SECRETION OF PROLACTIN--REGULATION BY ENDOTHELIN

这项建议的长期目标是确定内皮素(ETS)在调节生殖激素分泌中的作用。这项建议的目的是围绕阐明ETS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，以响应NICHD的特别强调领域的征集。这项建议的第一个具体目标是表征内皮素在发情周期中控制催乳素功能的生理作用。这将通过检测体内特定的ET拮抗剂对PRL分泌的影响来实现。第二个具体目的是研究ETS在控制PRL分泌中的自分泌和旁分泌作用。这将在体外(A)通过在发情周期不同阶段收集的垂体细胞上使用反向溶血空斑分析方法在单细胞水平上进行评估，以及(B)在多细胞环境中通过在细胞灌流系统中使用垂体细胞微聚体培养来进行评估。ET受体拮抗剂对哺乳细胞分泌活性的影响将在基础条件和促分泌剂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催乳素的垂体细胞和/或已建立的表达ETA受体的催乳素来源的垂体细胞系。我们将使用被认为参与调节催乳素分泌的蛋白激酶级联反应的特定抑制剂。重点是研究蛋白激酶C(PKC)和丝裂原活化蛋白激酶(MAPK)依赖的信号通路的作用。第四个具体目的是建立内皮素对垂体促乳素细胞作用的离子基础。在这些研究中，我们将试图证实我们的假设，即ET诱导的多条信号通路汇聚在一组独特的BKCa通道上，这些通道的调节是ETS对PRL分泌的大部分影响的原因。利用培养的乳酸菌，我们将在细胞贴附和内向外配置中使用膜片钳方法，以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。研究的重点将集中在多巴胺将乳酸菌对ET的反应从抑制转化为刺激的机制上。这些研究将第一次为ETS在调节脑下垂体激素分泌中的生理作用提供一个彻底的表征。这些特定目标的实现将阐明迄今尚未描述的调节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的治疗方法。

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.