Neurokinin 3 Receptor: Nuclear Localization in Supraoptic Neurons

描述（由申请人提供）：本申请的目的是发展背景信息，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）诱导下丘脑视上核（SON）神经元核中NK3-R免疫反应性（ir）的意外观察的生理意义。由于NK3-Rs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，这一观察结果可能对广泛的中枢神经系统功能很重要，因为NK3-Rs广泛分布在中枢神经系统中，并与多种重要的中枢神经系统功能和病理生理有关，包括动机行为，如盐、可卡因和酒精摄入；焦虑、抑郁等情绪状态；运动和肌张力障碍；液体、电解质和心血管内稳态；和镇痛。因此，这一观察结果有可能影响各种衰弱的神经、精神和体内平衡疾病的治疗。为了评估这一观察的潜在重要性，我们必须1)充分表征在SON神经元核中观察到的NK3-R-ir， 2)确定同样的现象是否发生在其他中枢神经系统区域。因此，本提案的具体目标将验证以下假设：假设1：配体诱导的NK3-R-ir在SON神经元细胞核中的出现代表了在受体内化后易位到细胞核的全部或部分NK3-R。为了验证这一假设，我们在NK3-R的n端和c端区域产生了针对不同表位的抗体。这些抗体将用于SON的免疫组织化学（IHC）和western blot分析。将比较正常、低血压和senktide（一种选择性NK3-R激动剂）治疗大鼠的SON。Western blots将用于分析SON的胞浆和核提取物，以确定NK3-R-ir的大小和细胞位置。假设2：大脑其他区域的NK3-R激活诱导了NK3-R免疫反应性的核定位。侧脑室注射Senktide已被证明可以激活CNS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、杏仁核、下丘脑（正中视前核、室旁核、视上核和弓形核）、黑质、腹侧被盖区、导尿管周围灰质和孤束核等区域的核易位。所提出的研究对广泛的人类疾病具有潜在的意义，因为1)可能发现一种适用于其他g蛋白偶联受体的神经激肽3受体（NK3-R）调控基因表达的新方法，2)有证据表明NK3-R参与大量神经和精神疾病，包括成瘾、抑郁、焦虑、运动障碍和高血压。

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.