Effect of arachidonic acid reacylation on leukotriene biosynthesis in human neutrophils stimulated with granulocyte-macrophage colony-stimulating factor and formyl-methionyl-leucyl-phenylalanine.
Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.
Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, 12801 E. 17th Avenue, Aurora, CO 80045, USA.
Simona
S
Zarini
Miguel A
MA
Gijón
Giancarlo
G
Folco
Robert C
RC
Murphy
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D008401:Gas Chromatography-Mass Spectrometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D054513:Group IV Phospholipases A2; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007476:Ionophores / Q000378:metabolism; D015289:Leukotrienes / Q000378:metabolism*; D013058:Mass Spectrometry; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism; D013849:Thimerosal / Q000494:pharmacology; D013997:Time Factors
D006893:Hydroxyeicosatetraenoic Acids; D007476:Ionophores; D015289:Leukotrienes; D013849:Thimerosal; D016718:Arachidonic Acid; D000001:Calcimycin; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D001094:Arachidonate 5-Lipoxygenase; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D002118:Calcium