Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Abstract

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous 14C-AA (10 μM) to PGE 2 (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B 2 (TXB 2) (7.4%), PGD 2 (4.4%) and PGF 2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF 1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin ( ID 50=0.6 μM) while the synthesis of TXB 2 and HHT was selectively inhibited by benzylimidazole ( ID 50=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.