Inhibition of Transferrin Recycling and Endosome Tubulation by Phospholipase A2 Antagonists

Abstract

We report here that a broad spectrum of phospholipase A 2 (PLA 2 ) antagonists produce a concentrationdependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (ϳ1 M), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (ϳ10 M), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA 2 antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA 2 antagonists. And third, endosome tubule formation in a cell-free, cytosoldependent reconstitution system was equally sensitive PLA 2 antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA 2 and that PLA 2 -dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA 2 activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.

Inhibition of Membrane Tubule Formation and Trafficking by Isotetrandrine, an Antagonist of G-protein-regulated Phospholipase A2 Enzymes

“…ITD also inhibited endosome tubule formation with a resultant enlargement of Tf receptor-positive endosomes, similar to that previously observed with PLA 2 antagonists (de Figueiredo et al, 2001). In the case of PLA 2 antagonists, endosome enlargement appeared to result from the inhibition of receptor and membrane export from endosomes.…”

“…ITD inhibition of Golgi, TGN, and endosome tubule formation is similar to that exhibited by well-characterized PLA 2 antagonists (de Figueiredo et al, 1998(de Figueiredo et al, , 2001). An additional similarity is that ITD led to the fragmentation of Golgi ribbons into physically separate stacks, consistent with previous studies which concluded that the steady-state architecture of the interconnected Golgi complex requires the dynamic formation of PLA 2 -mediated membrane tubules .…”

“…Previous studies have demonstrated that PLA 2 antagonists also inhibit BFA-stimulated tubule formation from endosomes and the normal exit of transferrin (Tf) and Tf receptors, i.e., endocytic recycling, from endosomes (de Figueiredo et al, 2001). These studies suggest that PLA 2 activity is a general requirement for at least certain types of membrane tubule formation.…”

“…For example, a wide spectrum of PLA 2 antagonists, including those specific for cytoplasmic Ca ϩ -independent PLA 2 enzymes, are potent inhibitors of brefeldin A (BFA)-stimulated tubule formation from the Golgi complex, trans-Golgi network (TGN), and endosomes, and tubule-mediated trafficking pathways from these organelles (de Figueiredo et al, 1998(de Figueiredo et al, , 2000(de Figueiredo et al, , 2001Drecktrah and Brown, 1999;Polizotto et al, 1999). The mechanisms responsible for PLA 2 -mediated tubule formation likely include the localized accumulation of inverted coneshaped lysophospholipids (LPLs) that are generated by PLA 2 hydrolysis.…”

Phospholipase A2 Inhibitors or Platelet-activating Factor Antagonists Prevent Prion Replication

“…These observations raise the possibility that the activation of PLA 2 seen in prion infected cells and the production of PAF may encourage the formation of PrP res by enhancing propitious trafficking and sorting pathways. In some cell lines PAF antagonists prevent endocytosis (39), while in other studies, cPLA 2 inhibitors (AA-COCF 3 or BEL) prevent the maintenance of the Golgi network (40), endosome fusion, and endocytosis (41), and modulate the intracellular trafficking of some proteins (42). Together with the observation that the Golgi and the endosomal compartments are involved in the trafficking of a GFP-tagged PrP C (43), these observations suggest that treatment of neurons with PLA 2 inhibitors or PAF antagonists may inhibit PrP res formation by altering the intracellular trafficking of PrP C .…”