Ca²⁺-independent fusion of synaptic vesicles with phospholipase A₂-treated presynaptic membranes in vitro

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 µM 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 5 mM 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⁺.