Evidence for novel cannabinoid receptors

Transcription

1 Pharmacology & Therapeutics 106 (2005) Associate editor: D.M. Lovinger Evidence for novel cannabinoid receptors Malcolm Begg 1,Pál Pacher, Sándor Bátkai, Douglas Osei-Hyiaman, László Offertáler, Fong Ming Mo, Jie Liu, George Kunos* National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, MSC-9413 Bethesda, MD , United States Abstract Cannabinoids, including the bioactive constituents of the marijuana plant, their synthetic analogs, and endogenous lipids with cannabinoid-like activity, produce their biological effects by interacting with specific receptors. To date, two G protein-coupled cannabinoid receptors have been identified by molecular cloning, CB 1 receptors mainly expressed in the brain and mediating most of the neurobehavioral effects of cannabinoids and CB 2 receptors expressed by immune and hematopoietic tissues. Recent findings indicate that some cannabinoid effects are not mediated by either CB 1 or CB 2 receptors, and in some cases there is compelling evidence to implicate additional receptors in these actions. These include transient receptor potential vanilloid 1 (TRPV 1 ) receptors and as-yet-unidentified receptors implicated in the endothelium-dependent vasodilator effect of certain cannabinoids and in the presynaptic inhibition of glutamatergic neurotransmission in the hippocampus. The case for these additional receptors is being reviewed here. D 2004 Elsevier Inc. All rights reserved. Keywords: Endocannabinoids; Cannabinoid receptors; Endothelium; Vasodilation; Hippocampus Abbreviations: 2-AG, 2-arachidonoylglycerol; abn-cbd, abnormal cannabidiol; BK Ca, large conductance calcium-activated potassium channel; CGRP, calcitonin gene-related peptide; DSI or DSE, depolarization-induced suppression of inhibition or excitation; EDHF, endothelium-derived hyperpolarizing factor; GPCR, G protein-coupled receptor; HUVEC, human umbilical vein endothelial cell; IPSC or EPSC, inhibitory or excitatory postsynaptic current; LPS, lipopolysaccharide; NO, nitric oxide; PEA, palmitoylethanolamine; PTX, pertussis toxin; THC, D 9 -tetrahydrocannabinol; TRPV receptor, transient receptor potential vanilloid receptor. Contents 1. Introduction CB 1 and CB 2 receptors Non-CB 1 /non-cb 2 endothelial cannabinoid receptor Pharmacology of the endothelial cannabinoid receptor Signaling by the endothelial cannabinoid receptor Possible physiological functions of the endothelial cannabinoid receptor Atypical cannabinoid receptors in the central nervous system Pharmacology of the non-cb 1 hippocampal cannabinoid receptor Function of hippocampal non-cb 1 receptors Role of transient receptor potential vanilloid 1 receptors in endocannabinoid action Evidence for additional cannabinoid-sensitive receptors Conclusions References * Corresponding author. Tel.: ; fax: address: gkunos@mail.nih.gov (G. Kunos). 1 Current address: National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK /$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.pharmthera

2 134 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) Introduction The marijuana plant (Cannabis sativa) contains more than 60 bioactive ingredients, of which D 9 -tetrahydrocannabinol (THC) is mainly responsible for its psychoactive properties. As recently as the 1970s, it was believed that THC produced its effects by perturbing neuronal cell membranes due to its lipid-soluble, hydrophobic nature. However, the structural and steric selectivity of the actions of THC and its synthetic analogs suggested the involvement of specific receptors. This was subsequently demonstrated in radioligand-binding studies that documented the existence of saturable, stereoselective, high-affinity membranebinding sites for cannabinoids in the mammalian brain (Devane et al., 1988). In the early 1990s, 2 distinct G protein-coupled cannabinoid receptors were identified by molecular cloning: the brain-type cannabinoid receptor later termed CB 1, which is highly expressed in the brain but is also present in peripheral tissues, and CB 2 receptors whose expression is largely limited to cells of the immune and hematopoietic systems (see below). The existence in mammalian cells of specific membrane receptors for plant-derived substances triggered a search for an endogenous ligand. In 1992, this search culminated in the identification of arachidonoyl ethanolamide, named anandamide, a brain-derived lipid that binds to cannabinoid receptors and mimics the biological effects of THC (Devane et al., 1992). Three years later, a second endogenous cannabinoid, 2-arachidonoylglycerol (2-AG), was isolated from gut (Mechoulam et al., 1995) and brain tissue (Sugiura et al., 1995). In the ensuing years several other related lipids with endocannabinoid properties were identified, but have been characterized less extensively than anandamide or 2- AG (Hanus et al., 2001; Porter et al., 2002). The subsequent development of subtype-selective cannabinoid receptor antagonists (Rinaldi-Carmona et al., 1994, 1998; Gatley et al., 1997) played a major role in identifying a growing list of biological processes regulated by endocannabinoids. The role of endocannabinoids and their receptors in such processes was then further confirmed by the use of genetically altered mice strains lacking CB 1 (Ledent et al., 1999; Zimmer et al., 1999) orcb 2 receptors (Buckley et al., 2000). Studies using these receptor knockout mice also indicated, however, that some effects of endocannabinoids are mediated neither by CB 1 nor by CB 2 receptors, pointing to the existence of additional as-yetunidentified sites of action. Some of these effects may be linked to certain chemical properties of cannabinoid ligands and may not be mediated by specific receptors such as effects attributed to the antioxidant properties of certain cannabinoid ligands (Hampson et al., 1998) or to their high degree of lipophilicity, which may account for nonspecific interactions at the level of the plasma membrane or at intracellular sites. In contrast, in some other cases, there are reasonable grounds to postulate interactions with specific membrane receptors distinct from CB 1 or CB 2, based on the structural or steric selectivity of the response or its sensitivity to inhibition by pertussis toxin (PTX). The present review will discuss the growing evidence for novel cannabinoid receptors and their possible physiological roles. 