Brewer G J
Southern Illinois University School of Medicine, Springfield, Illinois 62794-1220, USA.
J Neurochem. 1996 Jul;67(1):119-30. doi: 10.1046/j.1471-4159.1996.67010119.x.
Culture of rat embryonic hippocampal neurons in serum-free B27/Neurobasal for 4 days enabled tests of the effect of added thrombin on differentiated cell morphology and processing of the amyloid precursor protein (APP). By fluorescence microscopy of neurons labeled with dil and by scanning electron microscopy, an increase in spreading of the neuron soma was clearly seen in cells treated with 1 microg/ml (27 nM) of thrombin for 24 h. This treatment also caused a dose-dependent increase in immunoreactive area/cell, detected with antibody 4G8 binding to the beta-amyloid region of APP. Thrombin treatment also produced a dose-dependent increase in immunoreactive brightness detected with the Alz-50 antibody. Thrombin did not affect viability or cause neurite retraction. The thrombin effect on 4G8 immunoreactivity required 24 h for full effect and could be blocked by the thrombin inhibitor antithrombin III or hirudin. A thrombin receptor appeared to be activated because a full immunoreactive response was observed by treatment of neurons with the thrombin receptor-activating peptide SFL-LRNPNNKYEPF. When cytoplasmic extracts were analyzed by western immunoblots or by pulse-chase radiolabeling, no thrombin-dependent changes in processing of 127- and 120-kDa bands were seen. Material migrating in the region of synthetic betaA4 was not found. Together, these results suggest that thrombin acts on neurons through a thrombin receptor to stimulate cell spreading and redistribution of APP without amyloidogenic changes. The adhesion responsible for this spreading could be important in altering synaptic connections in the brain.
将大鼠胚胎海马神经元在无血清的B27/Neurobasal培养基中培养4天，能够测试添加的凝血酶对分化细胞形态和淀粉样前体蛋白（APP）加工的影响。通过对用dil标记的神经元进行荧光显微镜观察和扫描电子显微镜观察，在用1微克/毫升（27纳摩尔）凝血酶处理24小时的细胞中，明显可见神经元胞体的伸展增加。这种处理还导致与APP的β淀粉样区域结合的抗体4G8检测到的免疫反应面积/细胞呈剂量依赖性增加。凝血酶处理还使Alz-50抗体检测到的免疫反应亮度呈剂量依赖性增加。凝血酶不影响细胞活力或导致神经突回缩。凝血酶对4G8免疫反应性的影响需要24小时才能完全显现，并且可以被凝血酶抑制剂抗凝血酶III或水蛭素阻断。似乎凝血酶受体被激活了，因为用凝血酶受体激活肽SFL-LRNPNNKYEPF处理神经元可观察到完全的免疫反应。当通过蛋白质免疫印迹或脉冲追踪放射性标记分析细胞质提取物时，未发现凝血酶依赖性的127 kDa和120 kDa条带加工变化。在合成βA4区域迁移的物质未被发现。总之，这些结果表明，凝血酶通过凝血酶受体作用于神经元，刺激细胞伸展和APP的重新分布，而不发生淀粉样变。这种伸展所涉及的黏附可能对改变大脑中的突触连接很重要。
