LIPOXIN BIOSYNTHESIS BY TROUT MACROPHAGES INVOLVES THE FORMATION OF EPOXIDE INTERMEDIATES

Rainbow trout macrophages incubated with calcium ionophore A23187 or z ymosan synthesize a range of lipoxygenase products, including lipoxins from endogenous arachidonic and eicosapentaenoic acids. The profile o f products formed was consistent with the presence of 5- and 12-lipoxy genase activity in intact cells, whereas freeze-thaw disruption of mac rophages revealed a further 15-lipoxygenase activity. To examine the m echanism of lipoxin biosynthesis in these cells, macrophages from the hemopoietic head kidney were incubated with potential intermediates an d substrates, including 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hyd roperoxyeicosatetraenoic acid (5-HPETE), 15-HETE, 15-HPETE, 5,15-dihyd ro-peroxyeicosatetraenoic acid (5,15-diHPETE), 5,15-dihydroxyeicosatet raenoic acid (5,15-diHETE), and LTA4. Only 5-HPETE caused an increase in LXA4 formation, while incubation with 15-HETE resulted in the appea rance of LXB4, a product not formed from endogenous substrates. Alcoho l trapping experiments were conducted to evaluate the formation of epo xide-containing intermediates during lipoxin biosynthesis. Both 12-O-m ethoxy and 6-O-methoxy derivatives of LTA4/5 were formed, together wit h 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 i n trout macrophages that converted the 5(6)-epoxy tetraene to LXA4. Th e results of this series of experiments suggest that lipoxin biosynthe sis in trout macrophages involves the cooperation of 5- and 12-lipoxyg enases to yield an epoxy tetraene-containing intermediate, or its equi valent, that is specifically converted to LXA4. Furthermore, there is no evidence for the direct conversion of LTA4 to lipoxins, suggesting the existence of a novel situation in trout macrophages with two disti nct pathways for the generation of lipoxins and leukotrienes.