Lipoxin biosynthesis by trout macrophages involves the formation of epoxide intermediates

Rainbow trout macrophages incubated with calcium ionophore A23187 or zymosan synthesize a range of lipoxygenase products, including lipoxins from endogenous arachidonic and eicosapentaenoic acids. The profile of products formed was consistent with the presence of 5- and 12-lipoxygenase activity in intact cells, whereas freeze-thaw disruption of macrophages revealed a further 15-lipoxygenase activity. To examine the mechanism of lipoxin biosynthesis in these cells, macrophages from the hemopoietic head kidney were incubated with potential intermediates and substrates, including 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 15-HETE, 15-HPETE, 5,15-dihydroperoxyeicosatetraenoic acid (5,15-diHPETE), 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE), and LTA4. Only 5-HPETE caused an increase in LXA4 formation, while incubation with 15-HETE resulted in the appearance of LXB4, a product not formed from endogenous substrates. Alcohol trapping experiments were conducted to evaluate the formation of epoxide-containing intermediates during lipoxin biosynthesis. Both 12-O-methoxy and 6-O-methoxy derivatives of LTA4/5 were formed, together with three groups of tetraene-containing trapping products, one of which co-chromatographed with the methanol trapping products generated from a synthetic 5(6)-epoxy tetraene. The time course of the appearance of tetraene and triene trapping products was similar. Preliminary results are also consistent with the presence of epoxide hydrolase activity in trout macrophages that converted the 5(6)-epoxy tetraene to LXA4. The results of this series of experiments suggest that lipoxin biosynthesis in trout macrophages involves the cooperation of 5- and 12-lipoxygenases to yield an epoxy tetraene-containing intermediate, or its equivalent, that is specifically converted to LXA4.(ABSTRACT TRUNCATED AT 250 WORDS)