2. CB 1 and CB 2 receptors The biological effects of endogenous, plant-derived and synthetic cannabinoids are mediated through specific G protein-coupled cannabinoid (CB) receptors. To date, 2 such receptors have been identified by molecular cloning, CB 1 and CB 2 receptors. There is an extensive literature on the pharmacology, molecular biology, and functional roles of CB 1 and CB 2 receptors, and only some salient features will be summarized here. The CB 1 receptor was originally cloned as an orphan G protein-coupled receptor (GPCR; Matsuda et al., 1990) and its functional identity was revealed by the perfect overlap between the brain distribution of its mrna and of the specific binding sites for a radiolabeled cannabinoid (Herkenham et al., 1990). The human homolog of the CB 1 was identified shortly thereafter (Gerard et al., 1991), and a second cannabinoid receptor was cloned from rat spleen and named CB 2 (Munro et al., 1993). A splice variant of the mrna encoding the CB 1 receptor has also been identified in human and rat tissues (Shire et al., 1996b), but the existence of the protein product of this mrna has not yet been demonstrated. CB 1 receptors are expressed predominantly in the CNS with particularly high levels in cerebellum, hippocampus and basal ganglia. In fact, of all known neurotransmitter and hormone receptors, the CB 1 receptor is by far the most abundant in the mammalian brain. CB 1 receptors are also expressed, albeit at much lower levels, in the peripheral nervous system as well as on the cells of the immune system, in the heart, vascular tissues, and the testis (Herkenham et al., 1990; Gerard et al., 1991; Ishac et al., 1996; Pertwee, 1997; Gebremedhin et al., 1999; Liu et al., 2000; Bonz et al., 2003). CB 1 receptors are present and evolutionarily conserved in many species including hydra, mollusk, leech, see urchin, fish, rodents, and humans (reviewed in Pertwee, 1997; Reggio, 2003). In tissues naturally expressing CB 1 receptors and in transfected cell lines, both CB 1 (Bouaboula et al., 1995; Pan et al., 1998; Meschler et al., 2000; Mato et al., 2002; Losonczy et al., 2004) and CB 2 receptors (Bouaboula et al., 1999) have been shown to have a high level of ligand-independent activation (i.e., constitutive activity). It was estimated that in the population of wild-type CB 1 receptors only 30% exist in the activated state, while 70% are inactive (Kearn et al., 1999). The expression of the CB 2 receptor is more restricted, limited primarily to immune and hematopoietic cells (Munro et al., 1993). The human CB 2 receptor shows 68% amino acid homology with the CB 1 receptor in the transmembrane domains and a 44% overall homology (Munro et al., 1993). Despite this surprisingly low

3 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) homology, the pharmacology of the 2 receptors is remarkably similar, with most plant-derived, endogenous, and classical synthetic cannabinoids having similar affinities for the 2 receptors (Showalter et al., 1996), although synthetic agonists with greater than 100-fold selectivity for CB 1 (Hillard et al., 1999) orcb 2 receptors (Hanus et al., 1999; Huffman et al., 1999; Malan et al., 2001) have been developed. In contrast to the CB 1 receptor, which is highly conserved in mice, rats, and humans, the CB 2 receptor is much more divergent. The amino acid homology between mouse and rat is 93%, while between rat and human it is only 81% (Munro et al., 1993; Shire et al., 1996a; Griffin et al., 2000). The molecular biology of cannabinoid receptors has been recently reviewed by Lutz (2002). Both CB 1 and CB 2 receptors are coupled through G i and G o proteins to inhibit adenylyl cyclase and regulate calcium and potassium channels (Mackie & Hille, 1992; Mackie et al., 1995). Moreover, various intracellular kinases, including the mitogen-activated protein kinases, extracellular signalregulated kinases type 1 and 2, JUN N-terminal kinase, focal adhesion kinase, and protein kinase B/Akt, are also activated by CB 1 receptors (Bouaboula et al., 1995, 1997; Derkinderen et al., 1996, 2001, 2003; Gomez del Pulgar et al., 2000; Rueda et al., 2000). There is evidence for agonist selectivity for CB 1 receptors coupled to different subtypes of G i proteins (Houston & Howlett, 1998) ortog i vs. G o proteins (Glass & Northup, 1999). Cannabinoid receptor signal transduction has been recently reviewed by Howlett (2004). 3. Non-CB 1 /non-cb 2 endothelial cannabinoid receptor The first indication that cannabinoid receptors other than CB 1 or CB 2 may exist came from studies of the mesenteric vasodilator effect of cannabinoids. In the rat isolated perfused mesenteric arterial bed preparation, anandamide, and R-methanandamide elicit long-lasting vasodilation, whereas synthetic cannabinoids potent at both CB 1 and CB 2 receptors or THC do not have a dilator effect (Wagner et al., 1999). Although the vasodilator response to anandamide and R-methanandamide could be inhibited by the selective CB 1 receptor antagonist SR (Rinaldi- Carmona et al., 1994), somewhat higher concentrations were needed (1 5 AM) than concentrations sufficient to inhibit CB 1 receptors (Chaytor et al., 1999; Járai et al., 1999; Wagner et al., 2001a, 2001b; White et al., 2001; Mukhopadhyay et al., 2002). Furthermore, the inhibitory activity of SR depended on intact vascular endothelium