6 R.G. Pertwee
(reviewed in the chapter by Vaughan and Christie, this volume). There is also evi-
dence that following their release, anandamide and 2-arachidonoyl glycerol enter
cells by a combination of simple diffusion and facilitated, carrier-mediated trans-
port (reviewed in Hillard and Jarrahian 2003) and are then metabolized by intra-
cellular enzymes, anandamide by fatty acid amide hydrolase and 2-arachidonoyl
glycerol mainly by monoacylglycerol lipase (monoglyceride lipase) but also by
fatty acid amide hydrolase (reviewed in Cravatt and Lichtman 2002; Dinh et
al. 2002; Ueda 2002; van der Stelt and Di Marzo 2004; see also the chapter by
Di Marzo et al., this volume). Noladin ether also seems to be a substrate for
anandamide/2-arachidonoyl glycerol membrane transporter(s) (Fezza et al. 2002).
The processes responsible for the production, membrane transport and enzymic
inactivation of endocannabinoids are all pharmacological targets through which
the activity of the endocannabinoid system can or might be modulated to ex-
perimental or therapeutic advantage (reviewed in the chapters by Howlett and
by Di Marzo et al., this volume). There is evidence that such modulation may
also take place naturally as a result of the co-release of endogenous fatty acid
derivatives such as palmitoylethanolamide and oleamide, which can potentiate
anandamide, or of 2-linoleyl glycerol and 2-palmitoyl glycerol, which can poten-
tiate 2-arachidonoyl glycerol (Mechoulam et al. 1998). For anandamide, mecha-
nisms through which co-released ligands induce this “entourage effect” include
not only inhibition of its metabolism by fatty acid amide hydrolase but also in-
creases in the sensitivity of CB 1 or vanilloid receptors or of other pharmacological
targets for anandamide through allosteric or other mechanisms (De Petrocel-
lis et al. 2001b, 2002; Franklin et al. 2003; Mechoulam et al. 1998; Smart et al.
2002).
This chapter describes the in vitro and in vivo bioassays that have been most
widely used to characterize ligands for CB 1 and/or CB 2 receptors and reviews the
ability of compounds commonly used in cannabinoid research as experimental
tools to activate or block these receptors. The likelihood that the most widely
used cannabinoid receptor antagonists are inverse agonists rather than neutral
antagonists is also discussed, as is evidence for the presence in mammalian tissues
of non-CB 1 , non-CB 2 pharmacological targets for cannabinoids.
2 Bioassays for Characterizing CB 1 and CB 2 Receptor Ligands..........
2.
In Vitro Binding Assays
Several cannabinoid receptor ligands have been radiolabelled with tritium, and
thesehavebeenusedbothtodeterminetheCB 1 and CB 2 receptor affinities of unla-
belled cannabinoids in displacement assays and to establish the tissue distribution
patterns of these receptors (reviewed in Howlett et al. 2002; Pertwee 1999a). As
indicated in Tables 1, 2 and 3, some of these compounds bind more readily to CB 1
or to CB 2 receptors, whilst the others bind more or less equally well to both these