EFFECTS OF SELECTIVE-INHIBITION OF PROTEIN-KINASE-C, CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, AND CA2+-CALMODULIN-DEPENDENT PROTEIN-KINASE ON NEURITE DEVELOPMENT IN CULTURED RAT HIPPOCAMPAL-NEURONS
EFFECTS OF SELECTIVE-INHIBITION OF PROTEIN-KINASE-C, CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, AND CA2+-CALMODULIN-DEPENDENT PROTEIN-KINASE ON NEURITE DEVELOPMENT IN CULTURED RAT HIPPOCAMPAL-NEURONS
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DOI：
10.1016/0736-5748(93)90007-z
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发表时间：
1993-06-01
影响因子：
1.8
通讯作者：
AUDESIRK, G
中科院分区：
文献类型：
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作者：
CABELL, L;AUDESIRK, G
许多实验证据表明，钙调蛋白和蛋白激酶，尤其是蛋白激酶C，可能参与调节培养神经元的轴突发育，特别是轴突的起始。然而，结果有些矛盾。此外，钙调蛋白和蛋白激酶在轴突发育的许多方面的作用，如轴突和树突的分支或延长，还没有得到广泛的研究。培养的胚胎大鼠海马锥体神经元发育出易于识别的轴突和树突。我们使用这种培养系统和新一代高度特异的蛋白激酶抑制剂来研究蛋白激酶和钙调蛋白在轴突发育中的作用。神经元在持续存在的条件下培养2天，分别加入Caphostin C(蛋白激酶C的特异性抑制剂)、KT5720(环磷酸腺苷依赖的蛋白激酶的抑制剂)、KN62(钙调素依赖的蛋白激酶II的抑制剂)或Calmidazolium(钙调素的抑制剂)，每种药物的浓度约为文献报道浓度的1-10倍，使每种蛋白激酶的抑制率达到50%。我们还测试了佛波醇12-肉豆蔻酸盐13-乙酸酯(蛋白激酶C的激活剂)和4α-佛波醇12，13-十二酸(一种非活性佛波醇酯)的作用。在对神经元活性没有影响的浓度下，Calphostin C减少了神经突起的起始和轴突的分支，而对每个神经元的树突数量、树突分支、树突长度或轴突长度没有显著影响。与非活性的4α-佛波醇12，13-十二酸相比，佛波醇12-肉豆蔻酸13-乙酸酯增加了轴突分支和每个细胞的树突数量。KT5720仅抑制轴突分支。KN62减少了轴突长度、每个神经元的树突数量以及轴突和树突分支。在低浓度时，卡咪唑对轴突发育的任何方面都没有影响，但在高浓度时，卡咪唑抑制了所测量的每一个参数(包括活力)。这些结果表明，这三种蛋白激酶选择性地调节轴突发育的不同方面。由于钙调蛋白抑制作用的普遍性，因此不可能确定是否有特定的钙调蛋白作用靶点参与轴突的发育。最后，我们的数据表明，一些表面上相似的神经元分化特征，如轴突的起始和分支，可能受到完全不同的分子机制的控制。
A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca2+-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested.At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability).These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The universality of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.