and was lost following endothelial denudation (Chaytor et al., 1999; Járai et al., 1999; Wagner et al., 2001a, 2001b; Mukhopadhyay et al., 2002). As a result, it was proposed that an endothelial site distinct from CB 1 or CB 2 receptors, yet somewhat sensitive to inhibition by SR141716, is involved in the vasodilator effect of anandamide and R-methanandamide in the rat mesenteric circulation (Járai et al., 1999). A similar mechanism may operate in the rat coronary circulation (Ford et al., 2002). Within the mesenteric vascular bed, these receptors appear to be localized selectively in resistance, but not in conduit, vessels (O Sullivan et al., 2004b). SR sensitive effects that persist in CB 1 knockout mice have also been described in in vivo paradigms. Bacterial endotoxin (lipopolysaccharide [LPS]) increases anandamide synthesis in circulating macrophages, and the acute hypotensive response to LPS or to LPS-treated macrophages is inhibited by SR (Varga et al., 1998; Liu et al., 2003), suggesting endocannabinoid involvement in endotoxic hypotension. However, another CB 1 antagonist (AM251) fails to inhibit LPS-induced hypotension in rats, which is due to a decrease in cardiac contractility rather than a decrease in vascular resistance, and the inhibitory effect of SR in wild-type mice remains unchanged in mice deficient in CB 1 or in both CB 1 and CB 2 receptors (Bátkai et al., 2004). The relationship between the SR sensitive AM251-insensitive cardiac site and the putative vascular endothelial receptor or a non- CB 1 /CB 2 site mediating the negative inotropic effect of anandamide in isolated rat hearts (Ford et al., 2002) is not yet clear. Interestingly, CB 1 receptors also appear to be involved in some effects of LPS; they have been implicated in the presynaptic sympathoinhibitory effect of LPS that probably does not contribute to the overall blood pressure response (Godlewski et al., 2004). In another in vivo paradigm, blockade of milk suckling in newborn mice by SR persists in CB 1 receptor deficient mice, illustrating another CB 1 -independent effect of this antagonist (Fride et al., 2003) Pharmacology of the endothelial cannabinoid receptor The endothelium-dependent response is selectively activated by anandamide and R-methanandamide, but not by THC or synthetic agonists, which suggests distinct structure-activity relationships for ligands recognized by the putative endothelial receptor. We were intrigued by an earlier study published by Adams et al. (1977), in which the authors reported that abnormal cannabidiol (abn-cbd), a structural analog of the behaviorally inactive marijuana constituent cannabidiol (see Fig. 1), was inactive in 2 behavioral tests in mice used at the time to screen cannabinoids, but elicited a major drop in blood pressure in dogs, suggesting that it may be a nonpsychotropic agonist at cardiovascular receptors. The parent compound, cannabidiol, was similarly inactive in the behavioral paradigms and was also devoid of an effect on blood pressure (Adams et al., 1977). Indeed, in a study in the rat isolated, buffer-perfused mesenteric bed we found that abn-cbd caused SR sensitive, endothelium-dependent vasodilation, although it did not bind to either CB 1 or CB 2 receptors (Járai et al., 1999). Thus, abn-cbd appears to

4 136 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) Fig. 1. Chemical structure of the endocannabinoids anandamide and 2-arachidonoyl glycerol, and of synthetic ligands used for the pharmacological characterization of non-cb 1 /non-cb 2 cannabinoid receptors. be a selective agonist of the endothelial cannabinoid receptor. Cannabidiol itself did not elicit vasodilation in the same preparation, but at micromolar concentrations it antagonized the dilation caused by abn-cbd, thus acting as an antagonist. In these studies, 1 5 AM of the CB 1 antagonist SR was needed to antagonize the vasodilator effects of anandamide and abn-cbd, which is higher than the submicromolar concentrations sufficient to block CB 1 receptor-mediated responses, and a similar difference was noted by others (Mukhopadhyay et al., 2002; Ho & Hiley, 2003; O Sullivan et al., 2004a, 2004b). Furthermore, the compound AM251, which only differs from SR in one of the phenolic chloride side groups being substituted with iodine (Fig. 1) and which has similar nanomolar potency as a CB 1 receptor antagonist (Gatley et al., 1997), failed to inhibit the vascular effect of anandamide (White et al., 2001; O Sullivan et al., 2004a, 2004b) or abn-cbd (Ho & Hiley, 2003; Mo et al., 2004). A similar dichotomy was evident regarding blockade of the vasorelaxant effect of the endogenous ligands N-arachidonoyl dopamine (O Sullivan et al., 2004a, 2004b) and virodhamine (Ho & Hiley, 2004). This remarkable structural selectivity supports the involve-

5 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) ment of a common specific receptor in the vasodilator action of the above substances. In a subsequent study using rat isolated mesenteric artery segments set up in a wire myograph, abn-cbd caused similar vasorelaxation as a full agonist with an EC 50 of ~2 3 AM. Unexpectedly, cannabidiol and SR141716, both of which were effective antagonists in the resistance vessels of the mesenteric arterial bed preparation, potently relaxed the isolated artery segments, precluding their use as antagonists. This prompted us to develop structurally modified analogs of cannabidiol to search for a bsilentq antagonist. The compound O-1918 (Fig. 1) does not relax mesenteric arterial segments at concentrations up to 30 AM, but concentrationdependently inhibits the vasorelaxant response to abn-cbd without affecting vasorelaxation by carbachol or calcitonin gene-related peptide (CGRP; Offertáler et al., 2003). Analysis of the pharmacological properties of abn-cbd supported its selective interaction with an endothelial site distinct