Stimulation of different phospholipase A2 isoforms by TNF-α and IL-1β in adult rat ventricular myocytes

We previously showed that in adult rat ventricular myocytes interleukin (IL)-1 beta activates a membrane-associated, Ca2+-independent phospholipase A(2) (iPLA(2)). In this study, we examined the possible existence of different PLA(2) isoforms and effects of tumor necrosis factor (TNF)-alpha on iPLA(2) activities. Western blot analysis identified iPLA(2) in both membrane (similar to 82 kDa) and cytosolic (similar to 40 kDa) fractions and identified Ca2+-dependent PLA(2) (cPLA(2)) only in cytosolic fractions. With plasmenylcholine or alkylacyl glycerophosphorylcholine as substrate, TNF-alpha elicited a twofold transient increase in cytosolic iPLA(2) activity accompanied by an increase in arachidonic acid release and decreased membrane-associated iPLA(2) activity with plasmenylcholine. With phosphatidylcholine as substrate, TNF-alpha decreased both cytosolic and membrane-associated iPLA(2) activities. TNF-alpha-induced increases in cytosolic iPLA(2) activity and arachidonic acid release were completely blocked by methyl arachidonyl fluorophosphonate (MAFP) but not by bromoenol lactone (BEL). TNF-alpha and IL-1 beta together enhanced synergistically cytosolic and membrane PLA(2) activities and arachidonic acid release that were blocked differentially by MAFP and EEL, respectively, and inhibited completely by MAFP plus EEL. These results suggest that TNF-alpha and IL-1 beta act on different PLA(2) isoforms in ventricular myocytes.