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)