from CB 1 or CB 2 (see also Table 1). Abn-cbd does not bind to CB 1 receptors in rat cerebellar membranes or to human CB 2 receptor expressed in chinese hamster ovary (CHO) cells at concentrations up to 100 AM (Showalter et al., 1996; Offertáler et al., 2003) and does not induce analgesia, hypomotility, hypothermia, or catalepsy in mice at doses up to 60 mg/kg (Járai et al., 1999). Yet, abn-cbd elicits endothelium-dependent vasodilation in the bufferperfused mesenteric vascular bed of rats and mice including mice deficient in both CB 1 and CB 2 receptors, and these effects are inhibited by 1 5 AM SR (Járai et al., 1999). In the rat mesenteric vasculature, the vasodilator response to abn-cbd was unaffected by L-NAME+indomethacin, suggesting that endothelial nitric oxide (NO) and prostacyclin are not involved. On the other hand, a combination of apamin (100 nm) and charybdotoxin (100 nm), inhibitors of calcium-activated potassium channels, significantly attenuated the vasodilation caused by abn-cbd. As the same combination of potassium channel blockers inhibit mesenteric vasodilation induced by the endotheliumderived hyperpolarizing factor (EDHF; Randall & Kendall, 1998), these findings could suggest that activation of this novel endothelial receptor may trigger the release of EDHF (Járai et al., 1999; also see above), at least in the rat mesenteric vasculature. In view of the documented interaction of anandamide with transient receptor potential vanilloid 1 (TRPV 1 ) receptors (Zygmunt et al., 1999), it is important to distinguish between effects mediated by TRPV 1 receptors and abn-cbd-sensitive receptors. Capsazepine, a TRPV 1 receptor inhibitor, does not influence the mesenteric vasodilator response to abn-cbd at a concentration which blocks the capsaicin-induced vasodilation or vasodilation in response to anandamide in endothelium-intact preparations (Járai et al., 1999; Offertáler et al., 2003). Similarly, desensitization of TRPV 1 receptors by capsaicin treatment does not affect abn-cbd-induced mesenteric vasorelaxation (Ho & Hiley, 2003). On the other hand, cannabidiol, which Table 1 Differential interaction of cannabinoid ligands with CB 1, TRPV 1 and non- CB 1 /CB 2 endothelial and CNS receptors Agonists CB 1 TRPV 1 Non-CB 1 /non-cb 2 Endothelial CNS Anandamide ++ a,b + b + c,d,e n.d. 2-AG ++ a,f 0 g 0 h n.d. Noladin-ether ++ i 0 j + j,k n.d. WIN55, b 0 g 0 l,h + m NADA ++ n ++ o ++ e,k n.d. Virodhamine ++ p 0 q ++ q n.d. THC ++ b 0 g 0 h n.d. CP55, b 0 g 0 l + m HU b n.d. 0 n.d. Abn-cbd 0 b,c,d 0 d,k,l + c,d,r 0 s SR b,t 0 c,u + c,h,l + m AM v 0 0 e,r 0 m Cannabidiol 0 b 0 w + c n.d. O d n.d. + d,e,q,z n.d. Capsazepine n.d. u + u 0 c,e,l,r + m Symbols represent potency/binding affinity in nanomolar range (++), micromolar range (+), no interaction or N10 AM (0), or not determined (n.d.), which are documented in the indicated reference(s). a Mechoulam et al., b Showalter et al., c Járai et al., d Offertáler et al., e O Sullivan et al., 2004a. f Sugiura et al., g Zygmunt et al., h Wagner et al., i Hanus et al., j Duncan et al., k Offertáler, L., Bátkai, S., & Kunos, G., unpublished observations. l Mukhopadhyay et al., m Hájos & Freund, n Bisogno et al., o Huang et al., p Porter et al., q Ho & Hiley, r Ho & Hiley, s Begg, M., Lovinger, D. M., & Kunos, G., unpublished observations. t Rinaldi-Carmina et al., u Pacher et al., v Gatley et al., w McQueen et al., z Begg et al., inhibits the mesenteric vasodilator effect of abn-cbd, does not interact with TRPV 1 receptors (McQueen et al., 2004). These findings clearly indicate that TRPV 1 receptors are not involved in the endothelium-dependent vasodilator effect of abn-cbd. The ability of capsazepine to antagonize anandamideinduced mesenteric vasorelaxation (Zygmunt et al., 1999) reflects an interaction with the endothelium-independent, SR insensitive component of the effect of anandamide (Járai et al., 1999). Similar conclusions were reached in a study of the vasodilator action of anandamide in rabbit isolated aortic rings, which had a major endotheliumdependent component sensitive to SR and PTX but not to capsazepine, and a minor endothelium-independent

6 138 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) component insensitive to PTX and SR but inhibited by TRPV 1 or CGRP receptor antagonists (Mukhopadhyay et al., 2002). In contrast to the NO-independent mesenteric vasorelaxant effect of anandamide, its effect on the rabbit aorta is NO-dependent (Mukhopadhyay et al., 2002). Thus, the involvement of NO in effects mediated by the atypical endothelial cannabinoid receptor may depend on species and vessel type Signaling by the endothelial cannabinoid receptor The potential involvement of a GPCR in the vasorelaxant effect of anandamide is suggested by its PTX sensitivity, as first documented by White and Hiley (1997) in rat mesenteric arteries. Similarly PTX-sensitive is the endothelium-dependent vasodilator effect of abn-cbd in the rat mesentery (Járai et al., 1999; Offertáler et al., 2003) orof anandamide in the rabbit aorta (Mukhopadhyay et al., 2002). Together, these observations implicate a G i /G o - coupled endothelial receptor in the above effects. Ho and Hiley (2003) reported, however, that the mesenteric vasodilator effect of abn-cbd was unaffected by PTX, although its other properties, including its endothelium-dependence and susceptibility to inhibition by the compound O-1918 (see below), were similar to those reported by Offertáler et al. (2003). Signaling mechanisms activated by the endothelial non- CB 1 /non-cb 2 cannabinoid receptor have been explored using human umbilical vein endothelial cells (HUVECs). In HUVEC maintained in primary culture, abn-cbd was found to activate p42/44 MAP kinase and protein kinase B/Akt phosphorylation (Offertáler et al., 2003). Similar to the vasorelaxant effect of abn-cbd in isolated artery segments, the