▪ 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.

Four related genes encode four different secretory phospholipase A₂ (sPLA₂) 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 sPLA₂ 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 sPLA₂, i.e. the Group V sPLA₂, in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D₁. Abundant message for Group V sPLA₂ was detected in both resting and activated cells. In contrast, Group IIA sPLA₂ message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA₂ gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E₂ production as well as reduction of the amount of sPLA₂ protein at the cellular surface. Collectively, these results uncover Group V sPLA₂ as a novel effector involved in arachidonic acid-mediated signal transduction.

A major issue in lipid signaling relates to the role of particular phospholipase A₂ 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 Ca²⁺-independent phospholipase A₂ (iPLA₂) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA₂ 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 iPLA₂ in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA₂ 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 iPLA₂ activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA₂ plays a major role in regulating phospholipid fatty acyl turnover in P388D₁ macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA₂ does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA₂antisense oligonucleotide on PAF-stimulated arachidonate release.

Secretory phospholipase A₂(sPLA₂) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E₂(PGE₂) production during stimulation of P388D₁macrophages with the inflammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE₂in stimulated P388D₁cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE₂production in P388D₁macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA₂demonstrate that activation of the sPLA₂at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A₂(cPLA₂). Collectively these results demonstrate (i) that functional coupling exists between sPLA₂and COX-2 in activated cells, (ii) the critical role that cPLA₂plays in lipid mediator production, and (iii) that there is crosstalk between cPLA₂and sPLA₂in the cell.