SECRETION OF PROLACTIN--REGULATION BY ENDOTHELIN

催乳素的分泌——内皮素的调节

• 批准号: 6043640
• 负责人: MARC Edward FREEMAN
• 金额: $ 26.61万
• 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/6043640
• 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.

该提案的长期目标是表征内皮蛋白（ETS）在调节生殖激素分泌中的作用。 该提案的目的围绕着阐明ETS影响垂体催乳素（PRL）分泌的机制。 它是根据生殖内分泌学领域提交的，以应对NICHD的特殊强调区域征集。 该提案的第一个具体目的是表征内皮蛋白在发情周期中控制乳酸营养功能中的生理作用。这将通过检查特定ET拮抗剂对体内PRL分泌的影响来实现。 第二个具体目的是表征ET在控制PRL分泌过程中的自分泌和旁分泌作用。 这将在单个细胞水平上在体外（a）通过在垂体周期不同阶段收集的垂体细胞上的反向溶血斑块测定方法以及在多细胞环境中收集的反向斑块测定方法，并使用垂体细胞微细胞在细胞 - 渗透系统中使用垂体细胞微型凝聚力。 ET受体拮抗剂对乳营养素分泌活性的影响将在基底和促分泌诱导的条件下进行测试。 第三个具体目的是确定ETS影响PRL分泌的信号转导机制。 我们将使用富含乳腺营养的垂体细胞和/或乳腺营养起源表达ETA受体的已建立的垂体细胞系。 我们将采用被认为与PRL分泌有关的蛋白激酶级联反应的特定抑制剂。 将特别强调研究蛋白激酶C（PKC）和有​​丝分裂原激活的蛋白激酶（MAPK）依赖信号通路的作用。 第四个具体目的是建立内皮蛋白对垂体乳营养素的影响的离子基础。在这些研究中，我们将寻求确认我们的假设，即ET诱导的多个信号通路会汇聚在独特的BKCA通道群体上，并且对这些通道的调节构成了ETS对PRL分泌的大多数影响。使用培养的乳营养素，我们将在细胞连接和内而外的构型中采用斑块夹方法，以获取有关单个通道水平的门控动力学的信息。 第五个具体目的是建立多巴胺能D2和内皮素ETA受体之间的串扰机理。 该研究将重点放在多巴胺将乳突对ET的反应从抑制性转化为刺激性的机制上。这些研究将首次提供ETS在调节垂体激素分泌中的生理作用的彻底表征。 这些特定目标的完成将阐明迄今调节PRL分泌的未描述的细胞机制，并提出了用于控制下丘脑 - 垂体轴的分泌功能的新型治疗方法。