Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells Available to Purchase

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D₃. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP⁺ over NAD⁺ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE→5-HETE) is NADPH-dependent and is maximal at pH 6. Although the K_m for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the V_max is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP⁺ (K_m 139 nM), inhibited by NADPH (K_i 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP⁺ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP⁺ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP⁺ to NADPH.