▪ Abstract In recent years, there has been great interest in the study of phospholipid metabolism in intact cell systems. Such an interest arises mainly from the discovery that cellular membrane phospholipids serve not only in structural roles, but are also reservoirs of preformed second messenger molecules with key roles in cellular signaling. These second messenger molecules are generated by agonist-induced activation and secretion of intracellular and extracellular phospholipases, respectively, i.e. enzymes that cleave ester bonds within phospholipids. Prominent members of the large collection of signal-activated phospholipases are the phospholipase A₂s. These enzymes hydrolyze the sn-2 ester bond of phospholipids, releasing a free fatty acid and a lysophospholipid, both of which may alter cell function. In addition to its role in cellular signaling, phospholipase A₂has recently been recognized to be involved in a wide number of pathophysiological situations, ranging from systemic and acute inflammatory conditions to cancer. A growing number of pharmacologic inhibitors will help define the role of particular phospholipase A₂s in signaling cascades.

Receptor-stimulated arachidonic acid (AA) mobilization in P388D macrophages consists of a transient phase in which AA accumulates in the cell and a sustained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A (sPLA) is the enzyme responsible for most of the AA released to the incubation medium. By using selective inhibitors for each of the PLAs present in P388D macrophages, we demonstrate herein that the cytosolic group IV PLA (cPLA) mediates accumulation of cell-associated AA during the early steps of P388D cell activation. The contribution of both cPLA and sPLA to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase As (PLAs). Furthermore, the results suggest the possibility that a functionally active cPLA may be necessary for sPLA to act. cPLA action precedes that of sPLA, and overcoming cPLA inhibition by artificially increasing intracellular free AA levels restores extracellular AA release. Although this suggests cross-talk between cPLA and sPLA, selective inhibition of one other PLA present in these cells, namely the Ca-independent PLA, does not block, but instead enhances receptor-coupled AA release. These data indicate that Ca-independent PLA does not mediate AA mobilization in P388D macrophages. Collectively, the results of this work suggest that each of the PLAs present in P388D macrophages serves a distinct role in cell activation and signal transduction.

Jesús Balsinde, Edward A. Dennis

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 Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-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 Ca(2+)-independent PLA2--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 Ca(2+)-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.