2. CB₁- and CB₂-Selective Cannabinoid Receptor Agonists.

Compounds that are significantly more potent at activating CB₁ than CB₂ receptors include three synthetic analogs of anandamide (Table 1 and Fig. 2): R-(+)-methanandamide, arachidonyl-2′-chloroethylamide (ACEA), and arachidonylcyclopropylamide (Abadji et al., 1994; Hillard et al., 1999). Each of these compounds possesses significant potency and relative intrinsic activity as a CB₁ receptor agonist. ACEA and arachidonylcyclopropylamide are both substrates for the anandamide-metabolizing enzyme fatty acid amide hydrolase, whereas this enzyme does not readily hydrolyze R-(+)-methanandamide. Noladin ether (2-arachidonyl glyceryl ether) (Hanus et al., 2001) is also a CB₁-selective agonist. It has been reported to possess CP55940-like CB₁ receptor relative intrinsic activity but less potency as a CB₁ receptor agonist than either CP55940 or 2-AG (Suhara et al., 2000, 2001; Savinainen et al., 2001, 2003). As to CB₂-selective agonists (Table 1 and Fig. 2), those most frequently used as pharmacological tools are (6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d] pyran (JWH-133; a classical cannabinoid), {4-[4-(1,1-dimethylheptyl)-2,6-dimethoxy-phenyl]-6,6-dimethyl-bicyclo[3.1.1]hept-2-en-2-yl}-methanol (HU-308; a nonclassical cannabinoid), and (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015) and R-3-(2-iodo-5-nitrobenzoyl)-1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (aminoalkylindoles) (for review, see Pertwee, 1999, 2005a, 2008b; Howlett et al., 2002).

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Fig. 2.

The structures of the CB₁-selective agonists ACEA, arachidonylcyclopropylamide (ACPA), methanandamide, and noladin ether and of the CB₂-selective agonists JWH-133, HU-308, JWH-015, and AM1241.

3. CB₁-Selective Competitive Antagonists.

As discussed in greater detail elsewhere (Pertwee, 1999, 2005a, 2008b; Howlett et al., 2002; Fong et al., 2007), the diarylpyrazole rimonabant (SR141716A), N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281), 4-[[6-methoxy-2-(4-methoxyphenyl)-3-benzofuranyl]carbonyl]benzonitrile (LY320135), and taranabant (Fig. 3) can all block agonist-induced activation of cannabinoid CB₁ receptors in a competitive manner and bind with significantly greater affinity to cannabinoid CB₁ than cannabinoid CB₂ receptors (Table 1). Although these compounds lack any ability to activate CB₁ receptors when administered alone, there is evidence that in some CB₁ receptor-containing tissues, they can induce responses opposite in direction from those elicited by a CB₁ receptor agonist (Pertwee, 2005c; Fong et al., 2007). In some instances, at least, this may reflect an ability of these compounds to decrease the spontaneous coupling of CB₁ receptors to their effector mechanisms that it is thought can occur in the absence of exogenously added or endogenously released CB₁ agonists. There is also evidence that at least one of these compounds, rimonabant, can produce inverse cannabimimetic effects in a CB₁ receptor-independent manner (Breivogel et al., 2001; Savinainen et al., 2003; Cinar and Szücs, 2009).

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Fig. 3.

The structures of the CB₁-selective antagonists/inverse agonists, rimonabant, AM251, AM281, LY320135, and taranabant and of the CB₂-selective antagonists/inverse agonists SR144528 and AM630.

Some CB₁ receptor competitive antagonists have been developed that lack any detectable ability to induce signs of inverse agonism at the CB₁ receptor when administered alone. One example of such a “neutral” antagonist (Table 1) is N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS O327) (Fig. 4), which is a structural analog of rimonabant and displays markedly higher affinity for CB₁ than for CB₂ receptors. This compound behaves as CB₁ receptor antagonist both in vitro and in vivo and yet, by itself, does not affect [³⁵S]GTPγS binding to rat cerebellar membranes (Ruiu et al., 2003). Several other compounds have been reported to behave as neutral cannabinoid CB₁ receptor antagonists (Pertwee, 2005a). These include (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050) (Fig. 4), a sulfonamide analog of Δ⁸-tetrahydrocannabinol with an acetylenic side chain. It is noteworthy that the classification of this compound as a neutral antagonist is based on a very limited set of data, prompting a need for further research into its CB₁ receptor pharmacology.

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Fig. 4.

The structures of NESS O327 and O-2050.

4. CB₂-Selective Competitive Antagonists.

[6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (6-iodopravadoline) (AM630) and the diarylpyrazole N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (Fig. 3) are both more potent at blocking CB₂ than CB₁ receptor activation. They display much higher affinity for CB₂ than for CB₁ receptors (Table 1) and block agonist-induced CB₂ receptor activation in a competitive manner (for review, see Pertwee, 1999, 2005a, 2008b; Howlett et al., 2002). Both these compounds are thought to be CB₂ receptor inverse agonists rather than neutral antagonists, because when administered by themselves, they can produce inverse cannabimimetic effects in CB₂ receptor-expressing tissues (Rinaldi-Carmona et al., 1998; Ross et al., 1999). Other notable examples of CB₂-selective cannabinoid receptor antagonists/inverse agonists include N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE-907) (Iwamura et al., 2001) (Table 1) and the triaryl bis-sulfones N-[1(S)-[4-[[4-methoxy-2-[(4-methoxyphenyl)sulfonyl]phenyl]sulfonyl]phenyl]ethyl]methanesulfonamide) (Sch.225336), N-[1(S)-[4-[[4-chloro-2-[(2-fluorophenyl)sulfonyl]phenyl]sulfonyl]phenyl]ethyl]methanesulfonamide (Sch.356036), and N-[1(S)-[4-[[4-chloro-2-[(2-fluorophenyl)sulfonyl]phenyl]sulfonyl]phenyl]ethyl]-1,1,1-trifluoromethanesulfonamide (Sch.414319) (for review, see Lunn et al., 2008). A neutral antagonist that selectively targets the CB₂ receptor has not yet been developed.

2. GPR84 and GPR120.

The two orphan receptors, GPR84 and GPR120, seem to be receptors for medium-chain fatty acids (C9-C14; Table 3) (Wang et al., 2006a) and for long-chain fatty acids (C14-C22) (Hirasawa et al., 2005; Katsuma et al., 2005), respectively.

GPR84 is highly expressed in bone marrow, and in splenic T cells and B cells, and results from studies with GPR84 knockout mice suggest that GPR84 is involved in regulating early IL-4 gene expression in activated T cells (Venkataraman and Kuo, 2005) and that it is expressed in activated microglial cells and macrophages (Bouchard et al., 2007; Lattin et al., 2008). Medium-chain fatty acids activate GPR84 as can be seen from their ability to decrease intracellular cAMP and to stimulate [³⁵S]GTPγS binding to membranes from CHO cells stably expressing GPR84 (Wang et al., 2006a). Short- and long-chain fatty acids were inactive, and GPR84 has not been reported to be activated or inhibited by any known agonists or antagonists for cannabinoid CB₁ or CB₂ receptors.

