NEUROENDO SIGNAL TRANSDUCTION--REPRODUCTIVE PHYSIOLOGY

神经内信号转导——生殖生理学

• 批准号: 2742894
• 负责人: KAREN Ann GREGERSON
• 金额: $ 27.07万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2742894
• 关键词: Xenopus oocyte; animal puberty; biological signal transduction; calcium ion; dopamine; estrus; female; fertility; gene expression; gene targeting; genetic models; genetically modified animals; hormone regulation /control mechanism; immunocytochemistry; laboratory mouse; lactation; membrane potentials; model design /development; neuroendocrine system; pituitary gland; potassium channel; prolactin; pseudopregnancy; secretion; voltage /patch clamp

Prolactin (PRL), an anterior pituitary hormone, has profound effects on female fertility. In fact, hypersecretion of PRL is the major neuroendocrine-related pathology associated with female infertility. Unfortunately, the underlying defects which lead to the hypersecretion of PRL remain ill-defined. To provide a rational framework for understanding these defects and treating associated pathologies, it is essential to elucidate the cellular and molecular mechanisms of PRL secretion regulation. Normally PRL secretion is under tonic inhibition by hypothalamic dopamine (DA). Periodic, physiological surges of PRL in females require a withdrawal from this dopaminergic inhibition. Since reduced responsiveness to DA is a hallmark of hyperprolactinemic syndromes, our attention has focused on the cellular and molecular basis of DA action on the lactotrope. We have discovered and extensively characterized a DA-activated inwardly-rectifying potassium channel (KDA) in normal lactotropes. KDA activation by DA leads to hyperpolarization of the lactotrope membrane and cessation of calcium-dependent action potentials, the driving force for tonic PRL secretion. In vitro studies have demonstrated a critical role for this KDA channel in the regulation PRL release by DA. Furthermore, the functional expression of KDA is dependent upon estrogen, which may explain a long-recognized modulatory action of estrogen on this system. In these regards, the KDA channel lies at the top of a hierarchy of events in dopaminergic regulation of PRL secretion. As an initial step in elucidating the molecular basis for the physiological role of KDA in lactotropes, we have cloned three different K channel gene products from female anterior pituitary tissue. Based on functional similarities between the native channel and recombinant channels expressed in Xenopus oocytes, two of the gene products are excellent candidate subunits encoding a heteromultimeric KDA. As a logical extension of these studies, we propose to evaluate the role of this effector K channel within its physiological context- the whole animal-through transgenic technology. To this end, we will design and construct "dominant-negative" mutants, capable of inhibiting wild-type KDA function in lactotropes. We will then create a transgenic mouse model in which expression of this mutant is directed to pituitary lactotropes using the PRL promoter. These studies represent a timely and important extension of the P.I.'s work and will ultimately provide insight into the basis of disorders in PRL secretion and their impact on female fertility.

催乳素 (PRL) 是一种垂体前叶激素，对 女性生育能力。 事实上，PRL 分泌过多是主要原因。 与女性不孕相关的神经内分泌相关病理学。 不幸的是，导致分泌过多的潜在缺陷 PRL 的定义仍然不明确。 提供合理的框架 了解这些缺陷并治疗相关病理， 对于阐明 PRL 的细胞和分子机制至关重要 分泌调节。 正常情况下，PRL 分泌处于强直性抑制状态 通过下丘脑多巴胺（DA）。 PRL 周期性生理性激增 女性需要从这种多巴胺能抑制中退出。 由于对 DA 的反应性降低是高催乳素血症的标志 综合症，我们的注意力集中在细胞和分子基础上 DA 对催乳素的作用。 我们发现并广泛表征了 DA 激活的 正常催乳素中的内向整流钾通道（KDA）。 凯达 DA 激活导致乳素膜超极化 和钙依赖性动作电位的停止，驱动力 促进PRL分泌。 体外研究表明了一个关键的 该 KDA 通道在 DA 调节 PRL 释放中的作用。 此外，KDA 的功能表达取决于 雌激素，这可以解释长期以来公认的调节作用 这个系统上有雌激素。 在这些方面，KDA 渠道处于 PRL 多巴胺能调节事件层次的顶部 分泌。 作为阐明分子基础的第一步 KDA 在催乳素中的生理作用，我们克隆了三种不同的 来自女性垂体前叶组织的 K 通道基因产物。 基于 天然通道和重组通道之间的功能相似性 在非洲爪蟾卵母细胞中表达的通道，其中两个基因产物是 编码异源多聚体 KDA 的优秀候选亚基。 作为一个 这些研究的逻辑延伸，我们建议评估 该效应 K 通道在其生理背景下 - 整体 通过转基因技术动物。 为此，我们将设计并 构建“显性失活”突变体，能够抑制野生型 KDA 在催乳素中发挥作用。 然后我们将创造一只转基因小鼠 该突变体的表达针对垂体的模型 使用 PRL 启动子的催乳素。 这些研究代表了及时的 以及 P.I. 工作的重要延伸，最终将提供 深入了解 PRL 分泌紊乱的基础及其影响 关于女性生育能力。