An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity

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Abstract

2-Arachidonoyl-glycerol (2-Ara-Gl) has been isolated from various tissues and identified as an endogenous ligand for both cannabinoid receptors, CB1 and CB2. Here we report that in spleen, as in brain and gut, 2-Ara-Gl is accompanied by several 2-acyl-glycerol esters, two major ones being 2-linoleoyl-glycerol (2-Lino-Gl) and 2-palmitoyl-glycerol (2-Palm-Gl). These two esters do not bind to the cannabinoid receptors, nor do they inhibit adenylyl cyclase via either CB1 or CB2; however, they significantly potentiate the apparent binding of 2-Ara-Gl and its apparent capacity to inhibit adenylyl cyclase. Together these esters also significantly potentiate 2-Ara-Gl inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-Gl in mice. 2-Lino-Gl, but not 2-Palm-Gl, significantly inhibits the inactivation of 2-Ara-Gl by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-Gl can be increased by related, endogenous 2-acyl-glycerols, which alone show no significant activity in any of the tests employed. This effect (`entourage effect') may represent a novel route for molecular regulation of endogenous 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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