Esterification of an endogenously synthesized lipoxygenase product into granulocyte cellular lipids

Abstract

The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to Me2SO. [1-14C]Arachidonic acid incubated overnight with these cells was incorporated mainly into membrane phospholipids. Stimulation of these cells with the calcium ionophore, A23187, resulted in a rapid release of esterified arachidonic acid from phosphatidylethanolamine and phosphatidylcholine. The released arachidonic acid was metabolized via both the cyclooxygenase and lipoxygenase pathways into three major hydroxylated products, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 5(S)-hydroxy-6,8,11,14-icosatetraenoic acid (5-HETE), and 5-(S),12(R)-dihydroxy-6,8,10,14-icosatetraenoic acid (leukotriene B). Arachidonic acid was also incorporated into triacylglycerols and phosphatidylinositol. The lipoxygenase product, 5-HETE, was rapidly esterified into cellular lipids. Thirty minutes after ionophore stimulation, 55% of the total 5-HETE synthesized was esterified into phospholipids and 35% incorporated into acylglycerols. In contrast, the other hydroxylated derivatives of arachidonic acid (HHT and leukotriene B) were not incorporated into acylglycerols or phospholipids. Esterification of hydroxylated metabolites of arachidonic acid into membrane phospholipids may serve to regulate a number of granulocyte functions.

Characterization of the human 5-lipoxygenase gene promoter.

“…Substantial production of 5-lipoxygenase metabolites is well documented for cells of myeloid lineage such as granulocytes, basophils, and macrophages. Differentiation of immature cells is associated with enhanced 5-lipoxygenase activity, as shown in studies using HL-60 cells (16)(17)(18), chicken myelomonocytic cells (19), and mouse mastocytoma cells (20). Some of our findings may reflect the cell-specific expression of 5-lipoxygenase.…”

Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids.

“…These observations were not restricted to the deacylation and oxygenation of esterified sources of 15-HETE but were also demonstrable with 5-HETE ( Table 2). 5-HETE is esterified into PMN lipids (10,35,36 (4). Also, the ability of FMLP to stimulate the release and transformation of 5-HETE to its 15-hydroxy derivative provides further evidence that the 15-lipoxygenase activity of human PMNs can be stimulated by receptor-mediated signal transduction.…”

Glomerular arachidonate lipoxygenation in rat nephrotoxic serum nephritis.

“…During the heterologous phase (3-5 h and day 2), potential sources include injured glomerular, epithelial, or proliferating mesangial cells as well as recruited platelets and polymorphonuclear leukocytes. Leukocytes, however, are known to synthesize 5-, 12- [7][8][9][10][11][12][13][14], the inflammatory cellular infiltrates consist primarily of monocytes and macrophages (18,19). These cell types synthesize primarily 5-lipoxygenase products and are therefore unlikely sources of 1 2-HETE.…”

“…Extracts were combined, dried under N2, reconstituted in 300 Mul ofhexane, and injected in a gradient high pressure liquid chromatograph (Varian Associates, Inc., Instrument Group, Palo Alto, CA). In several experiments, we hydrolyzed membrane fatty acids using the technique of Bonser et al (13) to evaluate possible reacylation of HETE into membrane lipids over the 45-min incubation. Glomeruli were extracted by the addition of 2.4 ml chloroform/methanol (1:1 vol/vol) containing 0.001% butylated hydroxyanisole and hydroxytoluene.…”