Neuropeptide regulation of neurohypophyseal function

描述(申请人提供)：身体渗透压维持在一个狭窄的生理范围内，通过释放后叶加压素(VP)和催产素(OT)进入血液，荷尔蒙作用于促进水分保持和钠的排泄。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是由大多数大细胞VP神经元共同表达的。大细胞NK3R被激活是为了响应生理挑战，因为NK3R拮抗剂预先阻断了VP和OT对高渗透压和低血容量/低血压的释放。此外，NK3R在细胞信号传递和对高渗透压、低血容量/低血压的即时反应中扮演着传统的角色，并通过直接作用于基因转录而在调节大细胞神经元功能方面发挥更持久的作用。在高渗刺激后，共聚焦显微镜、免疫电子显微镜和Western印迹显示VP大细胞神经元表达的NK3R从细胞膜移位到细胞核。基础条件下未检测到核NK3R。激活的G蛋白偶联受体被认为是转录调控的一种新范式。然而，尽管NK3R的新颖性和核信号的重要性，我们对大细胞PVN神经元的NK3R信号知之甚少。这项建议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验证NK3R信号是室旁核大细胞神经元对高渗和低血压刺激反应的“共同途径”的假说。目的2将确定NK3R核转位的时间进程，并确定NKB是否转位到细胞核。实验假设是核NK3R通过蛋白质-蛋白质相互作用或通过结合DNA来影响基因转录。免疫共沉淀法和染色质免疫沉淀法将用于鉴定核NK3R的核靶点。目的3研究持续高渗对下丘脑室旁核NK3R和NKB基因表达的调节作用。AIM 4将确定NK3R配体(NKB)的释放来源。结果将确定NKB胞体是PVN固有的，或者投射到PVN大细胞神经元，并被高渗透压激活。总之，这些结果将为作为核转录因子的膜结合受体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的病理生理，如充血性心力衰竭，这一结果可能为新的治疗策略和/或与NK3R系统相关的人类病理的基因组基础提供新的见解。与公共卫生相关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疾病的神经内分泌激素失调。神经激肽受体到细胞核的核易位是新的，与大脑中的这种受体系统有关的大量病理变化，如癫痫，都有意义。

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.