PubMed · 2989193
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J Ophir, S Brenner, S Kivity. 1985. Leukotrienes.. https://doi.org/10.1111/j.1365-4362.1985.tb05759.x
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An assay was developed using electrospray ionization negative ion tandem mass spectrometry (MS) to identify and quantitate the major product in the reaction of leukotriene A(4) (LTA(4)) with deoxyguanosine (dGuo). A second quantitative assay was established using the same separation and detection techniques to determine the amount of dGuo isolated from enzymatically processed DNA. The amount of LTA(4)-dGuo adduct could then be analytically determined in DNA samples and normalized to the amount of dGuo that had been simultaneously derived from the DNA sample. Stable isotope-labeled internal standards used for these quantitative assays were readily synthesized from isotopically labeled [(15)N(5)(13)C(10)]deoxyguanosine triphosphate and analyzed for isotopic purity using MS. A comparison of fragment ions formed from stable isotope analogs of dGuo revealed the loss of deoxyribose and secondarily the loss of a series of stable neutral small molecules in a fashion similar to patterns described previously for the collisional fragmentation of protonated guanine determined by positive ion fast atom bombardment/MS/MS. The combined quantitative assays were used for the determination of the amount of endogenously formed LTA(4)-dGuo adducts observed in DNA when isolated human neutrophils that had been incubated with arachidonic acid were stimulated with calcium ionophore to initiate leukotriene biosynthesis.
It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.
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