Localization of Group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells

In murine P388D(1) macrophages, the generation of prostaglandin E-2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A(2) (PLA(2)), Group IV cytosolic PLA(2) (cPLA(2)), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA(2) that induces expression of Group V PLA(2), which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E-2 generation. Because Group V PLA(2) is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA(2) shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA(2) being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA(2), blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA(2) previously associated with the outer cellular surface. Localization of Group V PLA(2) in perinuclear granules is not observed if the cells are treated with the Group IV PLA(2) inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA(2) in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA(2). Collectively, these results suggest that encapsulation of Group V PLA(2) into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA(2) and COX-2 for efficient prostaglandin synthesis.