GPR120 is found mainly in the intestinal tract, although it is also expressed by a number of other tissues (e.g., adipocytes, taste buds, and lung) (Ichimura et al., 2009). More specifically, intestinal GPR120 is found in glucagon-like peptide-1 (GLP-1)-expressing endocrine cells in the large intestine (Hirasawa et al., 2005; Miyauchi et al., 2009) and in gastric inhibitory polypeptide-expressing K cells of the duodenum and jejunum (Parker et al., 2009). Dietary fatty acids may promote intestinal CCK and GLP-1 release via activation of intestinal GPR120 (Tanaka et al., 2008; Ichimura et al., 2009). Long-chain fatty acids (C14–C22; Table 3), especially unsaturated ones, activate GPR120 in cell lines stably expressing this receptor, as measured by an increase in intracellular Ca²⁺, whereas α-linolenic acid methyl ester lacks activity (Hirasawa et al., 2005). It is noteworthy that some plant-derived compounds, grifolin derivatives that do not contain a carboxylic group, can also activate GPR120 (Hara et al., 2009). GPR120 has not been reported to be activated or inhibited by any known agonists or antagonists for cannabinoid CB₁ or CB₂ receptors.

3. GPR3, GPR6, and GPR12.

GPR3, GPR6, and GPR12 are constitutively active proteins that signal through Gα_s to increase cAMP levels in cells expressing these receptors (Tanaka et al., 2007). They are mainly expressed in the central nervous system, where they may contribute to the regulation of neuronal proliferation (Tanaka et al., 2009), monoamine neurotransmission (Valverde et al., 2009), reward learning processes (Lobo et al., 2007), and energy expenditure (Bjursell et al., 2006). They may also be involved in the regulation of meiosis in oocytes (Hinckley et al., 2005). Their closest phylogenetic GPCR relatives are cannabinoid receptors, lysophospholipid receptors, and melanocortin receptors (Uhlenbrock et al., 2002).

It has been suggested that GPR3, GPR6, and GPR12 (Table 3) are all activated by sphingosine-1-phosphate and/or sphingosylphosphorylcholine at nanomolar concentrations (Uhlenbrock et al., 2002; Ignatov et al., 2003a,b; Lobo et al., 2007). Results obtained in a recent investigation using β-arrestin recruitment instead of G protein activation as an assay for receptor agonism do, however, challenge this hypothesis as no sign of agonism was seen in response to sphingosine-1-phosphate or sphingosylphosphorylcholine at 8 and 42 μM, respectively (Yin et al., 2009). A large number of endogenous lipids, including endocannabinoids, were screened in this investigation and none of these were found to activate GPR3, GPR6, or GPR12 (Yin et al., 2009). However, anandamide and 2-AG did show weak agonist activity at the S1P₁ receptor (edg1) at concentrations in the micromolar range (Yin et al., 2009).

4. GPR18 and GPR92.

The chromosomal location of GPR18 has been determined as 13q32.3; it is a 331-amino acid GPCR. GPR18 (Table 3) is highly expressed in spleen, thymus, and peripheral lymphocyte subsets (Gantz et al., 1997; Kohno et al., 2006). In GPR18-transfected cells, N-arachidonoyl glycine (NAGly) has been shown to induce intracellular Ca²⁺ mobilization at 10 μM (Kohno et al., 2006). Furthermore, the same study demonstrated inhibition of forskolin-stimulated cAMP production by NAGly with an EC₅₀ of 20 nM; the effect was absent in untransfected cells and was pertussis toxin-sensitive, suggesting G_i-coupling. A more recent study used the β-arrestin PathHunter assay system to examine the pharmacological interactions of various lipids with a range of recently deorphanized GPCRs (Yin et al., 2009). In this study, NAGly did not activate GPR18 but elicited a weak activation of GPR92, at concentrations above 10 μM. GPR92 mRNA is highly expressed in dorsal root ganglia, suggesting a role in sensory neuron transmission. Oh et al. (2008) have also demonstrated that NAGly mobilizes intracellular Ca²⁺ and activates [³⁵S]GTPγS binding in GPR92-expressing cells. However, the relative intrinsic activity of NAGly is significantly lower than that of the other putative endogenous GPR92 agonists, LPA and farnesyl pyrophosphate (Oh et al., 2008; Williams et al., 2009). In addition, farnesyl pyrophosphate and LPA activate both G_q/11- and G_s-mediated signaling, whereas NAGly activates only G_q/11-mediated signaling. To date, there are no published data to indicate whether cannabinoid CB₁ or CB₂ receptor ligands can activate or block GPR18 or GPR92. It is noteworthy, however, that GPR18 may be a receptor for abnormal-cannabidiol (section III.H.2).

5. GPR23.

The orphan receptor GPR23/p2y9 is closely related to the purinergic P2Y receptor and mRNA for this receptor in the mouse is mainly found in ovary, uterus, and placenta (Ishii et al., 2009). It has been found (Table 3) that GPR23 is activated by LPA in the nanomolar range as indicated by intracellular calcium mobilization and cAMP formation (Noguchi et al., 2003), probably through the activation of G_q/11 and G_s proteins (Ishii et al., 2009). Two other recent investigations have used β-arrestin recruitment to test the ability of LPA to activate GPR23. In one of these, activation was detected at 100 μM (Wetter et al., 2009), whereas in the other, no activation was induced by concentrations of up to 100 μM LPA (Yin et al., 2009). GPR23 has not been reported to be activated or inhibited by any known agonists or antagonists for cannabinoid CB₁ or CB₂ receptors.

6. GPR119.

The human orphan receptor GPR119, identified by a basic local alignment search tool search of the genomic database, is an intronless GPCR belonging to the MECA (melanocortin, endothelial differentiation gene, cannabinoid, adenosine) cluster of receptors (Fredriksson et al., 2003a). It is preferentially expressed in pancreatic and intestinal cells, where it is involved in the control of glucose-dependent insulin release and GLP-1 release, respectively (Soga et al., 2005; Chu et al., 2007; Lauffer et al., 2008). Although GPR119 is phylogenetically related to cannabinoid receptors, only fatty acid amides interact with GPR119 (Table 3), the potency order of four of these being N-oleoyl dopamine > oleoyl ethanolamide > palmitoyl ethanolamide > anandamide (Overton et al., 2006; Chu et al., 2010). Because only N-oleoyl dopamine and oleoyl ethanolamide have reasonably high (low micromolar) affinity for GPR119, and because neither of these lipids interacts with CB₁ or CB₂ receptors, GPR119 cannot be viewed as a cannabinoid receptor. Oleoyl ethanolamide also activates TRPV1 channels and PPARs (sections III.E and III.G).

