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（由申请人提供）：心血管疾病是发病率和死亡率的主要原因。已提出雌激素具有保护作用。除了与两个经典核受体的相互作用外，雌激素还激活了非核受体GPR30。我们的长期目标是了解GPR30在雌激素介导的神经元作用中的作用。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，作用于位于Ambiguus核神经元上的GPR30上的雌激素会增加心脏的迷走神经活性，这可能是心脏保护性的。在这种情况下，GPR30是开发新型心血管治疗剂的潜在目标。我们将使用一种多学科方法，包括免疫组织化学，共聚焦成像，体内监测血压和心率，荧光测量胞质钙和全细胞斑块钳记录以解决以下目标。首先，将GPR30定位于Ambiguus核的心脏前神经神经元。我们的初步结果表明，GPR30存在于Ambiguus核的胆碱能神经元中。我们将使用逆行标记，单和双重免疫组织化学染色，以鉴定Ambiguus核心神经元中GPR30的存在及其与芳香酶的共定位。其次，响应体内GPR30激动剂的心率和血压的测量。我们的初步结果表明，特定的GPR30激动剂或17-雌二醇（E2）的g-1的微注射会产生矛盾核会产生心动过缓。 GPR30拮抗剂G-36事先给药消除了效果。此外，试点实验表明，静脉注射G-1和E2的静脉内施用甲基氨基化大鼠的平均动脉压和心率降低。我们将确定GPR30激动剂静脉内给药对血压，心率以及迷走神经参与这些反应的影响。第三，体外单核神经元中GPR30激活的电生理反应。我们的初步结果表明，G-1会激发来自Ambiguus核的神经元。我们将使用脑干切片中的全细胞斑块钳记录确定GPR30激动剂对膜性特性的影响，诱发和自发的突触电流。第四，定义了与GPR30介导的神经元反应有关的钙途径。我们的初步结果表明，GPR30的激活从培养的大鼠下丘脑神经元中的外部和内部来源产生钙动员。用G-36预处理消除了效果。我们将确定前心脏迷走神经元中GPR30信号传导涉及的钙池。这项研究的结果将扩展我们对雌激素对参与心血管调节的神经元作用机制的理解，对心血管疾病中有效治疗方法的发展产生影响。公共卫生相关性：我们将研究新的雌激素受体在心血管功能中心控制中的作用。更好地理解雌激素作用机制对于开发心血管疾病的新治疗方法至关重要。