Neurokinin 3 Receptor: Nuclear Localization in Supraoptic Neurons

神经激肽 3 受体：视上神经元的核定位

• 批准号: 7471320
• 负责人: CELIA D SLADEK
• 金额: $ 20.07万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7471320
• 关键词: Absence of pain sensation; Agonist; Alcohol consumption; Amygdaloid structure; Analgesics; Antibodies; Antihypertensive Agents; Anxiety; Appearance; Area; Binding; Brain region; C-terminal; Cardiovascular system; Cell Nucleolus; Cell Nucleus; Cell membrane; Cell physiology; Cocaine; Cytoplasm; DAPI; Data; Disease; Dystonia; Electrolytes; Emotional; Epitopes; Figs - dietary; Functional disorder; G-Protein-Coupled Receptors; Gene Expression Regulation; Goals; Homeostasis; Hypertension; Hypotension; Hypothalamic structure; Injection of therapeutic agent; Lateral; Ligands; Liquid substance; Location; Locomotion; Membrane; Mental Depression; Mental disorders; Messenger RNA; Methods; Movement Disorders; N-terminal; Neurologic; Neuromedin K Receptor; Neurons; Nuclear; Nuclear Extract; Nuclear Receptors; Nuclear Translocation; Nucleus solitarius; Numbers; Optics; Physiological; Public Health; Range; Rattus; Receptor Activation; Receptor Cell; Research; Specimen; Staining method; Stains; Stimulus; Structure of nucleus infundibularis hypothalami; Substantia nigra structure; Testing; Ventral Tegmental Area; Western Blotting; addiction; extracellular; human CCR10 protein; human disease; immunoreactivity; lateral ventricle; locus ceruleus structure; magnocellular; midbrain central gray substance; motivated behavior; normotensive; preoptic nucleus; receptor; receptor internalization; response; senktide; size; supraoptic nucleus

DESCRIPTION (provided by applicant): The goal of this application is to develop background information to determine the physiological significance of the unexpected observation that a physiological stimulus (hypotension) and a selective neurokinin 3 receptor (NK3-R) agonist (senktide) induce the appearance of NK3-R immunoreactivity (ir) in the nucleus of neurons in the supraoptic nucleus (SON) of the hypothalamus. Since NK3-Rs are G-protein coupled receptors (GPCR), this suggests the intriguing possibility that extracellular NK3-R ligand induces translocation of NK3-R from the cell membrane to the nucleus for direct regulation of gene expression by a GPCR. This is exciting, because it has important implications for other GPCRs, and therefore could impact on numerous GPCR-regulated cellular functions. Also, the observation could be important to a wide range of CNS functions, because NK3-Rs are widely distributed in the CNS and have been implicated in diverse and important CNS functions and pathophysiology including motivated behaviors such as salt, cocaine and alcohol intake; emotional states such as anxiety and depression; locomotion and dystonia; fluid, electrolyte, and cardiovascular homeostasis; and analgesia. Therefore, this observation has the potential to impact the treatment of diverse and debilitating neurologic, psychiatric, and homeostatic disorders. In order to assess the potential importance of this observation, we must 1) fully characterize the NK3-R-ir observed in the nucleus of SON neurons, and 2) determine if the same phenomenon occurs in other CNS regions. Therefore, the Specific Aims for this proposal will test the following hypotheses: Hypothesis 1: The ligand-induced appearance of NK3-R-ir in the nuclei of SON neurons represents all or a portion of NK3-R that translocates to the nucleus following receptor internalization. To test this hypothesis, we have generated antibodies to different epitopes in the N-terminal and C-terminal regions of NK3-R. These antibodies will be used for immunohistochemical (IHC) and western blot analysis of SON. SON from normotensive, hypotensive, and senktide (a selective NK3-R agonist)-treated rats will be compared. Western blots will be used to analyze cytosolic and nuclear extracts of SON to determine the size and cellular location of NK3-R-ir. Hypothesis 2: NK3-R activation in other brain regions induces nuclear localization of NK3-R immunoreactivity. Senktide injection into the lateral ventricle has been shown to activate cfos in numerous regions of the CNS that also express H3-senktide binding and NK3-R mRNA and immunoreactivity. This approach will be used to screen for nuclear translocation of NK3-R-ir in areas such as lateral septum, amygdala, hypothalamus (median preoptic nucleus, paraventricular, supraoptic, and arcuate nuclei), substantia nigra, ventral tegmental area, periaqueductal gray, and nucleus tractus solitarius. PUBLIC HEALTH RELEVANCE The proposed studies have potential significance to a wide range of human diseases as a result of 1) the possibility that a new method of regulation of gene expression by neurokinin 3 receptors (NK3-R) may be identified that is applicable to other G-protein coupled receptors and 2) the evidence that NK3-Rs are involved in a large number of neurological and psychiatric disorders including addiction, depression, anxiety, movement disorders, and hypertension.

