八肽胆囊收缩素对小鼠神经元蛋白质磷酸化的影响

Effects of CCK_8 on protein phosphorylation in mice neurons

目的:从蛋白质可逆磷酸化修饰的角度,对小鼠神经元在八肽胆囊收缩素(CCK8)刺激下的信号转导通路进行初步研究。方法:将培养的小鼠神经元分为对照组(加入无磷培养基)、实验组(加入CCK8)、拮抗剂组(分别加入CCKA受体拮抗剂L-364718和(或)CCKB受体拮抗剂L-365260)。采用双向电泳法观察CCK8和受体拮抗剂作用下蛋白质磷酸化水平的改变。结果:CCK8作用不同时间后,神经元蛋白质磷酸化状态发生明显改变,涉及的信号转导相关磷酸化蛋白质包括多种蛋白激酶、胞内信号分子、激素受体、转录因子等;动力学分析结果显示,主要的信号转导相关蛋白的磷酸化时间动力学曲线多数呈峰型;应用CCK受体拮抗剂作用后的结果表明,在皮质神经元中同时存在CCK的2种类型受体,其中B型受体介导的信号转导途径更复杂。结论:CCKA型、B型受体均可介导CCK8在神经元的信号转导,其中B型受体介导的信号途径可能包括肌醇磷脂信使系统、cAMP-PKA途径、MAPK途径和JNK途径等。

Aim： To explore the intracellular signaling transduction pathways in mice neurons stimulated by octapeptide cholecystokinin（ CCK8 ）. Methods：The cultured neurons were divided into control group（ DMEM , experiment group （ CCK8 ） , and antagonist groups（ L-364 718 and （or） L-365 260）. The changes of protein phosphorylation were analyzed in response of CCK8 and CCK8 receptor specific antagonists. Results： The levels of phosphorylation changed significantly with CCK8 stimulation for different time in mice neurons. The qualitative analysis revealed that various protein phosphorylation modifications were involved in CCK signaling transduction in neuron, including protein kinase, intracellular signal molecular, hormone receptor, transcription factor et al. The time courses of proteins phosphorylation modification demonstrated that most of the curves were peak form. The change of protein phosphorylation took place in neurons when treated with CCK receptor antagonist. This evaluated that there were two types of CCK receptor simultaneous in mice neurons, but CCKB probably played a more important role. Conclusion： Both CCKA and CCKB lead to the signaling transduction of CCK8 in neuron. It is suggested that phosphoinositide signal system, cAMP-PKA,MAPK, and JNK pathways are involved in CCKB mediated signaling transduction.