Abstract
In the present study we have presented data on the regulation of LT (leukotriene) and 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) syntheses in human neutrophils upon interaction with OZ (opsonized zymosan) or Salmonella typhimurium. Priming of neutrophils with PMA (phorbol 12-myristate 13-acetate) and LPS (lipopolysaccharide) elicits 5-oxo-ETE formation in neutrophils exposed to OZ, and the addition of AA (arachidonic acid) significantly increases 5-oxo-ETE synthesis. We found that NO (nitric oxide)-releasing compounds induce 5-oxo-ETE synthesis in neutrophils treated with OZ or S. typhimurium. Exposure of neutrophils to zymosan or bacteria in the presence of the NO donor DEA NONOate (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium) considerably increased the conversion of endogenously formed 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) to 5-oxo-ETE. To our knowledge, this study is the first to demonstrate that NO is a potent regulator of 5-oxo-ETE synthesis in human polymorphonuclear leucocytes exposed to Salmonella typhimurium and zymosan.
Highlights
INTRODUCTIONThe 5-LOX (5-lipoxygenase) pathway converts AA (arachidonic acid) to 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) and LTs (leukotrienes)
The 5-LOX (5-lipoxygenase) pathway converts arachidonic acid (AA) to 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) and LTs
The 5-LOX pathway in neutrophils metabolizes AA to LTB4, which mediates host defence and inflammatory responses [1]. 5-LOX is activated during the phagocytosis of microorganisms and foreign particles by neutrophils [2], which is accompanied by a considerable increase in NADPH-oxidase activity that generates superoxide anion O2 − [3]
Summary
The 5-LOX (5-lipoxygenase) pathway converts AA (arachidonic acid) to 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) and LTs (leukotrienes). Despite the Abbreviations: 5-LOX, 5-lipoxygenase; 5-HEDH, 5-hydroxyeicosanoid dehydrogenase; 5-HETE, 5S-hydroxy-6,8,11,14-eicosatetraenoic acid; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; AA, arachidonic acid; ANF, atrial natriuretic peptide; DEA NONOate, 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium; Dnp-Cl, 1-chloro-2,4-dinitrobenzene; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; HBSS, Hanks balanced salt solution; HBSS/Hepes, Hanks balanced salts medium containing. High level of 5-HEDH activity in the microsomes of neutrophils, intact cells convert 5-HETE to only trace amounts of 5-oxo-ETE, suggesting that the production of this substance is highly regulated [5]. Non-enzymatic conversion of intracellular NADPH to NADP + by the addition of phenazine methosulfate activates 5-oxo-ETE synthesis from exogenously added 5-HETE, suggesting that the stimulatory effect of NADPH oxidase activation is due to the generation of NADP + , the cofactor required by 5-HEDH [17]. Stimulation of PMNLs with zymosan or bacteria in the presence of an NO donor significantly increased the conversion of endogenously formed 5-HETE to 5-oxo-ETE. PMNLs were washed twice with PBS, resuspended at 107 cells/ml (purity, 96–97 %; viability 98–99 %) in Dulbecco’s PBS containing 1 mg/ml glucose (without CaCl2), and stored at room temperature
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