Neurokinin 3 Receptor: Nuclear Localization in Supraoptic Neurons

描述(申请人提供)：本申请的目的是开发背景信息，以确定意外观察到的生理意义，即生理刺激(降压)和选择性神经激肽3受体(NK3-R)激动剂(Senktie)诱导下丘脑视上核(SON)神经元核出现NK3-R免疫反应性(Ir)。由于NK3-R是G蛋白偶联受体，这表明细胞外NK3-R配体诱导NK3-R从细胞膜转位到细胞核，通过GPCR直接调节基因表达的可能性是有趣的。这是令人兴奋的，因为它对其他GPCRs有重要的影响，因此可能会影响许多GPCRs调节的细胞功能。此外，NK3受体广泛分布于中枢神经系统，参与多种重要的中枢神经系统功能和病理生理过程，包括盐、可卡因和酒精摄入等动机行为；焦虑和抑郁等情绪状态；运动和肌张力障碍；液体、电解质和心血管稳态；以及镇痛。因此，这一观察结果有可能影响各种和衰弱的神经、精神和内环境平衡疾病的治疗。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，以及2)确定同样的现象是否发生在其他CNS区域。因此，这一提议的特定目的将检验以下假设：假设1：配体诱导的NK3-R-ir出现在SON神经元的细胞核中，代表了受体内化后移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)和蛋白质印迹分析。我们将比较血压正常、血压较低和刺五加(一种选择性NK3-R激动剂)处理的大鼠的SON。Western blotting将用于分析SON的胞浆和核提取液，以确定NK3-R-ir的大小和细胞位置。假设2：NK3-R在其他脑区的激活诱导NK3-R免疫反应的核定位。侧脑室注射刺五加可激活中枢神经系统许多区域的CFO，这些CFO也表达H3-刺五加结合和NK3-R的mRNA和免疫反应性。这种方法将被用来筛选NK3-R-ir在外侧隔、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、中脑导水管周围灰质和孤束核的核转位。公共卫生相关性拟议的研究对广泛的人类疾病具有潜在的意义，结果是：1)神经激肽3受体(NK3-R)调节基因表达的新方法可能被识别出来，也适用于其他G蛋白偶联受体；2)证据表明NK3-R参与了大量的神经和精神疾病，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.