Role of nonnuclear estrogen receptor GPR30 in preganglionic vagal neurons

非核雌激素受体 GPR30 在节前迷走神经元中的作用

• 批准号: 8015225
• 负责人: EUGEN BRAILOIU
• 金额: $ 30万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015225
• 关键词: Address; Agonist; Aromatase; Blood Pressure; Blood Pressure Monitors; Bradycardia; Brain Stem; Calcium; Cardiac; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cells; Clinical; Conjugated Equine Estrogens; Development; Estradiol; Estrogen Nuclear Receptor; Estrogen Receptors; Estrogens; Goals; Health; Heart; Heart Rate; Hormone replacement therapy; Hypothalamic structure; Image; Immunohistochemistry; In Vitro; Knowledge; Label; Link; Measurable; Measurement; Mediating; Membrane; Microinjections; Morbidity - disease rate; Neurons; Nuclear Receptors; Pathway interactions; Postmenopause; Premenopause; Progesterone; Property; Rattus; Regulation; Relative (related person); Role; Signal Transduction; Slice; Source; Staining method; Stains; Synapses; Techniques; Testing; Therapeutic; Therapeutic Agents; Urethane; Vagus nerve structure; Woman; cholinergic neuron; design; extracellular; immunocytochemistry; in vivo; insight; interdisciplinary approach; intravenous administration; mortality; neural circuit; novel; nucleus ambiguus; patch clamp; pressure; protective effect; release of sequestered calcium ion into cytoplasm; research study; response

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.

描述（由申请方提供）：心血管疾病是发病率和死亡率的主要原因。雌激素被认为具有保护作用。除了与两种经典的核受体相互作用外，雌激素还激活一种非核受体GPR 30。我们的长期目标是了解GPR 30在雌激素介导的神经元效应中的作用。本应用的目的是确定GPR 30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GPR 30增加了心脏的迷走神经活动，这可能是心脏保护性的。在这种情况下，GPR 30是开发新型心血管治疗药物的潜在靶点。我们将使用多学科的方法，包括免疫组织化学，共聚焦成像，在体内监测血压和心率，荧光测量的细胞溶质钙和全细胞膜片钳记录，以解决以下目标。第一，GPR 30定位于疑核的心脏节前神经元。我们的初步结果表明，GPR 30是存在于疑核的胆碱能神经元。我们将使用逆行标记，单，双免疫组化染色，以确定存在的GPR 30心脏投射神经元的疑核和它的共定位与芳香化酶。第二，测量响应于GPR 30激动剂的体内心率和血压。我们的初步研究结果表明，微量注射G-1，一个特定的GPR 30激动剂，或17-雌二醇（E2）到疑核产生心动过缓的效果被取消了先前的G-36，GPR 30拮抗剂。此外，初步实验表明，静脉注射G-1和E2可降低麻醉大鼠的平均动脉压和心率。我们将确定静脉注射GPR 30激动剂对血压、心率和迷走神经参与这些反应的影响。第三，离体疑核神经元GPR 30激活的电生理反应。我们的初步结果表明，G-1兴奋疑核神经元。我们将使用脑干切片中的全细胞膜片钳记录来确定GPR 30激动剂对疑核神经元的膜特性、诱发和自发突触电流的影响。第四，定义参与GPR 30介导的神经元反应的钙通道。我们的初步研究结果表明，激活GPR 30产生钙动员从外部和内部来源的培养大鼠下丘脑神经元的效果被取消预处理与G-36。我们将确定在运动前心脏迷走神经元中参与GPR 30信号传导的钙池。这项研究的结果将扩大我们的了解雌激素对参与心血管调节的神经元的作用机制，为心血管疾病的有效治疗方法的发展带来影响。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。