TLR-4 and Sustained Calcium Agonists Synergistically Produce Eicosanoids Independent of Protein Synthesis in RAW264.7 Cells

Arachidonic acid is released by phospholipase A2 and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and syst...

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Veröffentlicht in:The Journal of biological chemistry 2007-08, Vol.282 (31), p.22834-22847
Hauptverfasser: Buczynski, Matthew W., Stephens, Daren L., Bowers-Gentry, Rebecca C., Grkovich, Andrej, Deems, Raymond A., Dennis, Edward A.
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Sprache:eng
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Zusammenfassung:Arachidonic acid is released by phospholipase A2 and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist-stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca2+ influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca2+ and TLR pathways was detected and discovered to be independent of NF-κB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A2-mediated eicosanoid production work through two distinct pathways.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M701831200