INHIBITION OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A(2) PREVENTS ARACHIDONIC-ACID INCORPORATION AND PHOSPHOLIPID REMODELING IN P388D(1) MACROPHAGES

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca2+-independent process and that it is blocked up to 60-70% by a bromoenol-lactone (BEL) that is a selective inhibitor of a newly discovered Ca2+-independent phospholipase A(2) (PLA(2)) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca2+-independent PLA(2) activity in these cells. This inhibition is specific for the Ca2+-independent PLA(2) in that neither group IV PLA(2), group II PLA(2), arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with EEL. Moreover, two EEL analogs that are not inhibitors of the Ca2+-independent PL(A2)-namely a bromo-methyl ketone and methyl-BEG-do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by EEL, indicating that de novo synthetic pathways are not inhibited by EEL. Collectively, the data suggest that the Ca2+-independent PLA(2) in P388D(1) macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.