Pitts G R, Nunemaker C S, Moenter S M
Departments of Internal Medicine, Cell Biology, and the National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908.
Endocrinology. 2001 May;142(5):1858-64. doi: 10.1210/endo.142.5.8137.
Neural control of reproduction is achieved through episodic GnRH secretion, but little is known about the molecular mechanisms underlying pulse generation. The ultradian time domain of GnRH release suggests mechanisms ranging from macromolecular synthesis to posttranslational modification could be involved. We tested if messenger RNA (mRNA) or protein synthesis are components of the pulse generator by determining the effects of transcription and translation inhibitors on episodic GnRH release from immortalized GT1-1 GnRH neurons. Time course and efficacy of transcription and translation blockade were assessed by determining the ability of specific inhibitors to block the robust, rapid induction of c-fos mRNA or protein accumulation by forskolin (10 microM). The transcription inhibitors actinomycin D (ACT-D, 20 microM) or 5,6-dichlorobenzimidazole riboside (DRB, 100 microM), or the translation inhibitors anisomycin (ANI, 10 microM) or puromycin (PUR, 10 microM) were applied to GT1-1 cells 30, 15, or 0 min before forskolin. Northern and Western blots revealed blockade of transcription and translation was rapid and essentially complete. GT1-1 cells were perifused for a 90- to 120-min control period then for 100-130 min with vehicle or inhibitor to examine pulsatile GnRH secretion. GnRH interpeak intervals, peak amplitude, and peak area were not different between control and experimental periods of cells treated with vehicle (n = 15), ACT-D (n = 10), DRB (n = 6), ANI (n = 8), and PUR (n = 6; P > 0.05). This study presents the first clear evidence that the series of reactions resulting in secretion of a GnRH pulse do not include cycles of transcription and translation. Although these mechanisms would be required to replenish components of the pulse generator, they are not integral components of this oscillator. We hypothesize that posttranslational events underlie episodic GnRH release in GT1-1 cells.
生殖的神经控制是通过促性腺激素释放激素（GnRH）的间歇性分泌实现的，但对于脉冲产生的分子机制知之甚少。GnRH释放的超日时间域表明，从大分子合成到翻译后修饰等机制都可能参与其中。我们通过确定转录和翻译抑制剂对永生化GT1-1 GnRH神经元间歇性GnRH释放的影响，来测试信使核糖核酸（mRNA）或蛋白质合成是否为脉冲发生器的组成部分。通过测定特定抑制剂阻断福斯高林（10微摩尔）对c-fos mRNA或蛋白质积累的强烈、快速诱导的能力，评估转录和翻译阻断的时间进程和效果。在加入福斯高林前30、15或0分钟，将转录抑制剂放线菌素D（ACT-D，20微摩尔）或5,6-二氯苯并咪唑核糖苷（DRB，100微摩尔），或翻译抑制剂茴香霉素（ANI，10微摩尔）或嘌呤霉素（PUR，10微摩尔）应用于GT1-1细胞。Northern印迹和Western印迹显示转录和翻译阻断迅速且基本完全。对GT1-1细胞进行90至120分钟的对照灌注期，然后用溶媒或抑制剂灌注100至130分钟，以检查GnRH的脉冲式分泌。用溶媒（n = 15）、ACT-D（n = 10）、DRB（n = 6）、ANI（n = 8）和PUR（n = 6；P>0.05）处理的细胞，其对照期和实验期之间的GnRH峰间间隔、峰值幅度和峰面积没有差异。本研究首次明确证明，导致GnRH脉冲分泌的一系列反应不包括转录和翻译循环。虽然这些机制是补充脉冲发生器组件所必需的，但它们不是该振荡器的组成部分。我们推测翻译后事件是GT1-1细胞中GnRH间歇性释放的基础。