above effects in HUVEC were inhibited by O-1918 or by preincubation of HUVEC with PTX (Offertáler et al., 2003), suggesting that the same or similar receptors are involved. In recent studies, O-1918 was reported to inhibit the mesenteric vasorelaxant effect of 2 putative novel endogenous ligands, the endovanilloid N-arachidonoyl dopamine (O Sullivan et al., 2004a, 2004b) and the endocannabinoid virodhamine (Ho & Hiley, 2004). Anandamide has relatively low potency in eliciting mesenteric vasorelaxation or Akt phosphorylation in HUVEC (Offertáler et al., 2003), so these findings raise the possibility that the primary endogenous ligand at the endothelial receptor is not anandamide. The ability of charybdotoxin to inhibit the vasodilator effect of abn-cbd (Ho & Hiley, 2003; Offertáler et al., 2003; see also above) suggests the involvement of a calciumactivated K + channel. We have described a voltage-dependent outward current in HUVEC that is carried by K + ions and is blocked by charybdotoxin and iberiotoxin, indicating the involvement of the large conductance calcium-activated potassium channel (BK Ca ) channel (Begg et al., 2003). Abncbd, which does not elicit a K + current on its own, concentration-dependently increases the voltage-gated K + current and this effect is blocked by PTX, implicating a G i / G o -coupled receptor. However, potentiation of the K + current by abn-cbd is unaffected by 1 AM SR or 1 AM SR144528, discounting the role of CB 1 or CB 2 receptors, respectively (Begg et al., 2003). Accordingly, the K + current is also unaffected by the potent synthetic CB 1 /CB 2 receptor agonist HU-210, which is devoid of mesenteric vasodilator activity. O-1918, which produces no effect by itself, inhibits the potentiation of the K + current by abn-cbd, suggesting that the receptor involved may be the same as that mediating mesenteric vasorelaxation by abncbd. The same concentration of O-1918 has no effect on the iberiotoxin-sensitive current induced by the selective BK Ca opener NS-1619, indicating that O-1918 blocks the effect of abn-cbd at a site proximal to the channel, likely at the receptor (Begg et al., 2003). The parallel effects of O-1918 and PTX in HUVEC and in the rat mesenteric artery (Offertáler et al., 2003) suggest that activation of BK Ca channels is involved in the mesenteric vasodilation mediated by the abn-cbd-sensitive endothelial receptor. This conclusion is supported by the finding that iberiotoxin significantly inhibits abn-cbd-induced vasorelaxation in intact, but not in endothelium-denuded, rat mesenteric arteries (Begg et al., 2003). Anandamide also increases the K + current evoked in HUVEC by a single voltage step, but it is ~10 times less potent than abn-cbd and its effect is partially inhibited by O-1918 or PTX (Begg et al., 2003). In contrast, anandamide acts as a full agonist in the rat isolated mesenteric artery preparation with an EC 50 comparable to that of abn-cbd (Offertáler et al., 2003), suggesting subtle differences between the rat and human receptors, or a nonlinear relationship between K + channel activation and vasodilation. As mentioned above, the low potency and efficacy of anandamide at the human endothelial receptor could suggest the existence of endogenous ligand(s) other than anandamide. In HUVEC, the K + current potentiated by abn-cbd is similarly potentiated by intracellularly applied cgmp or by YC-1, a soluble guanylyl cyclase activator. The effects of both abn-cbd and cgmp are antagonized by the protein kinase G inhibitor KT-5823, whereas the guanylyl cyclase inhibitor ODQ blocks the effect of abn-cbd, but not of cgmp. Furthermore, cgmp increases the K + current under conditions where intracellular Ca 2+ is held constant, indicating that its action is not due to modulation of [Ca 2+ ] i. The effects of abn-cbd, cgmp, and YC-1 on K + currents are not additive, suggesting that these compounds utilize a common intracellular pathway. Finally, abn-cbd increases intracellular cgmp, and this effect is blocked by O-1918 (Begg et al., 2003). Together, these data suggest that the novel, G i /G o -coupled receptor activated by abn-cbd is positively coupled to guanylyl cyclase to raise intracellular cgmp, which activates protein kinase G. NO is considered to be an obligatory intermediate in receptor-mediated

7 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) activation of soluble guanylate cyclase. Although inhibition of NO synthesis does not appear to affect the vasorelaxant effect of abn-cbd in the rat mesenteric artery (Ho & Hiley, 2003; Offertáler et al., 2003), NO involvement in the effects of abn-cbd in HUVEC has not been ruled out. The proposed signaling pathway activated by the putative endothelial receptor is illustrated in Fig. 2. TRPV 4 Ca 2+ entry channels in vascular endothelial cells have recently been implicated in the vasorelaxant effect of anandamide (Watanabe et al., 2003). However, for anandamide to activate TRPV 4 channels, it needs to be enzymatically degraded yielding arachidonic acid, which is then further metabolized by P450 epoxygenase. Correspondingly, the metabolically stable anandamide analog, R-methanandamide, is inactive at TRPV 4 channels (Watanabe et al., 2003), whereas it is equipotent with Fig. 2. Proposed signalling cascade of the endothelial cannabinoid receptor (for details, see Begg et al., 2003). anandamide in eliciting mesenteric vasodilation in rats (Wagner et al., 1999). An additional reason why TRPV 4 channels are unlikely to be involved in the effects of abncbd on the outward current or on vascular tone is that these effects are sensitive to PTX, whereas TRPV 4 - mediated calcium entry is not. Furthermore, in HUVEC there was no sign of an abn-cbd-induced inward current, and potentiation of the outward current by abn-cbd persisted in the presence of clamped intracellular calcium (Begg et al., 2003) Possible