1. Opioid Receptors.

There is evidence that certain phytocannabinoids or synthetic cannabinoids can act allosterically at concentrations in the low micromolar range to accelerate the dissociation of ligands from the orthosteric sites on μ- and/or δ-opioid receptors. Thus, it has been found that the rate of dissociation of [³H]DAMGO ([³H][d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin), presumably from μ-opioid receptors, and of [³H] naltrindol, presumably from δ-opioid receptors, can be increased by Δ⁹-THC (EC₅₀ = 21.4 and 10 μM, respectively) (Kathmann et al., 2006). In contrast, rimonabant seems to displace [³H]DAMGO in a competitive manner (IC₅₀ = 4.1 μM). Results obtained from equilibrium binding experiments with rat whole-brain membranes also suggest that Δ⁹-THC is a noncompetitive inhibitor of ligand binding to μ- and δ-opioid receptors (IC₅₀ = 7 and 16 μM, respectively), although not to κ-opioid receptors or σ/phencyclidine receptors, and that inhibition of ligand binding to μ-opioid receptors can be induced by certain other cannabinoids (Vaysse et al., 1987). In addition, it has been found by both Fong et al. (2009) and Cinar and Szücs (2009) that rimonabant can induce radioligand displacement from μ-opioid receptors (IC₅₀ = 3 and 5.7 μM, respectively), and by Fong et al. (2009) that this CB₁ receptor antagonist can induce radioligand displacement from κ-opioid receptors (IC₅₀ = 3.9 μM).

2. Muscarinic Acetylcholine Receptors.

At concentrations in the micromolar range, both anandamide and R-(+)-methanandamide have been shown to modulate tritiated ligand binding to muscarinic acetylcholine receptors, probably by targeting allosteric sites on these receptors. Thus, Lagalwar et al. (1999) found that [³H]N-methylscopolamine and [³H]quinuclidinyl benzilate could be displaced from binding sites on adult human frontal cerebrocortical membranes in a noncompetitive manner by both anandamide (IC₅₀ = 44 and 50 μM, respectively) and R-(+)-methanandamide (IC₅₀ = 15 and 34 μM, respectively) but not R-(+)-WIN55212 (up to 5 μM). Both ethanolamides stimulated [³H]oxotremorine binding to these membranes at concentrations below 50 or 100 μM, although they did inhibit such binding at higher concentrations. It was concluded that these effects of anandamide did not require its conversion to arachidonic acid. It has also been found that anandamide and R-(+)-methanandamide but not R-(+)-WIN55212 can displace tritiated ligands from human M₁ and M₄ muscarinic acetylcholine receptors transfected into CHO cells (Christopoulos and Wilson, 2001). IC₅₀ values for the displacement of [³H]N-methylscopolamine and [³H]quinuclidinyl benzilate from M₁ receptors were 2.8 and 13.5 μM, respectively, for anandamide and 1.45 and 6.5 μM, respectively, for R-(+)-methanandamide. Corresponding IC₅₀ values for the displacement of these tritiated ligands from M₄ receptors were 8.3 and 6.9 μM for anandamide and 9.8 and 3.1 μM for R-(+)-methanandamide. The effect of anandamide on tritiated ligand binding seemed to be noncompetitive and hence possibly allosteric in nature. It is noteworthy, however, that anandamide (10 μM) was subsequently found by the same research group not to affect the rate of dissociation of [³H]N-methylscopolamine from M₁ binding sites (Lanzafame et al., 2004).

3. Other Established G Protein-Coupled Receptors.

It has been reported that at 10 μM, both rimonabant and AM251 can oppose the activation of adenosine A₁ receptors in rat cerebellar membranes and that these CB₁ receptor antagonists can inhibit basal [³⁵S]GTPγS binding to these membranes, probably by blocking A₁ receptor activation by endogenously released adenosine (Savinainen et al., 2003). In addition, evidence has been obtained, first, that at 10 μM, both anandamide and 2-AG, but not AM251, R-(+)-WIN55212, or CP55940 can act as allosteric inhibitors at the human adenosine A₃ receptor although not at the human adenosine A₁ receptor (Lane et al., 2010), and second, that both rimonabant (IC₅₀ = 1.5 μM) and taranabant (IC₅₀ = 3.4 μM) can induce radiolabeled ligand displacement from adenosine A₃ receptors (Fong et al., 2009). In contrast to taranabant (IC₅₀ > 10 μM), rimonabant can also displace radiolabeled ligands from α_2A- and α_2C-adrenoceptors, from 5-HT₆ and angiotensin AT₁ receptors, and from prostanoid EP₄, FP, and IP receptors with IC₅₀ values ranging from 2 to 7.2 μM (Fong et al., 2009). Taranabant, however, has been found to displace radiolabeled ligands from dopamine D₁ and D₃ receptors (K_i = 3.4 and 1.9 μM, respectively) and from melatonin MT₁ receptors (K_i = 5.4 μM) (Fong et al., 2007). In addition, both rimonabant (IC₅₀ = 2 μM) and taranabant (IC₅₀ = 0.5 μM) can induce radiolabeled ligand displacement from tachykinin NK₂ receptors (Fong et al., 2009).

There is also evidence that at 3 or 10 μM, but not higher or lower concentrations, both Δ⁹-THC and 11-hydroxy-Δ⁹-THC increase the affinity of [³H]dihydroalprenolol for β-adrenoceptors in mouse cerebral cortical membranes (Hillard and Bloom, 1982). There is evidence too that [³H]5-HT binding to 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, and/or 5-HT_2C receptors in bovine cerebral cortical synaptic membranes can be reduced by 11-hydroxy-Δ⁸-THC and 11-oxo-Δ⁸-THC although not Δ⁸-THC at 10 μM, and by anandamide at 1 and 10 μM (Kimura et al., 1996, 1998). The same concentrations of these cannabinoids did not decrease [³H]ketanserin binding to 5-HT_2A or 5-HT_2B receptors, although evidence was obtained that anandamide can reduce radioligand binding to 5-HT₁ and 5-HT₂ receptors at 100 μM. In addition, there has been a report that [³H]ketanserin binding to 5-HT₂ receptors in rat cerebral cortical membranes is enhanced by HU-210 at 500 nM (Cheer et al., 1999).

1. 5-HT₃ Receptors.

There has been one report that antagonism of the activation of 5-HT₃ receptors by 5-HT can be induced by CP55940, R-(+)-WIN55212, and anandamide with IC₅₀ values of 94, 310, and 190 nM, respectively (Fan, 1995), and another report that these compounds induce antagonism of this kind with IC₅₀ values of 648, 104, and 130 nM, respectively (Barann et al., 2002). The second of these research groups also found 5-HT₃ receptor activation to be potently antagonized by two other cannabinoid receptor agonists, Δ⁹-THC and JWH-015 (IC₅₀ = 38 and 147 nM, respectively) and by the CB₁ receptor antagonist/inverse agonist LY320135 (IC₅₀ = 523 nM), although not by rimonabant at 1 μM. In other investigations, anandamide has been found to antagonize 5-HT₃ receptor activation with IC₅₀ values of 239 nM (Xiong et al., 2008) or 3.7 μM (Oz et al., 2002). There is evidence, at least for anandamide, CP55940, and R-(+)-WIN55212, that this antagonism is noncompetitive in nature and, indeed, that at least some of these cannabinoids may be targeting an allosteric site on the 5-HT₃ receptor (Fan, 1995; Barann et al., 2002; Oz et al., 2002; Xiong et al., 2008).

