An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity
Introduction
We have identified anandamide (arachidonoylethanolamide) in porcine brain and 2-arachidonoyl-glycerol (2-Ara-Gl) in canine gut (Devane et al., 1992b; Mechoulam et al., 1995). Both ligands bind to the CB1 and CB2 cannabinoid receptors and exhibit cannabinoid-type activities. Later Sugiura et al. (1995)and Stella et al. (1997)reported the presence of 2-Ara-Gl in brain, while Bisogno et al. (1997b)found that 2-Ara-Gl is biosynthesized and released in a Ca2+-dependent fashion by mouse neuroblastoma cells. 2-Ara-Gl inhibits forskolin-stimulated adenylyl cyclase in mouse spleen cells (Mechoulam et al., 1995) and rat neurons (Stella et al., 1997). In mice, 2-Ara-Gl is active in a tetrad of assays, which together have been shown to be highly predictive of cannabinoid-induced activity (Mechoulam et al., 1995; Fride and Mechoulam, 1993; Martin et al., 1991).
In view of the identification of CB2 cannabinoid receptor in immune cells (Munro et al., 1993) and of the inhibition by 2-Ara-Gl of T- and B-cell proliferation (Lee et al., 1995), we decided to look for the presence of active endogenous ligands in the spleen, an organ with well established immune functions, using a fractionation guided by a binding assay.
Section snippets
Isolation of fatty acid esters of glycerol
Mouse spleen tissue (280 mg from three mice) was homogenized in chloroform/methanol (2:1 v/v) with a Kontex glass tissue grinder. The homogenate was filtered via a sintered glass and the residue reextracted. The chloroform layer, which contained the extracted lipids, was partitioned against 0.8% aqueous NaCl, dried under a stream of nitrogen and redissolved in 1 ml of chloroform. Ten volumes of acetone were added to the solution and after 20 min (at −20°C) the mixture was centrifuged at 3500×g
Isolation and identification
Mouse spleen was extracted with methanol/chloroform (1:2) and the extract was chromatographed to yield a fraction that was found to bind to both CB1 and CB2 cannabinoid receptors. The active fraction was silylated with bis-TMS trifluoroacetamide and the resulting mixture was analysed by GC–MS. Several of the single peaks observed before silylation were transformed into pairs of compounds which, on the basis of our previous work, are the silylated derivatives of 1- and 2- monoacylglycerols (
Discussion
The above results indicate that in spleen, as in canine gut and rat brain (Mechoulam et al., 1995; Sugiura et al., 1995), 2-Ara-Gl is present together with additional 2-acyl-glycerols, two of which, 2-Lino-Gl and 2-Palm-Gl, showed neither binding activity to CB1 or CB2 cannabinoid receptors in membranes of CHO and/or COS-7 cells nor in vivo cannabinoid effects in mice. However, both 2-Lino-Gl and 2-Palm-Gl separately or together (in the ratio present in the spleen) potentiated the apparent
Acknowledgements
This work was supported by NIDA grant DA 9789 (to R.M.), a grant by the Israel Science Foundation (to Z.V. and R.M.), and a grant RG26/95 from The Human Frontier Science Program Organization (to V.D.M.). R.M. is associated with the David Bloom Centre for Pharmacy at the Hebrew University.
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