physiological functions of the endothelial cannabinoid receptor Extracellular calcium is known to have a potent vasodilator effect, particularly in the mesenteric circulation where it is thought to contribute to the postprandial vasodilation associated with the intestinal absorbtion of nutrients. Bukoski and coworkers found that SR can inhibit Ca 2+ -induced mesenteric vasodilation through a sensory nerve-dependent mechanism, which led them to suggest that anandamide may be a sensory nerve-derived vasodilator mediator (Ishioka & Bukoski, 1999). Interestingly, O-1918 also inhibits calcium-induced mesenteric vasorelaxation, which is similar in wild-type and CB 1 receptor knockout mice (Bukoski et al., 2002), suggesting that the vasodilation by extracellular calcium is most likely mediated by the endothelial abn-cbd-sensitive receptor. A recent study using newborn mouse microglial cells in primary culture indicates that exposure of these cells to various stimuli induces the production of 2-AG, which promotes their migration via a PTX-sensitive mechanism (Walter et al., 2003). In this study, abn-cbd also promoted microglial migration and its effect could be antagonized by cannabidiol or by O-1918, whereas the effect of 2-AG could be antagonized either by a CB 2 antagonist, by cannabidiol or by O This suggested the involvement of CB 2 receptors and a receptor similar to the abn-cbd-sensitive endothelial receptor. Interestingly, stimulation of microglial migration by arachidonoyl cyclopropylamide, a potent CB 1 - selective agonist (Hillard et al., 1999), also involved the above 2 and not CB 1 receptors (Franklin & Stella, 2004). Microglial migration has an important role in neuroinflammatory diseases where microglia migrating to the site of lesion releases cytokines to kill dying neurons, thus amplifying the inflammatory response. Interference with this process by cannabidiol may contribute to the beneficial effect of marijuana-smoking in multiple sclerosis (Consroe et al., 1997). An abn-cbd-sensitive receptor may also be involved in endothelial cell migration. Mo et al. (2004) reported that migration of HUVEC in culture is stimulated by abn-cbd, and this effect is inhibited by O-1918 or by PTX. Migration of vascular endothelial cells occurs during angiogenesis, a process activated by ischemia or by tumor growth. This highlights the potential pathological and

8 140 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) therapeutic importance of the receptors and their ligands involved in angiogenesis. 4. Atypical cannabinoid receptors in the central nervous system Cannabinoid-activated receptors distinct from CB 1 or CB 2 have been also postulated to exist in the central nervous system. Cannabinoids are known to inhibit GABA A -mediated inhibitory postsynaptic currents (IPSCs) in the hippocampus via a presynaptic action at CB 1 receptors located on GABAergic terminals (Hájos et al., 2000; Wilson et al., 2001). CB 1 receptors have also been implicated in the inhibition of glutamatergic excitatory postsynaptic currents (EPSCs; Stella et al., 1997; Ameri et al., 1999; Misner & Sullivan, 1999). However, Hájos et al. (2001) reported that the synthetic cannabinoid agonist WIN55,212-2 inhibited monosynaptically evoked EPSCs in CA1 pyramidal cells not only from wild-type but also from CB 1 receptor knockout mice. This and their inability to detect CB 1 receptor protein on glutamatergic terminals by light- and electronmicroscopic immunohistochemistry led the authors to postulate the existence of a presynaptic receptor distinct from CB 1 or CB 2 on glutamatergic terminals in the mouse hippocampus. The existence of such receptors is also supported by the findings of Rouach and Nicoll (2003) who reported that activation of group 1 metabotropic receptors releases endocannabinoids and causes short-term depression of excitatory transmission in the CA1 region of the hippocampus of both wild-type and CB 1 knockout mice. Breivogel et al. (2001) reported that anandamide and WIN55,212-2, but not other cannabinoid agonists, stimulated GTPgS binding in brain plasma membranes from CB 1 receptor knockout mice, in further support of a G protein-coupled cannabinoid receptor in the brain, other than CB 1. Depolarization of postsynaptic neurons is known to retrogradely suppress both inhibitory and excitatory neurotransmission from presynaptic terminals, and the mediator of these effects has been proposed to be an endocannabinoid (Kreitzer & Regehr, 2001; Wilson & Nicoll, 2001). Ohno-Shosaku et al. (2002) reported that depolarizationinduced suppression of inhibition (DSI) or excitation (DSE) can both be induced in rat hippocampal slices, and WIN55,212-2 inhibits both IPSCs and EPSCs. However, in contrast to the above studies they found that both effects of WIN55,212-2 were absent in preparations from CB 1 deficient mice. Ohno-Shosaku et al. used neonatal CB / 1 mice developed by Zimmer et al. (1999), whereas Hájos et al. (2001) used adult animals developed by Ledent et al. (1999). It is possible that the non-cb 1 receptors implicated in the latter findings are developmentally expressed (Al-Hayani & Davies, 2000). If this can be confirmed by using preparations from CB 1 deficient mice of different age but from the same strain, a subtraction cloning strategy using cdna from the CA3 and CA1 regions of the hippocampus of neonatal vs. adult CB / 1 mice may be a viable strategy to identify this receptor Pharmacology of the non-cb 1 hippocampal cannabinoid receptor Further study of this putative non-cb 1 receptor involved in inhibition of glutamatergic neurotransmission in the hippocampus indicated that it has a distinct pharmacological profile (Hájos & Freund, 2002). Similar to the endothelial non-cb 1 receptor, the cannabinoidinduced decrease in EPSCs could be antagonized by micromolar concentrations of SR (Breivogel et al., 2001; Hájos & Freund, 2002), but not by AM251 (Hájos & Freund, 2002). Another similar feature is the sensitivity to PTX of both the endothelial (see above) and the hippocampal response (Misner & Sullivan, 1999), suggesting the involvement of a G i /G o -coupled receptor in both cases. However, the synthetic agonists WIN55,212-2 and CP55,940 were able to inhibit glutamatergic EPSCs (Hájos & Freund, 2002), whereas these agonists had no effect at the endothelial receptor (Wagner et al., 1999; Mukhopadhyay et al., 2002). On the other hand, the selective endothelial agonist abncbd had no effect on glutamatergic EPSCs in hippocampal slices (M. Begg, D.M. Lovinger, and G. Kunos, unpublished observations). Another difference is the sensitivity of the 2 receptors to the TRPV 1 receptor antagonist capsazepine. Unlike the endothelial response to abn-cbd, which is unaffected by capsazepine (Járai et al., 1999; Mukhopadhyay et al., 2002; Ho & Hiley, 2003), capsazepine inhibits the suppression of glutamatergic EPSCs by WIN55,212-2 in the hippocampus (Hájos & Freund, 2002). Similar receptors may be present in projection neurons of the basolateral amygdala of the rat, inhibition of which by WIN55,212-2 could be antagonized by SR and capsazepine, but not by AM251 (Pistis et al., 2004). The capsazepine sensitivity of a response could suggest the involvement of TRPV 1 receptors. Indeed, TRPV 1 receptor mrna has been detected in various regions of the CNS including the hypothalamus in some (Mezey et al., 2000) but not other studies (Caterina et al., 1997), and both anandamide and capsaicin were found to cause a capsazepine-sensitive facilitation of glutamatergic synapses in rat hippocampus (Al-Hayani et al., 2001) and substantia nigra (Marinelli et al., 2003). However, WIN55,212-2 does not interact with the cloned TRPV 1 receptor (Zygmunt et al., 1999), which is a nonselective cation channel not known to be affected by PTX. Overall, the pharmacological differences between the endothelial and hippocampal non-cb 1 receptors suggest that they are different molecular entities. Table 1 summarizes the unique pharmacology of endothelial and hippocampal non-cb 1 receptors, in comparison with CB 1 and TRPV 1 receptors.

9 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) Function of hippocampal non-cb 1 receptors Haller et al. (2002) reported that treatment of both wildtype and CB 1 deficient mice with the CB 1 antagonist SR paradoxically decreased anxiety in the elevated / plus-maze test, although the increased anxiety of CB 1 vs. CB +/+ 1 mice (Haller et al., 2002) and the antianxiety effect of pharmacological inhibition of anandamide degradation (Kathuria et al., 2003) suggest that endocannabinoids reduce anxiety. In a more recent study Haller et al. (2004) propose to resolve this paradox by demonstrating that the CB 1 -selective antagonist AM251, which does not appear to affect the non-cb 1 receptor in the hippocampus and amygdala (see above), increases anxiety-like behavior in wild-type but not in CB / 1 mice, and blocks the antianxiety effects of low doses of WIN55,212-2 in the former. Together, these findings suggest that CB 1 and hippocampal non-cb 1 receptors mediate opposite, anxiolytic and anxiogenic effects, respectively, and antagonism of the latter may dominate in the net response to the mixed antagonist SR This may be a therapeutically useful side effect of SR141716, also called rimonabant, a promising drug for the treatment of obesity and the related metabolic syndrome. 5. Role of transient receptor potential vanilloid 1 receptors in endocannabinoid action An important interaction between the endocannabinoid and vanilloid systems was uncovered by the report that in rat hepatic, rat mesenteric, and guinea-pig basilar arteries the endothelium-independent vasodilator effect of anandamide (but not 2-AG, palmitoylethanolamide, or synthetic cannabinoid receptor agonists) was inhibited by the TRPV 1 receptor antagonist capsazepine or by a CGRP receptor antagonist (Zygmunt et al., 1999). This study was prompted by structural similarities between anandamide and the capsaicin analog olvanil, as noted earlier by Di Marzo et al. (1998). The above findings of Zygmunt et al. implied that anandamide can evoke the release of vasodilatory peptide CGRP from sensory neurons through the activation of TRPV 1 receptors. In further support of this conclusion it was also demonstrated that anandamide binds to the cloned rat TRPV 1 receptor with micromolar affinity, and at nanomolar concentrations it releases immunoreactive CGRP from sensory nerve terminals located in the vascular adventitia (Zygmunt et al., 1999). This observation was also confirmed for the human TRPV 1 receptor in a later study (Smart et al., 2000). A similar involvement of TRPV 1 receptors in the mesenteric vasodilator action of R-methanandamide, a metabolically stable anandamide analog, has also been demonstrated (Ralevic et al., 2000). Some other studies documented both endotheliumdependent and endothelium-independent components in the anandamide-induced vasodilation, and in these studies the role of TRPV 1 receptors was only confirmed for the endothelium-independent component (Járai et al., 1999; Mukhopadhyay et al., 2002). The endothelium-dependent vasodilator effect of anandamide in the rabbit aorta or the similar effect of abn-cbd in rat mesenteric arteries is unaffected by capsazepine (Járai et al., 1999; Mukhopadhyay et al., 2002; Ho & Hiley, 2003; Offertáler et al., 2003). Interestingly, the stimulation of CB 1 receptors on sensory nerve terminals by very low concentrations of anandamide or by the synthetic cannabinoid HU-210, which do not activate TRPV 1 receptors, inhibits sensory neurotransmission (reviewed in Ralevic et al., 2002). Furthermore, a recent study by Zygmunt et al. (2002) implies that cannabinol and THC, but not other psychotropic cannabinoids, elicit