2. Nicotinic Acetylcholine Receptors.

An allosteric mechanism may also underlie the antagonism of nicotinic acetylcholine receptors that is reportedly induced by anandamide, 2-AG, R-(+)-methanandamide, and CP55940 (IC₅₀ = 230, 168, 183, and 3400 nM, respectively), although not by Δ⁹-THC or R-(+)-WIN55212 (Oz et al., 2003, 2004a, 2005). Evidence that anandamide can antagonize nicotinic acetylcholine receptors with significant potency has also been obtained by Spivak et al. (2007) (IC₅₀ ~ 300 nM) and Butt et al. (2008) (IC₅₀ = 900 nM). Anandamide behaved as a noncompetitive antagonist in the second of these investigations, in which further evidence that Δ⁹-THC (30 μM) is not a nicotinic acetylcholine receptor antagonist was also obtained. Arachidonic acid, a metabolite of anandamide, has also been found to antagonize nicotinic acetylcholine receptors, although the ability of anandamide to inhibit this ligand-gated ion channel does not seem to depend on its conversion to this unsaturated fatty acid (Oz et al., 2003, 2004a).

3. Glycine Receptors.

At least some cannabinoid receptor agonists have been found to modulate glycine-induced activation of subunits of glycine receptors with significant potency in a positive or negative manner. Thus, for example, results obtained from experiments with transfected cells (Hejazi et al., 2006; Yang et al., 2008) suggest the following:

• Activation of human α1 subunits is enhanced by anandamide (IC₅₀ = 38 or 319 nM), Δ⁹-THC (IC₅₀ = 86 nM), and HU-210 (IC₅₀ = 270 nM) but weakly inhibited by HU-308 and unaffected by R-(+)-WIN55212 at 30 μM.

• Activation of neither human α2 nor rat α3 subunits is affected by anandamide at up to 30 μM, whereas activation of both is inhibited by HU-210 (IC₅₀ = 90 and 50 nM, respectively), R-(+)-WIN55212 (IC₅₀ = 220 and 86 nM, respectively), and HU-308 (IC₅₀ = 1130 and 97 nM, respectively).

• Activation of human α1β1 dimers is enhanced by anandamide and Δ⁹-THC (IC₅₀ = 318 and 73 nM, respectively).

• Activation of human α1β subunits is enhanced by anandamide (IC₅₀ = 75 nM) and HU-210 (30 μM) but unaffected by R-(+)-WIN55212 and inhibited by HU-308 at 30 μM.

Δ⁹-THC (IC₅₀ = 115 nM) and anandamide (IC₅₀ = 230 nM) have also been found to enhance the activation of native glycine receptors in rat isolated ventral tegmental area neurons (Hejazi et al., 2006). There is evidence too, from experiments with rat isolated hippocampal pyramidal neurons, that at 0.2 to 2 μM, anandamide and 2-AG can antagonize glycine receptor activation, and that this effect of anandamide is not TRPV1 channel-mediated (Lozovaya et al., 2005).

4. Other Ligand-Gated Ion Channels.

Evidence has also been obtained that anandamide (≥100 nM) and R-(+)-methanandamide (1 μM) but not Δ⁹-THC (≤10 μM) can enhance NMDA-induced activation of NMDA receptors (Hampson et al., 1998). In contrast, anandamide (1–100 μM) has been found not to affect binding of [³H]muscimol to GABA_A receptors in bovine cerebral cortical synaptic membranes or, indeed, binding of the benzodiazepine [³H]flunitrazepam to these membranes (Kimura et al., 1998). Binding of [³H]flunitrazepam to synaptic membranes, however, has been found to be decreased by 11-hydroxy-Δ⁸-THC at 10 μM (Yamamoto et al., 1992).

2. TRPV1 Channels.

TRPV1 (initially known as VR1) was the first TRP channel to be cloned as a receptor for capsaicin, the natural product responsible for the pungency of hot chilli peppers (Caterina et al., 1997). It is also activated by noxious stimuli, such as heat (>43°C), protons (pH <6.9), and various other natural toxins. Consistent with the hypothesis that it is involved in pain, nociception, and temperature sensing (Caterina et al., 2000; Davis et al., 2000), TRPV1 is predominantly expressed in sensory neurons of unmyelinated axons (C fibers) and thin myelinated axons (Aδ fibers) of dorsal root and trigeminal ganglia (Holzer, 1988; Caterina et al., 1997; Tominaga et al., 1998), where it can become up-regulated during nerve injury-induced thermal hyperalgesia (Rashid et al., 2003b) and diabetic neuropathy (Rashid et al., 2003a). Evidence has accumulated for the presence of TRPV1 not only in sensory neurons but also in brain neurons and in non-neuronal cells, including epithelial, endothelial, glial, smooth muscle, mast and dendritic cells, lymphocytes, keratinocytes, osteoclasts, hepatocytes, myotubes, fibroblasts, and pancreatic β-cells (for review, see Starowicz et al., 2007). It is noteworthy that TRPV1 colocalizes with CB₁ receptors in sensory (e.g., dorsal root ganglia and spinal cord) (Ahluwalia et al., 2003a; Price et al., 2004) and brain (Cristino et al., 2006) neurons and with CB₂ receptors in sensory neurons (Anand et al., 2008) and osteoclasts (Rossi et al., 2009). This colocalization makes possible several types of intracellular cross-talk (for review, see Di Marzo and Cristino, 2008) and might have important functional consequences for those endogenous and synthetic ligands that activate both cannabinoid and TRPV1 receptors (Hermann et al., 2003; Fioravanti et al., 2008).

In fact, it is now well established from experimental work described in more than 300 articles that endocannabinoids, such as anandamide and N-arachidonoyl dopamine but not 2-AG, noladin ether, or virodhamine, bind to both human and rat TRPV1, upon which they act as full agonists (Zygmunt et al., 1999; for review, see Starowicz et al., 2007; Tóth et al., 2009). Although the affinity of these compounds in binding assays with the human recombinant channel is similar to, or only slightly lower than, that of capsaicin (Ross et al., 2001b; Di Marzo et al., 2002), their relative intrinsic activity and particularly their potency (Table 6) depends on the type of TRPV1 functional assay in which these pharmacological properties are assessed (i.e., enhancement of intracellular Ca²⁺ levels, induction of cation currents in neurons, or release of algogenic/vasodilatory peptides from sensory nervous tissue preparations) and on the experimental conditions used to carry out these assays. Furthermore, TRPV1 gating by its ligands can be markedly altered by several regulatory events and signals, including post-translational modifications (such as phosphorylation by several protein kinases), allosteric modulation by temperature, acid, membrane potential, membrane phospholipids (phosphatidylinositol bisphosphate in particular), metabotropic (including cannabinoid) receptor activation, neurotrophins, etc., all of which can be modulated by inflammatory and other pathological conditions. As a result, apparent anandamide-induced activation of TRPV1 has often been found to increase or decrease under such conditions. It has been proposed, for example, that endogenously released anandamide has higher efficacy and potency (Ahluwalia et al., 2003b). Although anandamide may be less potent as a TRPV1 agonist than as a CB₁ receptor agonist, elevation of its endogenous levels with fatty acid amide hydrolase (FAAH) inhibitors [e.g., cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597)] can lead to effects that are mediated by TRPV1 (Maione et al., 2006; Morgese et al., 2007; Rubino et al., 2008). This, together with data indicating that exogenous anandamide can exert effects at TRPV1 in a way sensitive to inhibitors of anandamide cellular uptake in HEK293 cells (De Petrocellis et al., 2001), trigeminal neurons (Price et al., 2005b), and, ex vivo, in mesenteric arteries (Andersson et al., 2002) represents strong indirect evidence that both endogenous and exogenous anandamide reaches its intracellular binding site on this molecular target.

