CONTROL OF CELLS OF THE REPRODUCTIVE AXIS

生殖轴细胞的控制

• 批准号: 6272021
• 负责人: BERTIL HILLE
• 金额: $ 16.72万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272021
• 关键词: calcium flux; exocytosis; follicle stimulating hormone; gonadotropin releasing factor; gonadotropins; hormone regulation /control mechanism; inositol phosphates; luteinizing hormone; membrane activity; reproductive hormone; single cell analysis; spectrometry; sperm; tissue /cell culture; voltage /patch clamp; western blottings

The goal is to fill in gaps concerning our electrophysiological understanding of GnRH neurons and gonadotropes in order to learn how they are stimulated to secrete and if they have intrinsic mechanisms that would explain the rhythmicity and pulse shape of secretion. Three cell types will be used initially: a neuron-like GnRH-secreting cell line derived from a transgenic mouse tumor; a gonado trope-like alphaT-3 cell line derived from a transgenic mouse pituitary tumor; and juvenile and adult gonadotropes enzymatically dissociated from salmon anterior pituitaries. Responses will be studied primarily by membrane biophysical techniques including: whole-cell and patch clamp methods to characterize the ionic channels and their response to GnRH in these cells; changes of membrane capacitance as an index of exocytosis of secretory vesicles; and intracellular calcium changes with indo 1 fluorescent dye. The specific aims are: 1) To inventory the voltage-gated ionic channels and to determine the electrical activity they can support. 2) To inventory the fast ligand-gated channels. 3) To determine the actions of GnRH on channels, intracellular calcium, and secretion. 4) To determine modulatory actions of other peptides and neurotransmitters acting through G-protein- coupled receptors. 5) To explore the intracellular signaling pathways used for responses to GnRH and other modulatory transmitters. 6) To test hypotheses regarding the electrophysiological basis of excitation, inhibition, and ultradian rhythmicity.

目标是填补我们电生理学方面的空白 了解 GnRH 神经元和促性腺激素，以便了解它们如何 被刺激分泌，如果它们有内在的机制， 解释分泌物的节律性和脉冲形状。 三种细胞类型 最初将使用：源自神经元样 GnRH 分泌细胞系 来自转基因小鼠肿瘤；性腺样 alphaT-3 细胞系 源自转基因小鼠垂体瘤；以及青少年和成人 从鲑鱼垂体前叶酶解出的促性腺激素。 主要通过膜生物物理技术研究反应 包括：全细胞和膜片钳方法来表征离子 这些细胞中的通道及其对 GnRH 的反应；膜的变化 电容作为分泌囊泡胞吐作用的指标；和 indo 1 荧光染料会改变细胞内的钙。 具体的 目标是：1）清点电压门控离子通道并 确定它们可以支持的电活动。 2) 盘点 快速配体门控通道。 3) 确定GnRH对GnRH的作用 通道、细胞内钙和分泌。 4) 确定调制 其他肽和神经递质通过 G 蛋白发挥作用 偶联受体。 5) 探索所使用的细胞内信号通路 用于对 GnRH 和其他调制发射器的响应。 6) 测试 关于兴奋的电生理学基础的假设， 抑制和超电节律性。