Walaas S I
Department Group of Basic Medical Sciences, University of Oslo, Norway.
Neurochem Int. 1999 Mar;34(3):221-33. doi: 10.1016/s0197-0186(99)00007-8.
The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.
在体外分离的神经末梢中，研究了活性佛波酯、星形孢菌素以及肌动蛋白动力学变化，对由膜去极化剂或钙离子载体诱导的[3H] - 标记去甲肾上腺素的钙依赖性胞吐作用可能产生的影响。用30 mM KCl或1 mM 4 - 氨基吡啶使电压依赖性钙通道去极化诱导的开放，引起了[3H]去甲肾上腺素有限的胞吐作用，推测是从一个易于释放的囊泡池释放的。应用钙离子载体A23187（10 microM）诱导了更广泛的[3H]去甲肾上腺素释放，推测是从细胞内储备囊泡释放的。用佛波醇12 - 肉豆蔻酸酯、13 - 乙酸酯刺激蛋白激酶C增加了所有促分泌剂诱发的释放。星形孢菌素（1 microM）对去极化诱导的释放没有影响，但减少了离子载体诱导的释放，并逆转了佛波酯的所有作用。当释放由去极化诱导时，肌动蛋白不稳定剂脱氧核糖核酸酶I内化到突触体中使[3H]去甲肾上腺素释放略有增加，并强烈增强了佛波酯的增强作用。相反，当释放由钙离子载体诱导时，无论有无佛波酯，脱氧核糖核酸酶I都没有作用。结果表明，去甲肾上腺素能大鼠突触体的去极化诱导了来自对星形孢菌素不敏感的可释放囊泡池的钙依赖性释放。蛋白激酶C的激活通过对星形孢菌素敏感的机制增加了这种释放，并且肌动蛋白细胞骨架的不稳定进一步增强了蛋白激酶C的这种作用。相反，离子载体诱导的去甲肾上腺素释放起源于对星形孢菌素敏感的囊泡池，并且尽管蛋白激酶C的激活增加了从该池的释放，但脱氧核糖核酸酶I没有作用，也不改变蛋白激酶C的作用。这些结果支持去甲肾上腺素能中枢神经系统神经末梢中存在两个功能不同的分泌囊泡池，它们受到蛋白激酶C和肌动蛋白细胞骨架以不同方式的调节。
