When human neutrophils, prelabeled with [3H]arachidonic acid, were incubated with 5S,15S-dihydroxyeicosatetraenoic acid (5,15-diHETE), a dose-dependent increase in the 15-lipoxygenase product [3H]-15-HETE was observed relative to untreated cells. Typically, a fivefold increase in [3H]-15-HETE formation was obtained upon exposure of these cells to 3 muM 5,15-diHETE. There was no appreciable enhancement of the 5-lipoxygenase metabolite [3H]-5-HETE. Product identities were confirmed by comparing retention times on straight- and reversed-phase HPLC with authentic standards, and RIA. Other 5-hydroxyeicosanoids, such as 5-HETE, 5-HETE methyl ester, and leukotriene B4(5S,12R-diHETE), were equally effective in stimulating the formation of [3H]-15-HETE, but exogenously added lipoxin A4, lipoxin B4, 15-HETE, and 12-HETE were much less potent, whereas stearic acid was ineffective. The diHETEs also showed a greater selectivity in activating the 15-lipoxygenase relative to the 5-lipoxygenase. A likely source of substrate for the 15- and 5-lipoxygenases is a pool of cell-associated but noncovalently bound arachidonic acid. In [3H]arachidonic acid-prelabeled neutrophils, the amount of free [3H]arachidonic acid ranged between 50 and 700 fmol/10(7) cells, whereas unlabeled neutrophils contained 100-2,200 pmol/10(7) cells of nonesterified arachidonic acid. The exogenously added hydroxyeicosanoids induce a 0.5-3% conversion of this substrate pool to product. These findings indicate that the 15-lipoxygenase in human neutrophils is a cryptic enzyme that needs to be stimulated in order to metabolize endogenous substrate. It is possible that 5-hydroxyeicosanoids may mimic an as yet unidentified physiological activator of the 15-lipoxygenase.

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