描述（由申请人提供）：本申请的目的是开发背景信息，以确定生理刺激（低血压）和选择性神经激肽 3 受体（NK3-R）激动剂（senktide）诱导的意外观察的生理意义。下丘脑视上核 (SON) 神经元细胞核中出现 NK3-R 免疫反应性 (ir)。由于 NK3-R 是 G 蛋白偶联受体 (GPCR)，这表明细胞外 NK3-R 配体诱导 NK3-R 从细胞膜易位到细胞核，从而通过 GPCR 直接调节基因表达的有趣可能性。这是令人兴奋的，因为它对其他 GPCR 具有重要意义，因此可能影响许多 GPCR 调节的细胞功能。此外，这一观察结果对于广泛的中枢神经系统功能可能很重要，因为 NK3-R 广泛分布在中枢神经系统中，并且与多种重要的中枢神经系统功能和病理生理学有关，包括盐、可卡因和酒精摄入等动机行为；情绪状态，例如焦虑和抑郁；运动和肌张力障碍；液体、电解质和心血管稳态；和镇痛。因此，这一观察结果有可能影响多种神经系统、精神疾病和稳态疾病的治疗。为了评估这一观察结果的潜在重要性，我们必须 1) 充分表征在 SON 神经元细胞核中观察到的 NK3-R-ir，2) 确定其他 CNS 区域是否也发生相同的现象。因此，本提案的具体目标将检验以下假设： 假设 1：配体诱导 NK3-R-ir 在 SON 神经元细胞核中的出现代表 NK3-R 随受体转位至细胞核的全部或部分内化。为了检验这一假设，我们生成了针对 NK3-R N 端和 C 端区域不同表位的抗体。这些抗体将用于 SON 的免疫组织化学 (IHC) 和蛋白质印迹分析。将比较正常血压、低血压和senktide（一种选择性 NK3-R 激动剂）治疗的大鼠的 SON。蛋白质印迹将用于分析 SON 的胞质和核提取物，以确定 NK3-R-ir 的大小和细胞位置。假设 2：其他脑区的 NK3-R 激活诱导 NK3-R 免疫反应性的核定位。 Senktide 注射到侧脑室已被证明可以激活中枢神经系统许多区域的 cfos，这些区域也表达 H3-senktide 结合和 NK3-R mRNA 和免疫反应性。该方法将用于筛选 NK3-R-ir 在侧隔膜、杏仁核、下丘脑（正中视前核、室旁核、视上核和弓状核）、黑质、腹侧被盖区、导水管周围灰质、和孤束核。 公共卫生相关性 拟议的研究对广泛的人类疾病具有潜在意义，因为 1) 可能会发现一种神经激肽 3 受体 (NK3-R) 调节基因表达的新方法，该方法适用于其他疾病G 蛋白偶联受体；2) 有证据表明 NK3-R 与大量神经和精神疾病有关，包括成瘾、抑郁、焦虑、运动障碍和高血压。