Neuropeptide regulation of neurohypophyseal function

描述（由申请方提供）：通过将加压素（VP）和催产素（OT）释放到血液中，使身体渗透压维持在狭窄的生理范围内，在血液中激素起促进水潴留和钠排泄的作用。速激肽、神经激肽B（NKB）及其NK 3受体（NK 3 R）与VP有着非常独特的关系，因为NKB和NK 3 R在大多数VP大细胞神经元中共表达。大细胞NK 3R响应于生理挑战而被激活，因为用NK 3R拮抗剂预处理阻断VP和OT释放至高渗透压和低血容量/低血压。此外，NK 3R在细胞信号传导和对高渗透压和低血容量/低血压的即时反应中发挥传统作用，并且通过对基因转录的直接作用在调节大细胞神经元功能中发挥更持续的作用。共聚焦显微镜、免疫电镜和Western blot显示VP大细胞神经元表达的NK 3R在高渗刺激后从细胞膜转位到细胞核。在基础条件下未检测到核NK 3R。活化的g-蛋白偶联受体易位到细胞核中已被假设为转录调控的新范例。然而，尽管NK 3R的新奇和核信号的重要性，我们知道很少NK 3R信号通过大细胞PVN神经元。该建议的目的是确定内源性NKB和NK 3R激活在大细胞神经元功能中的作用。目的1将测试的假设，即NK 3R信号是一个“共同的路径”的大细胞神经元的激活PVN在高渗和膨胀的挑战。目的2将确定NK 3R核转位的时间过程，并确定NKB是否转位到细胞核。实验假设是核NK 3R通过蛋白质-蛋白质相互作用或通过结合DNA影响基因转录。免疫共沉淀和染色质免疫沉淀将用于鉴定核NK 3R的核靶点。目的3研究持续高渗对室旁核NK 3R和NKB mRNA表达的影响。目的4将确定NK 3R配体（NKB）的释放来源。结果将鉴定出PVN固有的或投射到大细胞PVN神经元并被高渗激活的NKB索马。总的来说，这些结果将为膜结合受体作为核转录因子和控制大细胞神经元功能的神经递质系统提供新的见解。VP释放的功能障碍有助于人类病理生理学，如充血性心力衰竭，并且结果可能提示新的治疗策略和/或对与NK 3R系统相关的人类病理学的基因组基础的新见解。 公共卫生相关性拟议的研究有可能有助于治疗神经内分泌激素失调，导致充血性心力衰竭和高血压等病理。神经激肽受体的核转位到细胞核是新颖的，并且对与大脑中的该受体系统相关的大量病理（例如癫痫）具有影响。

DESCRIPTION (provided by applicant): Bodily osmolarity is maintained in a narrow physiological range through the release of vasopressin (VP) and oxytocin (OT) into the blood where the hormones act to promote water retention and sodium excretion. The tachykinin, neurokinin B (NKB) and its NK3 receptor (NK3R) have a very unique relationship to VP because NKB and NK3R are co expressed by a majority of magnocellular VP neurons. Magnocellular NK3R are activated in response to physiological challenges because pretreatment with NK3R antagonists block both VP and OT release to both hyperosmolarity and hypovolemia/hypotension. Furthermore, NK3R play a traditional role in cellular signaling and immediate responses to hyperosmolarity and hypovolemia/hypotension and a more sustained role in regulating magnocellular neurons function through direct actions on gene transcription. Following a hyperosmotic challenge NK3R expressed by VP magnocellular neurons were shown to be translocated from the cell membrane to the cell nucleus by confocal microscopy, immuno-electron microscopy and Western blot. Nuclear NK3R was not detected under basal conditions. The translocation of activated g-protein coupled receptors into the nucleus has been hypothesized as a new paradigm for transcriptional regulation. Yet, in spite of the novelty and nuclear signaling importance of NK3R, we know little about NK3R signaling by magnocellular PVN neurons. The goals of this proposal are to identify the role of endogenous NKB and NK3R activation in magnocellular neuron function. Aim 1 will test the hypothesis that NK3R signaling is a "common path" for the activation of magnocellular neurons in the PVN in response to hyperosmotic and hypotensive challenges. Aim 2 will determine the time course of NK3R nuclear translocation and determine if NKB is translocated to the cell nucleus. The experimental hypothesis is that nuclear NK3R affects gene transcription through protein-protein interactions or by binding DNA. Co-immunoprecipitation and chromatin immunoprecipitation will be used to identify the nuclear targets of nuclear NK3R. Aim 3 will examine the effects of sustained hyperosmolarity on the regulation of NK3R and NKB mRNA in the PVN. Aim 4 will identify the source of release of the NK3R ligand (NKB). Results will identify NKB soma that are intrinsic to the PVN or that project to magnocellular PVN neurons and that are activated by hyperosmolarity. Collectively, the results will provide novel insights into membrane-bound receptors acting as nuclear transcription factors and the neurotransmitter systems controlling magnocellular neuron function. Dysfunction of VP release contributes to human pathophysiology, such as congestive heart failure, and the results may suggest novel treatment strategies and/or new insights into genomic underpinnings of human pathologies associated with NK3R system. PUBLIC HEALTH RELEVANCE The proposed research has the potential to contribute to therapies to deal with the dysregulation of neuroendocrine hormones that contribute to pathologies such as congestive heart failure and hypertension. The nuclear translocation of the neurokinin receptor to the nucleus is novel and has implications for a large number of pathologies, such as epilepsy, that are linked to this receptor system in the brain.