Dekker L V, De Graan P N, Spierenburg H, De Wit M, Versteeg D H, Gispen W H
Division of Molecular Neurobiology, Institute of Molecular Biology and Medical Biotechnology, Utrecht, The Netherlands.
Eur J Pharmacol. 1990 Mar 13;188(2-3):113-22. doi: 10.1016/0922-4106(90)90046-z.
Phosphorylation of the neuron-specific substrate of protein kinase C (PKC), B-50 (GAP-43), was studied parallel with noradrenaline release in rat brain synaptosomes. Both could be evoked by treating the synaptosomes with high K+ or veratridine. Phorbol 12,13-dibutyrate enhanced depolarization-induced B-50 phosphorylation and noradrenaline release. To investigate the involvement of PKC-mediated B-50 phosphorylation in noradrenaline release, we applied a variety of kinase inhibitors. Prior to measuring the effects of these inhibitors in intact synaptosomes, we determined their effectivity and specificity in a membrane phosphorylation assay. H-7 most specifically inhibited PKC-dependent phosphorylation, whereas calmidazolium inhibited calmodulin-dependent phosphorylation. Polymyxin B affected both protein kinase systems. Only polymyxin B effectively inhibited noradrenaline release in the intact synaptosomes. We conclude that PKC as well as calmodulin-dependent processes are important for the release event. Data are discussed in view of the presumed function of B-50 as a calmodulin-binding protein.
在大鼠脑突触体中，对蛋白激酶C（PKC）的神经元特异性底物B - 50（GAP - 43）的磷酸化与去甲肾上腺素释放进行了平行研究。用高钾或藜芦碱处理突触体均可诱发二者的变化。佛波醇12,13 - 二丁酸酯增强了去极化诱导的B - 50磷酸化和去甲肾上腺素释放。为了研究PKC介导的B - 50磷酸化在去甲肾上腺素释放中的作用，我们应用了多种激酶抑制剂。在测量这些抑制剂对完整突触体的作用之前，我们在膜磷酸化测定中确定了它们的有效性和特异性。H - 7最特异性地抑制PKC依赖性磷酸化，而钙调蛋白拮抗剂抑制钙调蛋白依赖性磷酸化。多粘菌素B影响这两种蛋白激酶系统。只有多粘菌素B有效地抑制了完整突触体中的去甲肾上腺素释放。我们得出结论，PKC以及钙调蛋白依赖性过程对释放事件很重要。鉴于B - 50作为钙调蛋白结合蛋白的假定功能，对数据进行了讨论。