It is noteworthy that anandamide and N-arachidonoyl dopamine seem to interact with TRPV1 at the same intracellular binding site as capsaicin. Jordt and Julius (2002) observed that the TM3/4 region of the mammalian TRPV1 receptor is responsible for its sensitivity to capsaicin and anandamide, whereas the avian receptor, which is activated only by heat or protons, lacks part of this region. The binding site is located on the inner face of the plasma membrane, thus opening up the possibility that when anandamide is biosynthesized by cells that express TRPV1, it will activate this receptor before being released, thereby regulating Ca²⁺ homeostasis as an intracellular messenger (van der Stelt et al., 2005). A crucial role for Tyr511 and Ser512, located at the transition between the second intracellular loop and TM3, has been demonstrated, and other critical residues seem to be in the TM4 segment (Jordt and Julius, 2002; Johnson et al., 2006). Tyr511 engages in a hydrophobic interaction with the hydrophobic tails of capsaicin, anandamide, and N-arachidonoyl dopamine, whereas the side-chain hydroxyl group of Thr550, as well as the aromatic region of Trp549, both present in the TM4 domain, might interact with the vanillyl, catecholamine, or ethanolamine moiety of TRPV1 ligands.

Synthetic CB₁ and CB₂ receptor ligands, such as HU-210, JWH-015, and rimonabant, have also been reported to interact with TRPV1, although usually with lower relative intrinsic activity and potency than anandamide, whereas N-arachidonoyl dopamine displays markedly greater potency than anandamide as a TRPV1 agonist (Table 6) (De Petrocellis et al., 2001; Qin et al., 2008). In contrast, phytocannabinoids that do not activate CB₁ receptors, such as cannabidiol and cannabigerol (section II.C.5), were shown to act as full agonists at TRPV1 receptors (Bisogno et al., 2001; Ligresti et al., 2006). Cannabidiol, in particular, exhibits almost the same K_i as capsaicin at the human TRPV1 receptor (Bisogno et al., 2001), but seems to be significantly less potent at rat TRPV1 (Qin et al., 2008). It is noteworthy that anandamide and N-arachidonoyldopamine congeners with little or no activity at CB₁/CB₂ receptors, such as oleoyl ethanolamide, linoleoyl ethanolamide, and N-oleoyl dopamine (section II.C.5), are also potent TRPV1 agonists (Movahed et al., 2005).

3. Other TRPV Channels.

Apart from TRPV1, five other TRPV channels, all insensitive to capsaicin, have been identified and cloned to date. TRPV2, -3, and -4 are involved in high-temperature sensing and nociception, whereas TRPV5 and -6 intervene in Ca²⁺ absorption/reabsorption (for review, see Vennekens et al., 2008). TRPV2 was previously known as growth factor-regulated Ca²⁺ channel or vanilloid receptor-like 1 and is activated by higher temperatures than TRPV1, but not by protons (Qin et al., 2008). Strong evidence (albeit obtained so far in only one laboratory) exists for the interaction of cannabidiol, Δ⁹-THC, cannabinol, and, to a smaller extent, 11-hydroxy-Δ⁹-THC, nabilone, CP55940, and HU-210, with TRPV2. These effects were observed by measuring elevation of intracellular Ca²⁺ in HEK293 cells transfected with rat recombinant TRPV2 cDNA. Apart from cannabidiol, these compounds exhibited EC₅₀ values much higher, or E_max values much lower, than those observed in functional assays that measure cannabinoid receptor-mediated responses (Table 6). It is noteworthy that cannabidiol has also been found to evoke TRPV2-mediated cation currents and release of calcitonin gene-related peptide in cultured dorsal root ganglion (DRG) neurons (Qin et al., 2008). Because TRPV2, like TRPV1, is immediately desensitized by its agonists, these findings might explain some of the anti-inflammatory and/or analgesic properties of cannabidiol.

TRPV4 is activated by a variety of physical and chemical stimuli, including heat and decreases in osmolarity (Everaerts et al., 2010). TRPV4 was originally reported to be activated by anandamide and 2-AG, probably via the formation of cytochrome P450 metabolites of arachidonic acid, such as epoxyeicosatrienoic acids (Watanabe et al., 2003). It is noteworthy that cytochrome P450 metabolites of anandamide activate cannabinoid receptors (Chen et al., 2008) but have never been tested on TRPV4.

4. Other TRP Channels: TRPM8 and TRPA1.

TRPM8 and TRPA1 belong to two subfamilies different from that of the capsaicin (TRPV1) receptor but are still involved in thermosensation. Indeed, TRPM8 and TRPA1 were suggested to be activated by cold temperatures as well as by natural compounds (such as menthol in the case of TRPM8) and by various irritants [(mustard oil isothiocyanates, acrolein, etc.) in the case of TRPA1] (for review, see McKemy 2005). Recent evidence has emerged that both anandamide and N-arachidonoyl dopamine, but not 2-AG, can efficaciously antagonize the stimulatory effect of two TRPM8 agonists, menthol and the synthetic compound icilin, on intracellular Ca²⁺ elevation in HEK293 cells transfected with rat recombinant TRPM8 (De Petrocellis et al., 2007), as well as in DRG neurons (De Petrocellis et al., 2008). It is noteworthy that they did this in a cannabinoid receptor-independent manner. The concentrations at which anandamide exerted this effect ranged from submicromolar to 10 μM, depending on the type of TRPM8 agonist and cellular system used for the Ca²⁺ assay. It is noteworthy that several nonpsychotropic phytocannabinoids exert a similar action at TRPM8, and at significantly lower concentrations. Cannabidiol, cannabigerol, Δ⁹-THC, and Δ⁹-THC-acid were almost equipotent (IC₅₀ = 70–160 nM in transfected HEK293 cells), whereas cannabidiolic acid was the least potent among the tested compounds (IC₅₀ = 0.9–1.6 μM) (De Petrocellis et al., 2008).

