Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Abstract

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%o by a bromoenollactone (BEL) that is a selective inhibitor ofa newly discovered Ca2+-independent phospholipase A2 (PLA2) in macrophages.The observed inhibition correlates with a decreased steadystate level of lysophospholipids as well as with the inhibition of the Ca2+-independent PLA2 activity in these cells. This inhibition is specific for the Ca2 -independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca2+-independent PIA2-namely a bromomethyl ketone and methyl-BEL-do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca2+-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A2-α

“…BEL demonstrated a reduction of 12-HETE synthesis by 52% at the highest dose tested (30 μM). However, the results obtained with 30 μM BEL is likely not due to a specific inhibition of iPLA 2 , since other intact cell assays demonstrate IC 50 values of less than 5 μM BEL on arachidonic acid release or eicosanoid synthesis [53,60]. In line with this, the broken cell assay demonstrated that 30 μM BEL inhibited the conversion of exogenous arachidonic acid into 12-HETE by 42.1 ± 3.7%.…”

Signaling Role for Lysophosphatidylcholine Acyltransferase 3 in Receptor-Regulated Arachidonic Acid Reacylation Reactions in Human Monocytes

“…It has traditionally been assumed that PL AA incorporation in activated cells may be secondary to the PLA 2 hydrolytic step because AA incorporates preferentially into the sn-2 position of PLs, and thus, for an enhanced AA reacylation to occur, 2-lysophospholipid acceptors, produced only by PLA 2 , should be provided. That lysophospholipid availability may limit AA reacylation in activated cells is also inferred from the finding that the specific activities of the enzymes of the reacylation pathway, ACSL and the CoA-dependent acyltransferases, are severalfold higher that that of PLA 2 in homogenates from resting and activated cells (23,(54)(55)(56)(57)(58).…”

“…Hence, inhibition of iPLA 2 results in reduced levels of lysophospholipid acceptors, which in turn leads to a decreased incorporation of AA into PLs under unstimulated conditions (14,(23)(24)(25). When the effect of zymosan on AA incorporation was assayed in monocytes treated with BEL, and hence exhibiting diminished lysophospholipid levels (44), the response was slightly but significantly reduced (Fig.…”

“…Our previous work has indicated that iPLA 2 is a significant contributor to the steady-state lysophospholipid level in resting phagocytic cells (23)(24)(25)43). Hence, inhibition of iPLA 2 results in reduced levels of lysophospholipid acceptors, which in turn leads to a decreased incorporation of AA into PLs under unstimulated conditions (14,(23)(24)(25).…”

“…By means of the former, low concentrations of free AA incorporate into PLs via direct acylation of preexisting PLA 2 -derived lysophospholipids. This pathway is believed to constitute the major pathway for AA incorporation into PLs in a variety of cells under physiological conditions; hence, the availability of lysophospholipid acceptors, particularly lysophosphatidylcholine (lysoPC), is a limiting factor (13,14,(23)(24)(25). The high-capacity/low-affinity pathway incorporates free AA via the de novo route, which ultimately results in the accumulation of AA into triacylglycerol and diarachidonoyl PLs (4,26).…”