REGULATION OF PITUITARY HORMONE SECRETION

垂体激素分泌的调节

基本信息

批准号：
6107981
负责人：
Kevin J Catt
金额：
--
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

The goals of this projects are to identify the mechanisms that control the secretion of gonadotropic hormones from the pituitary gland, and to clarify the molecular and cellular processes involved in the secretion and actions of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). A major component of this work addresses the structure-function properties and signal transduction mechanisms of the GnRH receptor in pituitary gonadotrophs and hypothalamic neurons. The GnRH receptor is unique among the G protein-coupled receptors in lacking a C-terminal cytoplasmic domain, and has several other unusual structural features including a highly basic first intracellular loop. The GnRH receptor is coupled primarily to the phosphoinositide/calcium signaling pathway through Gq/11, but also activates the adenylyl cyclase system through Gs. A mutational analysis of the amino acids in the first intracellular loop of the GnRH receptor expressed in COS-7 cells has identified four residues that are essential for coupling to Gs and activation of adenylyl cyclase. None of these amino acids was required for coupling to Gq and activation of phospholipase C. These and other findings have shown that signaling through Gs is mediated by the first intracellular loop of the GnRH receptor, while signaling through Gq to phospholipase C depends on interactions with the second and third intracellular loops. The presence of positively charged residues is important for the interaction between the first loop and Gs, and any imbalance of the overall charge at the C-terminus of the loop impairs the cyclic AMP response to receptor activation. The regulation of GnRH secretion in vivo has long been known to involve the cholinergic system, as well as the more recently studied aminergic and peptidergic systems. Current studies on the role of cholinergic action in the control of GnRH secretion have suggested that acetyl choline is produced by immortalized GnRH neurons (GT1 cells) and have shown that both GT1 cells and hypothalamic neurons are regulated by cholinergic receptors. The GnRH release profiles of cells stimulated with acetyl choline were shown to result from activation of both nicotine and muscarinic receptors, the former exerting stimulatory and the latter predominantly inhibitory effects on neurosecretion. Agonist-induced changes in Gq and Gi a-subunits in cell membranes during muscarinic receptor stimulation were correlated with activation of phospholipase C an adenylyl cyclase, respectively. The finding that acetyl choline differentially modulates GnRH release from hypothalamic neurons through nicotinic, and both M1 and M2 receptors, indicates the importance of cholinergic inputs in the complex control of neurosecretion.Further studies on the pulsatile release of GnRH in vitro were performed on immortalized GnRH neurons (GT1-7 cells) and cultured fetal hypothalamic cells. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons were found to co-express both GnRH peptide and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1-7 cells, treatment with GnRH receptor antagonists caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1-7 cells, GnRH antagonist analogs abolished the basal mode of pulsatile secretion and caused a sustained and progressive increase in GnRH release. In contrast, treatment with a GnRH receptor agonist reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1-7 cells. These findings have demonstrated that functional GnRH receptors are expressed in cultured hypothalamic GnRH neurons, as formerly shown for the GT1-7 line of immortalized GnRH neurons. In addition, the activation of such autoreceptors is required for pulsatile GnRH release from the hypothalamic GnRH neurons, and that their activation is required for pulsatile GnRH release from the hypothalamic GnRH neuronal network in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.

该项目的目标是确定控制促性腺激素的分泌的机制从垂体腺体阐明分子和细胞下丘脑的分泌和行动所涉及的过程脱肽，促性腺激素释放激素（GNRH）。专业这项工作的组成部分解决了结构功能的属性 GNRH受体的信号转导机制垂体促性腺营养和下丘脑神经元。 gnrh 受体在G蛋白偶联受体中是独一无二的缺乏C末端细胞质结构域，并且还有其他几个不寻常的结构特征，包括高度基本的第一细胞内环形。 GnRH受体主要耦合到通过GQ/11的磷酸肌醇/钙信号通路，但还通过GS激活腺苷酸环化酶系统。一个突变分析氨基酸在第一个细胞内环中的分析在COS-7细胞中表达的GnRH受体已鉴定出四个对于耦合到GS和激活至关重要的残留物腺苷酸环化酶。这些氨基酸都不需要耦合到GQ和磷脂酶C的激活。这些和其他调查结果表明，通过GS信号传导是由 GNRH受体的第一个细胞内环，信号传导通过GQ到磷脂酶C取决于与第二和第三个细胞内环。积极的存在充电残留物对于第一个之间的相互作用很重要循环和GS，以及整体费用的任何不平衡环的C末端会损害周期性AMP对受体的反应激活。长期以来，体内GnRH分泌的调节一直是已知涉及胆碱能系统，以及更多最近研究了AMINEMERGIC和肽吉尼系统。当前的研究关于胆碱能作用在控制GnRH分泌中的作用已经提出乙酰胆碱是通过永生化产生的 GnRH神经元（GT1细胞），并表明GT1细胞和下丘脑神经元受胆碱能受体调节。这用乙酰胆碱刺激的细胞的GnRH释放曲线为尼古丁和毒蕈碱激活所致受体，前者发挥刺激性和后者主要抑制神经分泌的作用。激动剂诱导的细胞膜中GQ和GI A-Subunits的变化在毒蕈碱受体刺激期间与磷脂酶C的激活分别激活腺苷酸环化酶。这发现乙酰胆碱会差异调节GnRH释放从下丘脑神经元到烟碱，以及M1和M2 受体，表明胆碱能输入的重要性神经分泌的复杂控制。在永生的GnRH上进行了GNRH的释放神经元（GT1-7细胞）和培养的胎儿下丘脑细胞。这样的培养物以及成年大鼠的下丘脑组织表达 GNRH受体的存在证明了高亲和力 GnRH结合位点和GNRH受体转录本。此外，发现单个GNRH神经元共表达二个GNRH 双重免疫染色显示的肽和GNRH受体下丘脑文化。在下丘脑神经元的静态培养物中和GT1-7细胞，用GnRH受体拮抗剂处理 GNRH释放显着增加。在下丘脑中细胞和GT1-7细胞，GnRH拮抗剂类似物废除了基础脉动分泌的模式，并引起了持续和进步的 GNRH释放的增加。相反，用GnRH治疗受体激动剂降低了频率并增加了幅度如先前在GT1-7细胞中观察到的脉冲GnRH释放。这些发现表明功能性GNRH受体在培养的下丘脑GNRH神经元中表达，以前为永生的GnRH神经元的GT1-7线显示。在此外，需要激活此类自身受体从下丘脑GNRH神经元中释放脉冲GnRH，并脉冲GnRH从体外下丘脑GNRH神经元网络。效果 GnRH激动剂和神经肽释放的拮抗剂类似物是与Ultrashort-loop自分泌的操作一致发挥正面和负面作用的反馈机制对于GNRH分泌的综合控制是必需的来自下丘脑。