Nishio H, Takeuchi T, Hata F, Yagasaki O
Department of Veterinary Pharmacology, College of Agriculture, Osaka Prefecture University, Sakai, Japan.
Biochem J. 1996 Sep 15;318 ( Pt 3)(Pt 3):981-7. doi: 10.1042/bj3180981.
To clarify the mechanism of exocytosis in neurotransmitter release, the fusion of synaptic vesicles with presynaptic membranes prepared from rat brain synaptosomes and concomitant acetylcholine (ACh) release induced by fusion of them were studied in vitro. Fusion of the synaptic vesicles with presynaptic membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B. Synaptic vesicles fused with presynaptic membranes which had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ and released ACh, whereas synaptic vesicles did not interact with non-pretreated membranes. The fusion followed by ACh release depended (i) on the activity of PLA2 during the membrane pretreatment, (ii) on the amount of pretreated membrane and (iii) on the duration of the pretreatment. The presence of Ca2+ ions during the pretreatment was essential for inducing a fusogenic activity of the membranes, but Ca2+ ions were not required for the fusion itself because the fusion experiment was carried out in the presence of 5mM EGTA without added Ca2+. The presence of quinacrine, an antagonist of PLA2, during the membrane pretreatment inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Presence of albumin during the pretreatment, which is an adsorbent of free fatty acids, also inhibited the fusogenic activity. Arachidonic acid, when added during the pretreatment, potentiated the fusogenic activity of the membrane. These findings suggest that the conformational change in the presynaptic membrane phospholipids induced by PLA2 and the presence of arachidonic acid produced by PLA2 are important in the process of fusion of synaptic vesicles with the presynaptic membranes of rat brain, and that the fusion process itself is independent of Ca2+.
为阐明神经递质释放过程中胞吐作用的机制，对大鼠脑突触体制备的突触前膜与突触小泡的融合以及融合诱导的乙酰胆碱（ACh）释放进行了体外研究。用十八烷基罗丹明B通过荧光猝灭测定法测量突触小泡与突触前膜的融合。在20微摩尔钙离子存在的情况下，突触小泡与经猪磷脂酶A2（PLA2）预处理的突触前膜融合并释放乙酰胆碱，而突触小泡不与未经预处理的膜相互作用。随后乙酰胆碱释放的融合取决于：（i）膜预处理期间PLA2的活性；（ii）预处理膜的量；（iii）预处理的持续时间。预处理期间钙离子的存在对于诱导膜的融合活性至关重要，但融合本身不需要钙离子，因为融合实验是在添加了5毫摩尔乙二醇双乙醚二胺四乙酸（EGTA）且未添加钙离子的情况下进行的。膜预处理期间，PLA2拮抗剂奎纳克林的存在抑制了它们的融合活性，这表明激活PLA2很重要。预处理期间白蛋白（游离脂肪酸的吸附剂）的存在也抑制了融合活性。预处理期间添加花生四烯酸可增强膜的融合活性。这些发现表明，PLA2诱导的突触前膜磷脂构象变化以及PLA2产生的花生四烯酸的存在在大鼠脑突触小泡与突触前膜融合过程中很重要，并且融合过程本身与钙离子无关。