Evidence that TRPA1 (ANKTM1) is activated by phytocannabinoid CB₁/CB₂ receptor agonists [i.e., Δ⁹-THC and cannabinol (sections II.C.1 and II.C.5)], was first described in the article reporting the cloning of this channel (Jordt et al., 2004). The authors of this article showed that concentrations of these two compounds ≥10 μM were necessary to induce TRPA1-mediated effects, including endothelium- and CB₁/CB₂-independent vasodilation of rat mesenteric arteries. Later, Akopian et al. (2008) showed that micromolar concentrations of R-(+)-WIN55212 as well as the CB₂-selective agonist AM1241 1) elicited TRPA1-mediated elevation of Ca²⁺ and currents in transfected CHO cells and trigeminal neurons; 2) desensitized TRPA1- and TRPV1-expressing cells to the action of capsaicin; 3) inhibited capsaicin-evoked nocifensive behavior in vivo in wild-type mice and much less so in TRPA1-null mutant mice (Akopian et al., 2008). Qin et al. (2008) recently confirmed that Δ⁹-THC and R-(+)-WIN55212 activate rat recombinant TRPA1 and also found that the cannabinoid receptor-inactive isomer of the latter compound, S-(−)-WIN55212, exerts a similar action, although at higher concentrations. Furthermore, the cannabinoid quinone 3S,4R-p-benzoquinone-3-hydroxy-2-p-mentha-(1,8)-dien-3-yl-5-pentyl (HU-331) was also quite potent. Almost at the same time, De Petrocellis et al. (2008) found that in HEK293 cells transfected with rat TRPA1, Δ⁹-THC could be much more potent at eliciting TRPA1-mediated elevation of intracellular Ca²⁺ than previously suspected and that several nonpsychotropic phytocannabinoids were even more potent, cannabichromene (EC₅₀ = 60 nM) being the most potent and cannabigerol and cannabidiolic acid (EC₅₀ = 3.4–12.0 μM) the least potent. Cannabichromene also activated mustard oil-sensitive (TRPA1-expressing) DRG neurons, although with lower potency (EC₅₀ = 34.3 μM). It is noteworthy that anandamide was also recently reported to weakly but efficaciously stimulate TRPA1-mediated elevation of intracellular Ca²⁺ in transfected HEK293 cells (EC₅₀ = 4.9 μM) (De Petrocellis and Di Marzo, 2009).

2. Potassium Channels.

Results obtained in a number of investigations suggest that several types of potassium channel can be targeted by certain cannabinoid receptor agonists (Table 7) and antagonists in a cannabinoid CB₁ receptor-independent manner at concentrations in the nanomolar or low micromolar range. These are members of the 2TM domain family of channels (K_ATP channels), the 4TM domain family of leak or background channels (TASK and TREK), and the 6TM domain family of voltage-gated channels (K_V and calcium-activated K_Ca channels).

Turning first to ATP-sensitive inward-rectifier (K_ATP) channels, there is evidence that anandamide is a noncompetitive inhibitor of such channels (IC₅₀ = 8.1 μM) and that this endocannabinoid also inhibits [³H]glibenclamide binding to K_ATP channels, again in a noncompetitive manner (IC₅₀ = 6.3 μM). These channels can be inhibited by R-(+)-methanandamide at 10 μM but not by rimonabant or SR144528 at 1 μM (Oz et al., 2007).

Moving on to the 4TM domain family, it has been found that human or rat TASK-1 channels can be inhibited by R-(+)-methanandamide (IC₅₀ = 700 nM), by anandamide at 3 and 10 μM, and by rimonabant (slight inhibition), CP55940, and R-(+)-WIN55212 but not Δ⁹-THC, HU-210, or 2-AG at 10 μM (Maingret et al., 2001; Berg et al., 2004; Veale et al., 2007; Zhang et al., 2009). There have also been reports that human, rat, or mouse TASK-3 channels can be inhibited by R-(+)-methanandamide and anandamide at 1 to 10 μM (Berg et al., 2004; Veale et al., 2007) and that bovine TREK-1 channels are inhibited by anandamide (IC₅₀ = 5.1 μM) (Liu et al., 2007).

As to K_V channels, there is evidence for the following:

• K_V1.2 channels are inhibited by anandamide (IC₅₀ = 2.7 μM), arachidonic acid (IC₅₀ = 6.6 μM), and Δ⁹-THC (IC₅₀ = 2.4 μM) (Poling et al., 1996).

• Human cardiac K_V1.5 channels are inhibited by anandamide (IC₅₀ = 0.9 μM), 2-AG (IC₅₀ = 2.5 μM), and, with a similar potency, by both R-(+)-methanandamide and arachidonic acid (Barana et al., 2010).

• Human K_V1.5 channels expressed on HEK293 cells are also blocked by anandamide, which displays significantly greater potency intracellularly (IC₅₀ = 213 nM) than extracellularly (IC₅₀ = 2.1 μM) (Moreno-Galindo et al., 2010).

• K_V3.1 channels are inhibited by anandamide (0.1–3 μM) and arachidonic acid (3 μM) (Oliver et al., 2004) and cardiac K_V4.3 channels are inhibited by anandamide (IC₅₀ = 400 nM), R-(+)-methanandamide (IC₅₀ = 600 nM), and 2-AG (IC₅₀ = 300 nM) (Amorós et al., 2010).

In addition, there has been a report that K_V channels can be inhibited not only by anandamide (IC₅₀ = 600 nM) and R-(+)-methanandamide (10 μM) but also by rimonabant (10 μM) and R-(+)-WIN55212 (20 μM), although not by arachidonic acid (Van den Bossche and Vanheel, 2000). It has been found too that both rimonabant (IC₅₀ = 2.5 μM) and taranabant (IC₅₀ = 2.3 μM) can induce radiolabeled ligand displacement from rapid delayed rectifier K_V channels (Fong et al., 2009). Evidence has also recently been obtained that anandamide (1 μM) can strongly inhibit K_V channel-mediated delayed rectifier outward potassium current (Vignali et al., 2009). A similar degree of inhibition was induced by R-(+)-methanandamide (1 μM). However, both R-(+)-WIN55212 and 2-AG induced less inhibition at 1 μM than anandamide.

Finally, there is evidence that anandamide (EC₅₀ = 631 nM or 4.8 μM) and R-(+)-methanandamide (EC₅₀ = 7.9 μM) but not 2-AG (up to 10 μM) can increase the activity of K_Ca (BK) channels (White et al., 2001; Godlewski et al., 2009). Results obtained in experiments with rat isolated coronary arteries also suggest that such activation can be produced by both anandamide and R-(+)-methanandamide at concentrations of 0.3 to 3 μM, although not by JWH-133 at 1 μM (Sade et al., 2006).

