nucleus ambiguus neurons using whole-cell patch-clamp recordings in brainstem slices. Fourth, define the calcium pathways involved in GPR30-mediated neuronal responses. Our preliminary results indicate that activation of GPR30 produces calcium mobilization from external and internal sources in cultured rat hypothalamic neurons; the effect was abolished by pretreatment with G-36. We will determine the calcium pools involved in GPR30 signaling in premotor cardiac vagal neurons. The result of this study will extend our understanding of the mechanisms of action of estrogen on neurons involved in cardiovascular regulation with implications for the development of effective therapeutic approaches in cardiovascular disorders. PUBLIC HEALTH RELEVANCE: We will study the role of a new estrogen receptor in the central control of cardiovascular function. A better understanding of the mechanisms of action of estrogen is critical for the development of new treatments for cardiovascular disease.