The role of phospholipase D associated with apical plasma membrabes in post-docking steps of exocytosis

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To clarify the involvement of phospholipase D (PLD) in producing fusogenic mediators during exocytosis, we investigated the regulatory mechanism of PLD activity associated with apical plasma membranes from rat parotid glands. Then, using a cell-free fusion system, coupling between conformational changes in the membranes induced by PLD and the fusion reaction was also investigatedPhosphatidylinositol 4, 5 ・ bisphosphate (PIP_2), which is an authentic activator of PLDs 1 and 2, markedly activated the apical PLD. GTP-y-S, another activator of PLD1, affected neither the basal activity of apical PLD nor its PIP_2, dependent activation. Oleic acid activated the apical PLD and the activation was amplified in the presence of PIP_2. Neomysin, an amino glycoside antibiotic with high affinity for PIP_2, partially inhibited this PLD activity. Pathways producing lipid activators such as PIP_2 and oleic acid were investigated in apical plasma membranes. Then we detected the activities of phosphatidy … More linositol 4-kinase and phosphatidylinositol-4-phosphate kinase in apical plasma membrabes, suggesting that PIP_2 was produced via the sequential pathway of the two enzymes. The Ca^<2+> independent phospholipase A_2 that selectively released unsaturated fatty acids from neutral phospholipids and the Ca^<2+> dependent deacylation from phosphatidylinositol were also found in apical plasma membranes. These enzymes may contribute to a full activation of the apical PLD. In addition, to clarify the post-docking role of the apical PLD, the fusion of exgenous PLD-treated membranes with secretory granules was examined by fluorescence dequenching assay. This study demonstrated the facilitation of fusion by PLD-trearment. To date, our data are insufficient to allow the conclusion that the apical PLD enhances membrane fusion in vivo. However, Dohke et al. [BBRC, 299, 699 (2002)] reported that neomysin partially suppressed Camp-dependent exocytosis in saponin- permeabilized parotid acinar cells. This finding seems to support our conclusion, since apical PLD was also inhibited by neomycin. Further investigation is needed to clarify the physiological role of apical PLD Less