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 A2 (PLA2) in the presence of 20 microM 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 Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 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 PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 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+.

ABSTRACT-The modulatory role of protein kinase C on phospholipase A2, activation of which had been suggested to result in acetylcholine release from cholinergic neurons, was studied in longitudinal muscle preparations with the myenteric plexus of guinea pig ileum. The relationship of muscarinic autoinhibition to the modulation was also examined. Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, dose-dependently increased spontaneous and electrical field stimulation-induced acetylcholine releases from the preparation. The inhibitors of protein kinase C, staurosporine and calphostin C, inhibited the stimu latory effects of PDBu, but neither inhibitor affected spontaneous or electrical field stimulation-induced acetylcholine release in the absence of PDBu. On the other hand, atropine significantly increased electrical field stimulation-induced release by blocking a muscarinic autoinhibitory mechanism. Under the auto inhibition blocked condition, U73122, an inhibitor of phospholipase C, and staurosporine significantly inhibited the effect of atropine on electrical field stimulation-induced release. An inhibitor of phospholipase A2, mepacrine, inhibited PDBu-induced acetylcholine release and also inhibited the effect of atropine on electrical field stimulation-induced release. An activator of phospholipase A2, melittin, and a product of the phospholipase, arachidonic acid, increased the spontaneous and electrical field stimulation-induced releases. These results suggest that the phospholipase C protein kinase C system modulates acetylcholine release from cholinergic neurons by activating phospholipase A2 in the myenteric plexus of guinea pig ileum, and the activation of muscarinic autoreceptor may negatively modulate acetylcholine release at a point upstream of the system.

No abstract

No abstract