SECRETION OF PROLACTIN--REGULATION BY ENDOTHELIN

催乳素的分泌——内皮素的调节

• 批准号: 6343255
• 负责人: MARC Edward FREEMAN
• 金额: $ 26.89万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343255
• 关键词: autocrine; biological signal transduction; dopamine receptor; endothelin; enzyme activity; estrus; hormone receptor; hormone regulation /control mechanism; immunocytochemistry; laboratory rat; mitogen activated protein kinase; paracrine; pituitary gland; plaque assay; potassium channel; prolactin; protein kinase C; secretion; tissue /cell culture; voltage /patch clamp

The long-term objective of this proposal is to characterize the role of endothelins (ETs) in regulating reproductive hormone secretion. The aims of this proposal revolve around elucidation of the mechanisms whereby ETs' affect pituitary prolactin (PRL) secretion. It is submitted under the area of Reproductive Endocrinology in response to the Special Emphasis Area solicitation of the NICHD. The first specific aim of this proposal is to characterize the physiological role of endothelins in the control of lactotroph function during the estrous cycle. This will be accomplished by examining the effects of specific ET antagonists on PRL secretion in vivo. The second specific aim is to characterize the autocrine and paracrine actions of ETs in controlling PRL secretion. This will be assessed in vitro (a) at the single cell level by using reverse hemolytic plaque assay methods on pituitary cells collected during different stages of the estrous cycle and (b) in a multicellular environment by using pituitary cell micro-aggregate cultures in a cell-perfusion system. The effects of ET receptor antagonists on the secretory activity of lactotrophs will be tested in basal and secretagogue-induced conditions. The third specific aim is to identify signal transduction mechanisms by which ETs affect PRL secretion. We will use dispersed pituitary cells enriched in lactotrophs and/or an established pituitary cell line of lactotroph origin expressing ETA receptors. We will employ specific inhibitors of protein kinase cascades thought to be involved in the regulation of PRL secretion. Specific emphasis will be made on investigating the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) dependent signaling pathways. The fourth specific aim is to establish the ionic basis for the effects of endothelins on pituitary lactotrophs. In these studies we will seek to confirm our hypothesis that ET-induced multiple signaling pathways converge upon a unique population of BKCa channels and that modulation of these channels is responsible for most of the effects of ETs on PRL secretion. Using cultured lactotrophs, we will employ patch clamp methods in cell-attached and inside-out configuration to gain information on gating kinetics at the single channel level. The fifth specific aim is to establish the mechanism of cross-talk between dopaminergic D2 and endothelin ETA receptors. The investigation will focus on the mechanisms by which dopamine transforms the response of lactotrophs to ET from inhibitory to stimulatory. These studies will provide, for the first time, a thorough characterization of the physiological role of ETs in regulating pituitary hormone secretion. Accomplishment of these specific aims will clarify a heretofore undescribed cellular mechanism regulating PRL secretion and suggest novel therapeutic approaches to control secretory functions of the hypothalamo-pituitary axis.

本建议的长期目标是表征内皮素（ETs）在调节生殖激素分泌中的作用。本建议的目的围绕阐明et影响垂体催乳素（PRL）分泌的机制。它是在生殖内分泌学领域下提交的，是应联合国人口与发展会议特别强调领域的邀请而提交的。本提案的第一个具体目的是表征内皮素在发情周期中控制嗜乳功能的生理作用。这将通过检查特异性ET拮抗剂对体内PRL分泌的影响来完成。第二个具体目的是表征ETs在控制PRL分泌中的自分泌和旁分泌作用。这将在体外进行评估(a)在单细胞水平上，通过对在发情周期的不同阶段收集的垂体细胞使用反向溶血斑块测定方法；(b)在多细胞环境下，通过在细胞灌注系统中使用垂体细胞微聚集体培养物。ET受体拮抗剂对乳养菌分泌活性的影响将在基础和分泌诱导条件下进行测试。第三个具体目标是确定ETs影响PRL分泌的信号转导机制。我们将使用分散的富含乳营养物的垂体细胞和/或已建立的表达ETA受体的乳营养物来源的垂体细胞系。我们将使用被认为参与PRL分泌调节的蛋白激酶级联的特异性抑制剂。特别强调将研究蛋白激酶C （PKC）和丝裂原活化蛋白激酶（MAPK）依赖的信号通路的作用。第四个具体目标是建立内皮素对垂体乳养细胞影响的离子基础。在这些研究中，我们将试图证实我们的假设，即et诱导的多种信号通路汇聚在一个独特的BKCa通道群上，这些通道的调节是et对PRL分泌的大部分影响的原因。使用培养的乳营养菌，我们将采用膜片钳方法在细胞附着和内向外配置中获得单通道水平的门控动力学信息。第五个具体目的是建立多巴胺能D2和内皮素ETA受体之间的串扰机制。研究将集中于多巴胺如何将乳营养体对ET的反应从抑制性转变为刺激性的机制。这些研究将首次提供et在调节垂体激素分泌中的生理作用的全面表征。完成这些特定目标将阐明迄今未描述的调节PRL分泌的细胞机制，并提出控制下丘脑-垂体轴分泌功能的新治疗方法。