Metabolic studies of synaptamide in an immortalized dopaminergic cell line

Sonti, Shilpa; Tolia, Mansi; Duclos, R.; Loring, Ralph H.; Gatley, S. John

doi:10.1016/j.prostaglandins.2019.02.002

Cited by 2 publications

(1 citation statement)

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“…This is most likely due to limited uptake of the DHEA, degradation via NAAA and FAAH, and conversion into novel metabolites via various lipoxygenases and CYP450s. Studies with radiolabeled DHEA in immortalized fetal mesencephalic cells also showed that the uptake of DHEA was limited, and that the uptake of DHEA is driven by its FAAH-dependent hydrolysis (51). Therefore, we expect that the reported values of 13- and 16-HDHEA most likely are an underestimation of the real biosynthetic capacity of the macrophage model used.…”

Section: Discussionmentioning

confidence: 99%

Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages

Bus

Zuilhof

Witkamp

et al. 2019

Journal of Lipid Research

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Supplementary key words cyclooxygenase • fatty acid amides • fatty acid oxidation • high-performance liquid chromatography • inflammation • mass spectrometry • prostaglandins • cyclooxygenase 2 Cyclooxygenase 2 (COX-2) is a nonconstitutional enzyme that is upregulated upon inflammation in order to generate inflammatory regulators (1-3). It is known to convert arachidonic acid (AA) into prostaglandin (PG)H 2 , the precursor of various inflammation-regulating PGs and thromboxanes (1, 2, 4). Although AA is considered the prototypical COX-2 substrate, the enzyme has a broad substrate specificity that includes other PUFAs and their derivatives, such as the endocannabinoid, arachidonoylethanolamide (AEA; also known as anandamide) (2, 5-11). Previous studies revealed that COX-2 converts AEA into PG ethanolamides (EAs), also called prostamides, and potent anti-inflammatory monohydroxylated AEAs (5, 6, 11-14). Although various amide-and ester-bound derivatives of AA are converted by COX-2 (5, 6, 13-17), the interaction between COX-2 and endocannabinoid-like molecules derived from PUFAs other than AA is barely investigated. Previous work revealed that docosahexaenoylethanolamide (DHEA), an n-3 PUFA endocannabinoid-like metabolite, Abstract Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE 3-ethanolamide (PGE 3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13-and 16-hydroxy-DHEA (13-and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.-de Bus, I., H. Zuilhof, R. Witkamp, M. Balvers, and B. Albada. Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages.

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Section: Discussionmentioning

confidence: 99%