Roth Christian L, McCormack Ashley L, Lomniczi Alejandro, Mungenast Alison E, Ojeda Sergio R
Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006, USA.
Mol Cell Endocrinol. 2006 Jul 25;254-255:51-9. doi: 10.1016/j.mce.2006.04.017. Epub 2006 Jun 5.
Mammalian puberty requires activation of luteinizing hormone-releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC-MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.
哺乳动物的青春期需要促黄体生成素释放激素（LHRH）神经元被激活。反过来，这些神经元受跨突触和胶质细胞到神经元的通讯通路控制，这些通路利用多种细胞蛋白来实现正常功能。我们现在使用了一种高通量相对定量蛋白质组学技术来鉴定此类蛋白质。我们选择了二维液相色谱串联质谱法（2DLC-MS/MS）和可裂解同位素编码亲和标签（cICAT），以鉴定和定量复杂蛋白质混合物中的单个蛋白质。所使用的蛋白质源自幼年（25日龄）和青春期前（首次发情前期，LP）雌性大鼠的下丘脑，通过数据库搜索分析它们的质谱来确定其身份。检测到五种参与谷氨酸代谢的蛋白质，其中两种似乎存在差异表达。由于它们在控制谷氨酸合成和降解方面的重要性以及在星形胶质细胞中的优先表达，因此选择它们进行进一步分析。一种是谷氨酸脱氢酶（GDH），催化谷氨酸合成；通过2DLC-MS/MS检测到其下丘脑含量在首次发情前期增加。另一种是谷氨酰胺合成酶（GS），催化谷氨酸代谢生成谷氨酰胺；其含量在发情前期减少。蛋白质印迹分析证实了这些结果。因为这些变化表明青春期时谷氨酸生成增加，我们测量了29日龄幼年大鼠下丘脑片段以及用孕马血清促性腺激素（PMSG）处理以诱导促黄体生成素（LH）过早出现发情前期激增的大鼠下丘脑片段释放的谷氨酸。为了确定在没有再摄取情况下谷氨酸的净输出，我们使用了兴奋性氨基酸转运体（EAAT）抑制剂L-反式吡咯烷-2,4-二羧酸（PDC）。PDC从发情前期下丘脑引发了显著更多的谷氨酸和LHRH释放。因此，由胶质细胞来源的谷氨酸能输入为LHRH神经元网络提供的兴奋性驱动增加，是促成LHRH分泌青春期激活的一个事件。
