Ohbayashi K, Fukura H, Inoue H K, Komiya Y, Igarashi M
Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi, Japan.
J Neurosci Res. 1998 Mar 15;51(6):682-96. doi: 10.1002/(SICI)1097-4547(19980315)51:6<682::AID-JNR3>3.0.CO;2-7.
Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca2+]i than that in adult synaptosomes. Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca2+-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of alpha-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca2+ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca2+-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca2+-dependent degradation of brain spectrin and the release of alpha-actinin by calpain.
尽管生长锥对多种神经突生长调节因子有反应，如神经营养因子、神经递质和细胞黏附分子，但这些调节因子在生长锥中的信号转导机制尚不清楚。由于最近的研究表明，这些调节因子的信号是由钙离子通过生长锥中的L型电压敏感性钙离子通道（VSCCs）内流介导的，我们使用发育中大鼠前脑分离的生长锥（IGCs）研究了L型VSCC依赖性信号通路。结合试验表明，L型VSCC在生长锥膜中富集，且在发育过程中数量逐渐减少，而N型VSCC则有相反的趋势。在完整的IGCs中，Bay K 8644（BK，一种L型激动剂）诱导的[Ca2+]i升高比成年突触体中快得多。BK增强了蛋白激酶C（PKC）对生长锥中GAP-43和MARCKS蛋白的钙离子依赖性磷酸化，导致这些蛋白从膜上释放，这与我们最近的报告一致。此外，BK或GABA也增强了钙蛋白酶对脑血影蛋白（血影蛋白）的钙离子依赖性降解，从而诱导α-辅肌动蛋白从生长锥的膜骨架上释放。PKC和钙蛋白酶的活性不受另一种抑制剂的抑制，表明这些反应是独立发生的。我们的结果表明，通过L型VSCCs的钙离子内流激活了两个不同的信号分支，可能在生长锥的不同区域，即GAP-43和MARCKS蛋白的钙离子依赖性磷酸化，以及脑血影蛋白的钙离子依赖性降解和钙蛋白酶介导的α-辅肌动蛋白的释放。
