Campenot R B, Draker D D, Senger D L
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
J Neurochem. 1994 Sep;63(3):868-78. doi: 10.1046/j.1471-4159.1994.63030868.x.
Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by > 50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca(2+)-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 microM PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca(2+)-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC50 value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.
此前，我们观察到，在分隔培养中用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）对大鼠交感神经元的远端神经纤维进行长期处理，会使神经突伸长速率降低超过50%。在本报告中，我们表明，通过对PKC特异性八肽底物的钙依赖性磷酸化测定，可以在远端神经突提取物中测量蛋白激酶C（PKC）活性。我们发现，以这种方式测量时，将1 microM PMA局部应用于远端神经突24小时会导致钙依赖性PKC活性几乎完全下调。我们确定PMA对神经突伸长的抑制是由神经突中的局部机制介导的，因为将PMA局部应用于含有细胞体和近端神经突的中央隔室不会抑制远端神经突的伸长速率。然后，我们研究了最近可用的PKC抑制剂钙泊三醇C和白屈菜红碱的作用，发现与PMA一样，将它们局部应用于远端神经突时会抑制其生长，而应用于细胞体和近端神经突时则没有效果。然而，钙泊三醇C对神经突生长的抑制发生在远低于其蛋白激酶抑制IC50值的浓度下，并且即使在经PMA预处理的神经元中，钙泊三醇C和白屈菜红碱也会抑制远端神经突生长。因此，似乎这些药物并非都通过相同的机制抑制神经突生长。尽管交感神经元中参与神经突伸长的PKC活性尚未精确界定，但本研究中呈现的这些数据表明，定位于生长锥的蛋白激酶在调节轴突生长中发挥着复杂而重要的作用。
