Biosynthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in circulating and tumoral macrophages.
The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 microM) and lipopolysaccharide (LPS, 200 microg x mL-1) cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2-AG levels. Extracellular [3H]2-AG was cleared from the medium of intact J774 macrophages (t1/2 = 19-28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2-AG hydrolysis, and (b) a 2-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2-AG hydrolase with apparent Km = 110 microM and Vmax = 7.9 nmol x min-1 x (mg protein)-1 was partially characterized in J774 cells and found to exhibit an optimal pH of 6-7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2-AG through metabolic mechanisms that are subject to regulation.
Di Marzo
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V
Istituto per la Chimica di Molecole di Interesse Biologico, CNR, Napoli, Italy.
Bisogno
T
T
De Petrocellis
L
L
Melck
D
D
Orlando
P
P
Wagner
J A
JA
Kunos
G
G
D000818:Animals; D001095:Arachidonic Acids; D001483:Base Sequence; D063385:Cannabinoid Receptor Modulators; D002460:Cell Line; D017931:DNA Primers; D063388:Endocannabinoids; D005989:Glycerides / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006868:Hydrolysis; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009374:Neoplasms, Experimental / Q000473:pathology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D017931:DNA Primers; D063388:Endocannabinoids; D005989:Glycerides; C094503:glyceryl 2-arachidonate