Cytosolic PLA₂ in Zymogen Granule Fusion and Amylase Release: Inhibition of GTP-induced Fusion by Arachidonyl Trifluoromethyl Ketone Points to cPLA₂ in G-Protein-mediated Secretory Vesicle Fusion

摘要

Previously we reported that the G-protein Gα_i3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A₂ (cPLA₂) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG–PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 μM reduces GTP-induced ZG–PM fusion by 25–50%. Anti-cPLA₂ antibody reduces ZG–PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48–0.64 ratios of cPLA₂ antibody to ZG proteins. PLAP, a cPLA₂ activator synthetic peptide increases ZG–PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG–PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG–PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG–PM fusion suggesting that cPLA₂ is involved in G-protein-mediated secretory vesicle fusion.