Neuropeptide regulation of neurohypophyseal function

描述（由申请人提供）：身体渗透压通过向血液中释放抗利尿激素（VP）和催产素（OT）来维持在一个狭窄的生理范围内，其中激素的作用是促进水潴留和钠排泄。速激肽、神经激肽B （NKB）及其NK3受体（NK3R）与VP有非常独特的关系，因为NKB和NK3R在大多数大细胞VP神经元中共同表达。由于预处理NK3R拮抗剂可阻断VP和OT对高渗透压和低血容量/低血压的释放，大细胞NK3R在生理挑战下被激活。此外，NK3R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，并通过直接作用于基因转录在调节大细胞神经元功能方面发挥更持久的作用。通过共聚焦显微镜、免疫电镜和Western blot观察发现，在高渗刺激后，VP大细胞神经元表达的NK3R从细胞膜转移到细胞核。基础条件下未检测到核NK3R。活化的g蛋白偶联受体易位进入细胞核已被假设为转录调控的新范式。然而，尽管NK3R具有新颖性和核信号的重要性，但我们对大细胞PVN神经元的NK3R信号知之甚少。本研究的目的是确定内源性NKB和NK3R激活在大细胞神经元功能中的作用。目的1将验证NK3R信号是PVN中大细胞神经元在应对高渗和低血压挑战时激活的“共同途径”的假设。Aim 2将确定NK3R核易位的时间过程，并确定NKB是否易位到细胞核。实验假设是核NK3R通过蛋白-蛋白相互作用或结合DNA影响基因转录。免疫共沉淀法和染色质免疫共沉淀法将用于鉴定核NK3R的核靶标。目的3将研究持续高渗对PVN中NK3R和NKB mRNA调控的影响。目的4将确定NK3R配体（NKB）的释放源。结果将确定NKB体是PVN固有的或投射到PVN大细胞神经元，并被高渗透压激活。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释放功能障碍有助于人类病理生理，如充血性心力衰竭，研究结果可能为NK3R系统相关人类病理的基因组基础提供新的治疗策略和/或新见解。拟议的研究有可能有助于治疗导致充血性心力衰竭和高血压等病理的神经内分泌激素失调。神经激肽受体向细胞核的核易位是一种新现象，它对许多疾病（如癫痫）都有影响，这些疾病与大脑中的这种受体系统有关。

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.