3. Sodium Channels.

There is evidence that voltage-gated sodium channels can be targeted by some cannabinoid receptor agonists at concentrations in the nanomolar or micromolar range (Table 7). Thus, for example, R-(+)-WIN55212 has been found by Fu et al. (2008) to induce a slight enhancement (11.5%) of voltage-gated sodium currents in rat cultured trigeminal ganglion neurons at 10 nM but to inhibit these currents at higher concentrations (IC₅₀ = 17.8 μM). In an earlier investigation, Nicholson et al. (2003) also found that R-(+)-WIN55212 can inhibit voltage-gated sodium channels. More specifically, they obtained evidence that this aminoalkylindole can act in a CB₁ receptor-independent manner to inhibit 1) depolarization of mouse brain synaptoneurosomes induced by the sodium channel-selective activator veratridine (IC₅₀ = 21.1 μM); 2) veratridine-dependent release of l-glutamic acid (IC₅₀ = 12.2 μM) and γ-aminobutyric acid (IC₅₀ = 14.4 μM) from mouse purified whole-brain synaptosomes; and 3) the binding of the sodium channel site 2-selective ligand [³H]batrachotoxinin A 20-α-benzoate, to mouse brain synaptoneurosomal voltage-gated sodium channels (IC₅₀ = 19.5 μM). Anandamide displayed similar inhibitory activity in these four bioassays (IC₅₀ = 21.8, 5.1, 16.5, and 23.4 μM, respectively) (Nicholson et al., 2003) as did AM251 (IC₅₀ = 8.9, 8.5, 9.2, and 11.2 μM, respectively) (Liao et al., 2004). CP55940, N-arachidonoyl dopamine, noladin ether, and 2-AG have also been found to displace [³H]batrachotoxinin A 20-α-benzoate from mouse brain synaptoneurosomal voltage-gated sodium channels (IC₅₀ = 22.3, 20.7, 51.2, and 90.4 μM, respectively) (Duan et al., 2008a,b). The mechanism underlying the inhibition of binding induced in this assay by these four compounds, and by AM251 and anandamide but not by R-(+)-WIN55212, could well be allosteric in nature (Nicholson et al., 2003; Liao et al., 2004; Duan et al., 2008a,b). It has been found too by Duan et al. (2008a) that the ability of R-(+)-WIN55212 and anandamide to inhibit veratridine-dependent depolarization of mouse synaptoneurosomes (Nicholson et al., 2003) extends to CP55940 (IC₅₀ = 3.2 μM). This inhibition was produced by CP55940 in a noncompetitive manner. There have also been reports that voltage-gated sodium channels can be inhibited by anandamide in rat DRG sensory neurons (apparent K_d = 5.4 and 38.4 μM for tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents, respectively) (Kim et al., 2005) and by R-(+)-WIN55212 (10 μM) and noladin ether (50 μM) in frog parathyroid cells (Okada et al., 2005).

1. Direct Evidence for Peroxisome Proliferator-Activated Receptor Activation or Occupancy by Cannabinoids and Related Molecules.

A number of cannabinoid CB₁/CB₂ agonists have also been reported to be PPAR agonists in in vitro experiments (Table 8). These include the archetypal endocannabinoids anandamide and 2-AG as well as the phytocannabinoids Δ⁹-THC and cannabidiol and the synthetic cannabinoids R-(+)-WIN55212 and ajulemic acid (Table 8). The potency of the majority of these agents is approximately 2 orders of magnitude lower at PPARs than at the conventional cannabinoid GPCRs (CB₁ and CB₂), although it is noteworthy that data from two investigations suggest that PPARγ can be activated by Δ⁹-THC (O'Sullivan et al., 2005) and R-(+)-WIN55212 (Giuliano et al., 2009) at 100 nM. There are also reports that rimonabant and AM251 can activate both PPARγ (at 10 μM) and PPARα (O'Sullivan et al., 2006; Sun et al., 2007b). However, it remains unclear whether this represents a direct or indirect phenomenon associated with high ligand concentrations. Two fatty acid ethanolamides (oleoyl ethanolamide and palmitoyl ethanolamide), which are essentially inactive at cannabinoid CB₁ and CB₂ receptors, are agonists with reasonable potency at PPARα (Table 8). This receptor seems to display some preference for the medium-chain mono- or diunsaturated fatty acid ethanolamides, such as oleoyl ethanolamide and linoleoyl ethanolamide, compared with the long-chain polyunsaturated fatty acid ethanolamides, such as anandamide, N-eicosapentaenoylethanolamine and N-docosohexaenoylethanolamine (Artmann et al., 2008).

Oxidative metabolism of endocannabinoids generates agents that are also active at PPARs. Thus, 15-lipoxygenase metabolism of 2-AG leads to the production of 15(S)-hydroxyeicosatetraenoic acid-glyceryl ester (Kozak et al., 2002). This agent was identified as a preferential agonist at PPARα in a reporter gene assay using NIH 3T3 cells as hosts, with an EC₅₀ value of ~3 μM, but was inactive up to 10 μM at PPARβ or PPARγ. It is noteworthy that the arachidonic acid metabolite 15(S)-hydroxyeicosatetraenoic acid at 10 μM is a preferential PPARγ agonist (Kozak et al., 2002). 2-(14,15-Epoxyeicosatrienoyl)glycerol (a product of 2-AG metabolism by epoxygenase) but not 2-AG itself also appeared to be a PPARα agonist (Fang et al., 2006). However, mass spectrometry analysis suggested that the active entity was 14,15-dihydroxyeicosatrienoic acid, produced through sequential hydrolysis of the glyceryl ester and epoxide bonds. One of the problems this study highlights is that the extended periods necessary for identification of agonist action at PPARs (4 h or more) increases the potential for conversion of the added agent into an entity with altered activity at the receptor.

2. Indirect Evidence for Cannabinoid Activation of Peroxisome Proliferator-Activated Receptors.

Indirect means of identifying an action of cannabinoid-related molecules at PPARs include 1) the use of reporter genes in model cells where PPARs are either endogenously expressed or overexpressed and 2) the use of pharmacological or genetic inhibition of PPARs.

3. Reporter Gene Assays and Metabolism of Endocannabinoids.

COX-2-mediated metabolism of 2-AG, noladin ether, and anandamide led to greater activation of PPARβ compared with PPARγ, with no discernible PPARα activation, in human umbilical vein endothelial cells transfected with peroxisome proliferator-activated response element coupled to a reporter gene (Ghosh et al., 2007). COX-2 inhibition alone did not alter PPAR activation, suggesting that conversion of endogenous COX-2 substrates was not sufficient to activate PPARs. 2-AG itself, in the presence of a COX-2 inhibitor, was ineffective, indicating that COX-2 metabolism was an obligatory step in the production of a PPAR ligand. siRNA knockdown of prostacyclin synthase (CYP8A1) inhibited PPAR activation, demonstrating that prostaglandin I₂-glyceride was probably the endogenous ligand, although attempts to identify endogenous levels of this agent using mass spectrometry were unsuccessful (Ghosh et al., 2007).

