TLR-4 and sustained calcium agonists synergistically produce eicosanoids independent of protein synthesis in RAW264.7 cells.
Arachidonic acid is released by phospholipase A(2) 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 Ca(2+) influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca(2+) and TLR pathways was detected and discovered to be independent of NF-kappaB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A(2)-mediated eicosanoid production work through two distinct pathways.
Buczynski
Matthew W
MW
Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
Stephens
Daren L
DL
Bowers-Gentry
Rebecca C
RC
Grkovich
Andrej
A
Deems
Raymond A
RA
Dennis
Edward A
EA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015777:Eicosanoids / Q000378:metabolism*; D005786:Gene Expression Regulation; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000502:physiology*
D015777:Eicosanoids; D008055:Lipids; D016328:NF-kappa B; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D000255:Adenosine Triphosphate; D001094:Arachidonate 5-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D002118:Calcium