Metabolism of Arachidonate-containing Phospholipid Molecular Species in the Murine Macrophage-like Cell Line, P388d1

Glycerophospholipids of mammalian cells exist as chemically diverse structures with various fatty acids at the sn-1 and sn-2 positions. Arachidonic acid, a polyunsaturated fatty acid, which may be converted to biologically active eicosanoids such as prostaglandins, thromboxanes, and leukotrienes, is found predominantly in the sn-2 position of glycerophospholipids. The purpose of this study was to examine, at the level of the individual molecular species, the incorporation of arachidonate into phospholipids and its release from phospholipids during stimulation. In this way, the specificity of the enzymes controlling arachidonate metabolism could be examined in order to clarify the processes that control the metabolism of this precursor of potent biological mediators. An investigation of the deacylation-reacylation mechanisms for the incorporation of arachidonic acid into the cellular phospholipids revealed that both the CoA-independent transacylation and CoA-dependent acylation mechanisms are active in the P388D1 macrophages. The CoA-independent transacylase preferentially acylated the alkyllysoglycero-phosphatidylcholine substrate with the polyunsaturates, arachidonate, and docosahexaenoate. The CoA-dependent pathways exhibited less selectively and acylated the alkyl-substrate with more saturated fatty acids. Supplementation of the P388D1 macrophages with the n-3 marine oil fatty acids, eicosapentaenoate and docosahexaenoate resulted in the enrichment of the cellular phospholipids with these polyunsaturates at the expense of arachidonate-containing molecular species. Using methodology, which permits the measure of both mass and specific radioactivity changes in the molecular species of phospholipids, it was determined that the arachidonate-containing species are preferentially degraded during stimulation with the calcium ionophore, A23187. Stimulation with calcium ionophore results in the activation of a calcium dependent phospholipase specific for the arachidonate-containing species. Together, these results demonstrate that the incorporation and release of arachidonic acid is regulated by enzymes that bear distinct substrate specificities. The specificities of these enzymes can be directly related to the trafficking of arachidonate and its various esterified forms in cell phospholipids