Temporal dissociation of COX-2-dependent arachidonic acid and 2-arachidonoylglycerol metabolism in RAW264.7 macrophages

• Published in: Journal of lipid research Vol. 65; no. 9; p. 100615
• Main Authors: Aleem, Ansari M., Mitchener, Michelle M., Kingsley, Philip J., Rouzer, Carol A., Marnett, Lawrence J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2024; Elsevier
• Subjects: 2-arachidonoylglycerol; Animals; arachidonic acid; Arachidonic Acid - metabolism; Arachidonic Acids - metabolism; Cyclooxygenase 2 - metabolism; DAG lipase; Endocannabinoids - metabolism; Glycerides - metabolism; Kdo2-lipid A; lipidomics; Lipopolysaccharides; macrophages; Macrophages - metabolism; Mice; prostaglandin glyceryl esters; prostaglandins; RAW 264.7 Cells; Time Factors; TLR4; KLA; 15d-PGJ2; PLC; PLD; Kdo2-lipid A; LPS; RPM; PGE2-G-d5; DAG lipase; macrophages; 15d-PGD2; qPCR; PGs; arachidonic acid; AA; prostaglandin glyceryl esters; cPLA2; PG-Gs; DAG; COX; 2-arachidonoylglycerol; TXA2; 2-AG; prostaglandins; TLR4; lipidomics; PIP2
• ISSN: 0022-2275; 1539-7262; 1539-7262
• DOI: 10.1016/j.jlr.2024.100615

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus. [Display omitted]