Abstract
Macrophage-derived endocannabinoids have been implicated in endotoxin (lipopolysaccharide (LPS))-induced hypotension, but the endocannabinoid involved and the mechanism of its regulation by LPS are unknown. In RAW264.7 mouse macrophages, LPS (10 ng/ml) increases anandamide (AEA) levels >10-fold via CD14-, NF-kappaB-, and p44/42-dependent, platelet-activating factor-independent activation of the AEA biosynthetic enzymes, N-acyltransferase and phospholipase D. LPS also induces the AEA-degrading enzyme fatty acid amidohydrolase (FAAH), and inhibition of FAAH activity potentiates, whereas actinomycin D or cycloheximide blocks the LPS-induced increase in AEA levels and N-acyltransferase and phospholipase D activities. In contrast, cellular levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are unaffected by LPS but increased by platelet-activating factor. LPS similarly induces AEA, but not 2-AG, in mouse peritoneal macrophages where basal AEA levels are higher, and the LPS-stimulated increase in AEA is potentiated in cells from FAAH-/- as compared with FAAH+/+ mice. Intravenous administration of 107 LPS-treated mouse macrophages to anesthetized rats elicits hypotension, which is much greater in response to FAAH-/- than FAAH+/+ cells and is susceptible to inhibition by SR141716, a cannabinoid CB1 receptor antagonist. We conclude that AEA and 2-AG synthesis are differentially regulated in macrophages, and AEA rather than 2-AG is a major contributor to LPS-induced hypotension.
Highlights
Macrophage-derived endocannabinoids have been implicated in endotoxin (lipopolysaccharide (LPS))-induced hypotension, but the endocannabinoid involved and the mechanism of its regulation by LPS are unknown
LPS Induction of AEA Involves CD14, p44/42 MAP Kinase, and NF-B—Preincubation of RAW264.7 cells with anti-CD14 antibodies (1:100) overnight or with the combination of the p44/42 kinase inhibitor PD98059 (50 M) and NF-B inhibitor MG132 (20 M) for 30 min almost completely blocked the effect of LPS, whereas incubation with PD98059, the phosphoinositide 3-kinase inhibitor wortmannin (100 nM), or MG132 alone partially reduced the increase in AEA caused by LPS
We have demonstrated that murine macrophages are capable of generating both AEA and 2-AG and that the cellular levels of these two endocannabinoids are differentially regulated
Summary
Vol 278, No 45, Issue of November 7, pp. 45034 –45039, 2003 Printed in U.S.A. Lipopolysaccharide Induces Anandamide Synthesis in Macrophages via CD14/MAPK/Phosphoinositide 3-Kinase/NF-B Independently of Platelet-activating Factor*. In RAW264.7 mouse macrophages, LPS (10 ng/ ml) increases anandamide (AEA) levels >10-fold via CD14-, NF-B-, and p44/42-dependent, platelet-activating factor-independent activation of the AEA biosynthetic enzymes, N-acyltransferase and phospholipase D. Cellular levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are unaffected by LPS but increased by platelet-activating factor. LPS induces AEA, but not 2-AG, in mouse peritoneal macrophages where basal AEA levels are higher, and the LPS-stimulated increase in AEA is potentiated in cells from FAAH؊/؊ as compared with FAAH؉/؉ mice. We used the RAW264.7 mouse macrophage cell line as well as peritoneal macrophages from FAAH knockout mice and their wild-type littermates to define the cellular mechanisms by which LPS induces the production of endocannabinoids in macrophages and to determine the relative role of AEA and 2-AG in LPS-induced hypotension
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