4. Antagonism.

In mouse primary splenocytes in vitro, anandamide evoked a concentration-dependent inhibition of interleukin-2 secretion that was not blocked by rimonabant or SR144528 but was inhibited by 2-chloro-5-nitro-N-4-pyridinyl-benzamide (T0070907), indicating a role for PPARγ in these effects (Rockwell and Kaminski, 2004). Sequential metabolism in HeLa human cervical carcinoma cells by COX-2 and lipocalin type-prostaglandin D synthase led to activation of PPARγ by R-(+)-methanandamide, as evidenced by siRNA and 2-chloro-5-nitro-N-phenylbenzamide (GW9662) inhibition (Eichele et al., 2009). In vitro, the PPARγ antagonist GW9662 was able to block relaxations evoked by cannabidiol (O'Sullivan et al., 2009b), Δ⁹-THC (O'Sullivan et al., 2005), N-arachidonoyl dopamine and anandamide (O'Sullivan et al., 2009a) in rat isolated aorta. R-(+)-WIN55212-evoked apoptosis of HepG2 human hepatoma cells was inhibited by GW9662 (Giuliano et al., 2009). Prolonged exposure of these cells to R-(+)-WIN55212 also induced increased expression of PPARγ, a response associated with PPARγ activation. Likewise, HU-210 exposure over several days increased PPARγ expression in 3T3-F442A mouse preadipocyte cells; however, this effect was blocked by rimonabant, indicating the involvement of cannabinoid CB₁ receptors (Matias et al., 2006).

5. Genetic Disruption.

An alternative method, although not without caveats, is to make use of animals in which the PPARs are genetically disrupted. These mice have been employed on a limited number of occasions in an attempt to study the effects of cannabinoid-related molecules, although, notably, not the archetypal endocannabinoids anandamide or 2-AG, or other ligands that are known to target CB₁ or CB₂ receptors. Thus, antinociceptive effects of palmitoyl ethanolamide, as well as the PPARα agonist 2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]-phenyl]thio]-2-methylpropanoic acid (GW7647), were lost in mice in which the ppara gene was disrupted (LoVerme et al., 2006; D'Agostino et al., 2007; Sasso et al., 2010). ppara gene disruption abrogated oleoyl ethanolamide-evoked feeding behaviors (Fu et al., 2003), oleoyl ethanolamide-evoked lipolysis (Guzmán et al., 2004) and oleoyl ethanolamide-mediated neuroprotection (Sun et al., 2007a), but not oleoyl ethanolamide effects on visceral pain (Suardíaz et al., 2007) or intestinal motility (Cluny et al., 2009). Genetic disruption of PPARγ in a homozygous manner results in embryonic lethality, so heterozygous mice have been generated and studied (Kishida et al., 2001), as have mice with conditional disruption of the pparg gene (Akiyama et al., 2002). However, the use of these mice or those in which PPARβ is genetically disrupted (Peters et al., 2000) has not been reported in investigations of endocannabinoids and their analogs.

6. Amplification of Endocannabinoid Levels and Peroxisome Proliferator-Activated Receptors.

Nicotine-induced elevation of firing of ventral tegmental area neurons in anesthetized rats was inhibited by prior administration (60–120 min) of URB597, an inhibitor of the anandamide-metabolizing enzyme FAAH (Melis et al., 2008). Rimonabant failed to alter the effects of URB597, whereas 1-[(4-chlorophenyl) methyl]-3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid (MK886) was able to prevent the effects of URB597. The effects of URB597 were mimicked in vitro by administration of oleoyl ethanolamide, palmitoyl ethanolamide, or the PPARα agonist WY14643 in a manner sensitive to MK886 (Melis et al., 2008).

Palmitoylallylamide, described as a low-potency inhibitor of FAAH (Vandevoorde et al., 2003), evoked an inhibition of nociceptive behaviors in rat models of neuropathic pain 40 to 100 min after administration (Wallace et al., 2007). These effects were reduced after administration of rimonabant, SR144528, or MK886, dependent on the paradigm under study. In a passive avoidance paradigm, URB597 administration to rats 40 min before testing enhanced memory acquisition (Mazzola et al., 2009), whereas Δ⁹-THC administration impaired memory. The URB597 enhancement was reduced by either rimonabant or MK886. Intraplantar administration of a low, but not high, dose of URB597 reduced pain behaviors in the carrageenan model of inflammatory pain (Jhaveri et al., 2008) up to 210 min after injection. At this time, levels of anandamide and 2-AG, but not oleoyl ethanolamide or palmitoyl ethanolamide, were elevated in URB597 and carrageenan-treated paws compared with tissue from animals treated with carrageenan alone. The antinociceptive effects of URB597 were prevented by coadministration of the PPARα-selective antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl)-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxyphenylpropyl]-carbamic acid ethyl ester (GW6471) but not the PPARγ-selective antagonist GW9662, indicating a role for PPARα in these responses.

Blockade of endocannabinoid uptake by (5Z,8Z, 11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) evoked neuroprotective effects in mouse mixed astrocytic neuronal cultures by activation of CB₁, CB₂, and PPARγ receptors (Loría et al., 2010). Application of CB₁ and CB₂ receptor antagonists, but not the PPARγ antagonist GW9662, led to an exacerbation of the excitotoxic effects of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid application. Administration of N-(4-hydroxyphenyl)-5Z,8Z, 11Z,14Z-eicosatetraenamide (AM404), a mixed inhibitor of FAAH and endocannabinoid uptake, enhanced bacterial lipopolysaccharide-evoked tumor necrosis factor-α levels in rat plasma. Antagonists of CB₁, CB₂, and TRPV1 receptors attenuated elevations of tumor necrosis factor-α levels in response to LPS and the combination of LPS and AM404, whereas PPARγ antagonism interfered only with the AM404-evoked elevation (Roche et al., 2008). Collectively, these data might be taken to indicate that PPARs are only activated significantly in vivo by endocannabinoids when endocannabinoid levels are pharmacologically amplified.

7. Regulation by Peroxisome Proliferator-Activated Receptors and Peroxisome Proliferator-Activated Receptor Ligands of the Endocannabinoid System.

There is some evidence that the functioning of the endocannabinoid system can be affected by PPARs and PPAR ligands. This has come primarily from experiments with adipocytes. For example, 24 h of activation of PPARγ by ciglitazone decreased levels of 2-AG, but not anandamide, in immature, but not mature, 3T3 F442A mouse adipocytes (Matias et al., 2006). It remains to be determined whether this effect is due to a direct action on endocannabinoid synthesizing and/or metabolizing enzymes, or a consequence of cellular differentiation; interestingly, however, Pagano et al. (2007) have found that the PPARγ agonist rosiglitazone up-regulates FAAH in human adipocytes. In 3T3 L1 mouse preadipocyte cells, a related cell type, ppard gene silencing increased CB₁ receptor gene expression (Yan et al., 2007). Treatment of human adipocytes in vitro with the PPARγ agonist rosiglitazone markedly down-regulated CB₁ receptor gene expression, whereas R-(+)-WIN55212 up-regulated PPARγ (Pagano et al., 2007). Treatment of human adipocytes in vitro with a different PPARγ agonist, ciglitazone, failed to alter endocannabinoids levels (Gonthier et al., 2007).

In a distinct series of investigations, a number of PPARγ agonists, including ciglitazone, pioglitazone, rosiglitazone, and troglitazone, were shown to inhibit FAAH activity in rat brain membranes, as well as in intact C6 rat glioma and RBL-2H3 rat basophilic leukemia cells (Lenman and Fowler, 2007). The low potency with which these inhibitions were induced (IC₅₀ values ~100 μM) suggests that this is an unlikely mechanism for influencing endocannabinoid levels.