促肾上腺皮质激素释放激素对下丘脑神经元CREB的调控

Regulation of P-CREB signals in rat hypothalamic neurons by exogeneous corticotropin—releasing hormone(CRH)

目的:研究促肾上腺皮质激素释放激素(Corticotropin-releasing hormone,CRH)对下丘脑神经元内CREB(CAMP-responsive element-binding protein)含量变化的调节作用。方法:取孕17d胎鼠下丘脑分散培养下丘脑神经元,采用PTI(Photon technology international,PTI)荧光成像系统测量下丘脑神经元胞浆内游离钙浓度([Ca2+]i)变化;测量和分析外源性CRH对培养下丘脑神经元内钙信号变化的影响,用Western blot方法测定神经元内P-CREB的含量变化,分别与用细胞膜L-型钙通道拮抗剂尼莫地平或CRH1受体(CRH1R)特异性拮抗剂CP-154526处理下丘脑神经元后[Ca2+]i和P-CREB的变化作比较。结果:正常对照组的下丘脑神经元[Ca2+]i含量较低,外源性CRH刺激后,[Ca2+]i立即升高;预先应用尼莫地平或CP-154526处理再用CRH刺激的神经元[Ca2+]i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,CRH可直接与下丘脑神经元CRH1R结合,开放膜L-型钙离子通道促使钙离子内流使[Ca2+]i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CRH可能起了重要的作用。

Objective：To study the function and regulation of CREB signaling in rat hypothalamic neurons by exogeneous corticotropin-releasing hormone（CRH）. Methods：Perifused primary cultures of rat hypothalamic neurons Intracelluar free Ca^2＋ concentr ation（[Ca^2＋]i） was assayed at 32℃ by fluorescence analysis,the coverslip was mounted in a Delta Scan spectrofluorimeter（Photon Technology International）;and phospho-CREB by western blot. Results：The basal lower intracellular Ca^2＋ level of perifused primary cultures of rat hypothalamic neurons cultured on coverslipy was markedly increased by CRH,and significantly attenuated by application of nifedipine（Nif） or CP-154526. CRH may cause the increase of phospho-CREB in the neuron of hypothalamus, while CP-154526,Nif can significantly inhibit the production of phospho-CREB. Conclusion： CRH may excite hypothalamic neurons directly suggest that phospho-and [Ca^2＋]i,suggesting a role of CRH type 1 receptor in the activation of CRH gene expression.