CA2+-INDEPENDENT FUSION OF SECRETORY GRANULES WITH PHOSPHOLIPASE A(2)-TREATED PLASMA-MEMBRANES IN-VITRO

The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A(2) (PLA(2)) in the presence of 20 mu M Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca2+-independent, On the other hand, the presence of EGTA or 100 mu M quinacrine, an inhibitor of PLA(2), during treatment of plasma membranes with PLA(2) inhibited their fusogenic activity, suggesting the importance of activation of PLA(2). Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA(2) treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA(2) treatment enhanced the membrane-fusion-inducing effect of PLA(2). Pre-treatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA(2) and the presence of arachidonic acid or linoleic acid produced by PLA(2) are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.