Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A₂ in COS-1 cells

Phospholipases A₂ (PLA₂) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A₂. We investigated coupling between PLA₂ and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca²⁺ ionophore ionomycin, generated trivial amounts of PGE₂. In cells co- transfected with cytosolic PLA₂ (cPLA₂) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE₂ production. There was no preferential coupling of cPLA₂ to either of the COX isoforms. In contrast, group IIA secretory PLA₂ (sPLA₂) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE₂ production, despite high levels of sPLA₂ enzymatic activity. Transfection of cPLA₂ did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA₂- transfected COS-1 cells, but not in untransfected cells, whereas sPLA₂ transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA₂ is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA₂ is not metabolized by COX. (C) 2000 Elsevier Science Inc.