促肾上腺皮质激素释放激素对下丘脑神经元钙信号和CREB的调节作用

背景在急性应激反应中肾上腺皮质激素释放激素可能通过何种信号途径调控下丘脑神经元的神经内分泌活性?目的探讨促肾上腺皮质激素释放激素对下丘脑神经元内CREB含量变化的调节作用.设计重复测量设计.单位解放军第三军医大学大坪医院野战外科研究所,首都医科大学附属天坛医院神经外科.材料实验于1999-12/2002-03在解放军第三军医大学大坪医院完成.选择孕17 d Wistar胎鼠.方法将培养的细胞分组①促肾上腺皮质激素释放激素(10-12,10-10,10-8,10-6 mol/L)刺激组.②预先分别用尼莫地平(5 μmol/L)或CP-154526(500 μmoL/L)处理再用促肾上腺皮质激素释放激素(10-12,10-10,10-8,10-6 mol/L)刺激组.③分别设置相应的对照组,对照组用等渗盐水刺激.用PTI荧光成像系统测量神经元胞浆内游离钙浓度变化,用Western blot方法测定神经元内P-CREB的含量变化.主要观察指标①神经元胞浆内游离钙浓度变化.②神经元内P-CREB的含量变化.结果正常对照组的下丘脑神经元胞浆内游离钙含量较低,外源性促肾上腺皮质激素释放激素刺激后,胞浆内游离钙立即升高;预先应用尼莫地平或CP-154526处理再用促肾上腺皮质激素释放激素刺激的神经元胞浆内游离钙含量的增加明显被抑制,同时也可明显抑制神经元内P-CREB含量的增加.结论在急性应激反应中,促肾上腺皮质激素释放激素可直接与下丘脑神经元促肾上腺皮质激素释放激素1受体结合,开放膜L-型钙离子通道促使钙离子内流使胞浆内游离钙明显增加,从而进一步激活神经元内P-CREB信号通路.说明在调节下丘脑神经元激活过程中促肾上腺皮质激素释放激素可能起了重要的作用.

Regulatory effects of corticotropin release hormone on hypothalamic neuronal calcium signals and CREB

BACKGROUND: Through what signal pathway does corticotropin release hormone (CRH) regulate hypothalamic neuronal neuroendocrine activity during acute stress?OBJECTIVE: To probe into the regulatory effects of CRH on CREB secretion in hypothalamic neurons.DESIGN: Repetitive measurement design.SETTING: At Field Surgery Research Institute of Daping Hospital, Third Military Medical University of Chinese PLA; Neurosurgery Department,Tiantan Hospital Affiliated to the Capital University of Medical Sciences.MATERIALS: This experiment was carried out in Daping Hospital of Third Military Medical University of Chinese PLA between December 1999and March 2002. Rat fetuses were selected from Wister rats of 17-day gestation.METHODS: In vitro cultured cells were divided into the following groups:① CRH (10-12, 10-10, 10-8 and 10-6 mol/L) stimulation groups. ② Pretreated with nimodipine (5 μmol/L) or CP-154526 (500 μmol/L) followed by CRH (10-12, 10-10, 10-8 and 10-6 mol/L ) stimulation groups. ③ Corresponding control groups stimulated with isotonic physiological saline. PTI fluorescence imaging system was used to detect the changes of neuronal cytoplasmic free calcium concentration; meanwhile, Western blot technique was used to determine the changes of neuronal P-CREB content.MAIN OUTCOME MEASURES: ① Changes of neuronal cytoplasmic free calcium concentration. ② Changes of neuronal P-CREB content.RESULTS: The content of cytoplasmic free calcium in hypothalamic neurons was lower in normal control group, and it increased immediately after exogenous CRH stimulation. However, such increase could be suppressed by pretreatment with nimodipine or CP-154526 before CRH stimulation,and the increase of neuronal P-CREB content was also obviously suppressed.CONCLUSION: During acute stress, the combination of CRH with hypothalamic neuronal CRH 1 receptor leads to the opening of membrane Ltype calcium ions channels, thus enhancing the influx of calcium ions and increasing cytoplasmic free calcium ions content, which would further activate P-CREB signal transduction pathway in neurons. It suggests that CRH may play a vital role in hypothalamic neuronal activation.