Inverse agonism and neutral antagonism at cannabinoid CB₁ receptors

Abstract

There are at least two types of cannabinoid receptor, CB₁ and CB₂, both G protein coupled. CB₁ receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB₂ receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these “endocannabinoids” together with their receptors constitute the “endocannabinoid system”. These discoveries were followed by the development of a number of CB₁- and CB₂-selective antagonists that in some CB₁ or CB₂ receptor-containing systems also produce “inverse cannabimimetic effects”, effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB₁-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB₁ receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB₁ receptors in which CB₁ receptors are shifted from a constitutively active “on” state to one or more constitutively inactive “off” states and (3) CB₁ receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A₁ receptors. Recently developed neutral competitive CB₁ receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB₁ receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have “autoprotective” roles.

Introduction

Mammalian tissues express at least two types of cannabinoid receptor, CB₁ and CB₂, both G protein coupled (reviewed in Howlett et al., 2002). CB₁ receptors are found predominantly at central and peripheral nerve terminals where they mediate inhibition of transmitter release. CB₂ receptors occur mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous ligands for these receptors also exist. These “endocannabinoids” are all eicosanoids, prominent examples including arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol, both of which are synthesized on demand, removed from their sites of action by tissue uptake processes and metabolized by intracellular enzymes (reviewed in De Petrocellis et al., 2004). Endocannabinoids and their receptors constitute the “endocannabinoid system”. The discovery of this system of receptors and endogenous messengers prompted the development of CB₁- and CB₂-selective agonists (reviewed in Howlett et al., 2002). However, these are used less widely as experimental tools than the mixed CB₁/CB₂ receptor agonists, CP55940, R-(+)-WIN55212 and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) (reviewed in Howlett et al., 2002). The discovery of cannabinoid receptors also prompted the development of selective antagonists the first of which were the CB₁-selective SR141716A, AM251, AM281 and LY320135, and the CB₂-selective SR144528 and AM630 (reviewed in Howlett et al., 2002). SR141716A, AM251, AM281 and LY320135 all behave as inverse agonists, producing effects in some CB₁ containing bioassay systems that are opposite in direction from those produced by agonists for these receptors. This paper describes these “inverse cannabimimetic effects” and discusses possible underlying mechanisms. The development of CB₁ receptor ligands that behave as “neutral” antagonists rather than inverse agonists is also described. SR144528 and AM630 behave as inverse agonists in at least some CB₂ receptor-containing bioassay systems (reviewed in Howlett et al., 2002). However, the mechanisms underlying the inverse agonist properties of these ligands has been little investigated. Consequently SR144528 and AM630 are not discussed further in this review which focuses on CB₁ receptor antagonists/inverse agonists.

The production of inverse cannabimimetic effects by SR141716A has been observed in experiments performed both in vivo and in vitro and selected examples of these effects are to be found in Table 1, Table 2, Table 3. As to inverse cannabimimetic effects that AM251, AM281 and LY320135 have been reported to produce, these include

• AM251-induced suppression of rat food intake and food-reinforced behavior (McLaughlin et al., 2003), inhibition of basal G-protein activity in rat cerebellar membranes (Savinainen et al., 2003) and enhancement of electrically-evoked glutamate release from rat cerebellar neurons (Kreitzer and Regehr, 2001);

• AM281-induced increases in mouse locomotor activity, in peristaltic ejection pressure in the guinea pig isolated ileum and in glutamatergic synaptic transmission in rat corticostriatal slices (Cosenza et al., 2000, Huang et al., 2001, Izzo et al., 2000);

• AM281-induced increases in evoked release of acetylcholine from rat hippocampal slices (Gifford et al., 1997) and of [³H]D-aspartate in primary cultures of rat cerebellar granule cells (Breivogel et al., 2004);

• AM281-induced inhibition of basal [³⁵S]GTPγS binding to primary cultures of rat cerebellar granule cells (Breivogel et al., 2004);

• AM251- and AM281-induced enhancement of electrically-evoked γ-aminobutyric acid release from rat hippocampal neurons (Ohno-Shosaku et al., 2001, Wilson and Nicoll, 2001);

• AM251- and AM281-induced increases in Ca²⁺ current and decreases in inward rectifier potassium current in HEK293 cells (Vásquez et al., 2003);

• LY320135-induced enhancement of forskolin-stimulated cyclic AMP accumulation in hCB₁-transfected Chinese hamster ovary (CHO-hCB₁) cells (Felder et al., 1998) and of the amplitude of electrically-evoked contractions of the rat isolated vas deferens (Christopoulos et al., 2001).

It is noteworthy that although AM251 and AM281 are structurally very similar to SR141716A and share its ability to block CB₁ receptors and to produce inverse cannabimimetic effects, several pharmacological differences between SR141716A and either or both AM251 and AM281 have unexpectedly been detected in vitro, for example in experiments with cardiovascular tissue (reviewed in Pertwee, 2004) and with rat hippocampal slices (Hájos and Freund, 2002, Hájos et al., 2001). It is also noteworthy that there are some reports that tolerance can rapidly develop to at least some of the inverse cannabimimetic effects of SR141716A, for example its ability to reduce food intake in rats (Colombo et al., 1998a, Vickers et al., 2003) and its ability to augment intestinal peristalsis in mice (Carai et al., 2004). However, the mechanisms that underlie this tolerance remain to be established.

Experiments with CB₁^-/- mice or with tissues from such animals lend support to the hypothesis that SR141716A produces it at least some of its inverse effects by binding to CB₁ receptors. Thus, for example, there is a report that whilst SR141716A enhances electrically-evoked release of noradrenaline in vasa deferentia from CB₁^+/+ mice, it does not produce this effect in vasa deferentia from CB₁^-/- mice (Schlicker et al., 2003). Similarly, SR141716A has been found to reduce food intake in CB₁^+/+ mice but not in CB₁^-/- mice (Table 1). There are also reports that signs of reduced food intake and increased severity of induced colitis, effects opposite to those produced by CB₁ receptor agonists, are observed both in SR141716A-treated CB₁^+/+ mice and in SR141716A-free CB₁^-/- mice (Di Marzo et al., 2001, Massa et al., 2004). Evidence that SR141716A has a number of CB₁ receptor-independent actions (see below) increases the need for such control experiments with knockout animals.