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 A2 has 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.

A major issue in lipid signaling relates to the role of particular phospholipase A 2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca 2؉ -independent phospholipase A 2 (iPLA 2 ) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA 2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527-8531). We have now further evaluated the role of the iPLA 2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA 2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA 2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA 2 plays a major role in regulating phospholipid fatty acyl turnover in P388D 1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA 2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA 2 antisense oligonucleotide on PAF-stimulated arachidonate release.

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Arachidonic acid (AA) and its oxygenated derivatives, collectively known as the eicosanoids, are key mediators of a wide variety of physiological and pathophysiological states. AA, obtained from the diet or synthesized from linoleic acid, is rapidly incorporated into cellular phospholipids by the concerted action of arachidonoyl-CoA synthetase and lysophospholipid acyltransferases. Under the appropriate conditions, AA is liberated from its phospholipid storage sites by the action of one or various phospholipase A(2) enzymes. Thus, cellular availability of AA, and hence the amount of eicosanoids produced, depends on an exquisite balance between phospholipid reacylation and hydrolysis reactions. This review focuses on the enzyme families that are involved in these reactions in resting and stimulated cells.

Four related genes encode four different secretory phospholipase A 2 (sPLA 2 ) enzymes in mammals, namely the well described Group I and IIA enzymes and the more recently described Groups IIC and V. A large body of research has putatively demonstrated that the Group IIA sPLA 2 is involved in diverse pathologic processes, such as rheumatoid arthritis, septic shock, intestinal neoplasia, and epidermal hyperplasia, as well as in cellular signaling by regulating the formation of arachidonate-derived lipid messengers. However, we demonstrate herein the involvement of another sPLA 2 , i.e. the Group V sPLA 2 , in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D 1 . Abundant message for Group V sPLA 2 was detected in both resting and activated cells. In contrast, Group IIA sPLA 2 message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA 2 gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E 2 production as well as reduction of the amount of sPLA 2 protein at the cellular surface. Collectively, these results uncover Group V sPLA 2 as a novel effector involved in arachidonic acid-mediated signal transduction.

Secretory phospholipase A 2 (sPLA 2 ) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E 2 (PGE 2 ) production during stimulation of P388D 1 macrophages with the inf lammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE 2 in stimulated P388D 1 cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE 2 production in P388D 1 macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA 2 demonstrate that activation of the sPLA 2 at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A 2 (cPLA 2 ). Collectively these results demonstrate (i) that functional coupling exists between sPLA 2 and COX-2 in activated cells, (ii) the critical role that cPLA 2 plays in lipid mediator production, and (iii) that there is crosstalk between cPLA 2 and sPLA 2 in the cell.

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A novel Ca 2؉-independent phospholipase A 2 (iPLA 2 ) has recently been purified and characterized from P388D 1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA 2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA 2 is equivalent to the mouse enzyme purified from P388D 1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D 1 cells using primers based on the CHO iPLA 2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D 1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D 1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [ 3