CGRP release from periarterial sensory nerves by a mechanism independent of CB 1,CB 2 and also TRPV 1 receptors. TRPV 1 receptors are not involved in the dilation of isolated coronary arteries by anandamide either in the rat, where the effect is endothelium-independent (White et al., 2001) or in the sheep, where the effect is endotheliumdependent (Grainger & Boachie-Ansah, 2001). Further complicating the picture, the role of sensory nerves and vanilloid receptors in the vasodilator effect of anandamide in the rat mesenteric artery was found to be conditional on the presence of NO (Harris et al., 2002). There are TRPV 1 -containing afferent nerve fibers on the epicardial surface of the heart and epicardially injected capsaicin evokes a sympathoexcitatory response with a brief increase in blood pressure (Zahner et al., 2003) through the activation of these receptors. Capsaicin infusion also induces a moderate pressor effect in pigs (Kapoor et al., 2003). Intravenous injection of 10 Ag/kg capsaicin evokes only a brief pressor response in wild-type mice, while the response to 100 Ag/kg has both a depressor and a pressor component (Pacher et al., 2004). In contrast, capsaicin does not affect blood pressure in TRPV / 1 mice suggesting that TRPV 1 receptors mediate the cardiogenic sympathetic or Bezold-Jarisch reflex in mice, which is in agreement with recent reports in which the cardiovascular effects of capsaicin were inhibited by TRPV 1 receptor antagonists (Smith & McQueen, 2001; Zahner et al., 2003). In the absence of anandamide-induced hypotension in CB 1 knockout mice, the physiological relevance of the interaction of anandamide with TRPV 1 receptors has been questioned (Szolcsányi, 2000). In a recent study, the detailed hemodynamic effects of anandamide have been analyzed in TRPV / 1 and TRPV +/+ 1 mice (Pacher et al., 2004). Similar to an earlier study in rats (Varga et al., 1995), bolus injections of anandamide (20 mg/kg i.v.) caused a triphasic effect in the wild-type control mice. A transient (b5 sec) drop in heart rate and cardiac contractility, associated with an increase in peripheral resistance (phase I), was followed by a brief pressor response associated with increased cardiac contractility (phase II). The third, prolonged hypotensive phase (phase III) lasting up to 10 min was associated with decreased cardiac contractility and total peripheral resist-

10 142 M. Begg et al. / Pharmacology & Therapeutics 106 (2005) ance (Pacher et al., 2004). Pretreatment of mice with CB 1 antagonist SR (3 mg/kg i.v.) had no effect on the first and second phases of the response to anandamide, but completely prevented the subsequent hypotension and the associated decreases in the cardiac contractility and total peripheral resistance. In TRPV 1 / mice the anandamideinduced initial component (phase I) was absent and the phase II pressor response was also largely diminished as compared to their wild-type littermates. In contrast, the subsequent prolonged hypotensive response accompanied by decreased cardiac contractility and vascular resistance were similar to the responses observed in TRPV 1 +/+ mice and could be antagonized by pretreatment with SR (Pacher et al., 2004). These findings are in agreement with those reported by Malinowska et al. (2001) who found that in rats the transient vagal activation to a bolus injection of anandamide was partially blocked by the TRPV 1 antagonists capsazepine or ruthenium red, whereas the CB 1 - mediated prolonged hypotension remained unaffected. Collectively, the above studies demonstrate that the prolonged hypotensive effect of anandamide, which consists of decreases in both cardiac contractility and total peripheral resistance, are mediated by CB 1 but not TRPV 1 receptors, and that the role of these latter is limited to the transient activation of the Bezold-Jarisch reflex by very high plasma concentrations of anandamide. 6. Evidence for additional cannabinoid-sensitive receptors Presynaptic receptors with low sensitivity to inhibition by SR141716, thus distinct from CB 1 receptors, have been also postulated to be present on nerve terminals in the mouse vas deferens (Pertwee, 1999) and the guinea-pig ileum (Mang et al., 2001), where their activation by anandamide inhibits noradrenaline or acetylcholine release, respectively. Whether or not such receptors bear pharmacological similarity to the receptors postulated to exist in vascular endothelium or on hippocampal glutamatergic neurons remains to be determined. Sensory nerve terminals may also express non-cb 1 / CB 2 receptors that have been shown to be activated by the putative endocannabinoid noladin ether, resulting in inhibition of sensory neurotransmission (Duncan et al., 2004). Unlike the effects described above, this effect was not sensitive to SR141716, and it was also insensitive to capsazepine. Franklin et al. (2003) recently reported that palmitoylethanolamide (PEA), a fatty acid amide which does not bind to either CB 1 or CB 2 receptors (Felder et al., 1995), increases in focal cerebral ischemia and potentiates microglial motility. However, unlike microglial migration stimulated by 2-AG, which appears to involve both CB 2 and abn-cbd-sensitive receptors (Walter et al., 2003), the role of CB 1,CB 2, TRPV 1, or abn-cbd-sensitive receptors could be ruled out in the effect of PEA, although it was sensitive to PTX, which implicates a G i /G o -coupled receptor. Earlier, PEA was found to produce potent antinociception by a peripheral action that could be antagonized by the CB 2 antagonist SR (Calignano et al., 1998). In the absence of a direct interaction of PEA with CB 2 receptors, the authors attributed this interaction to a bcb 2 -likeq receptor. A CB 2 -like receptor has been also postulated to be present in mouse vas deferens, but not in the guinea-pig myenteric plexus (Griffin et al., 1997). 7. Conclusions Endogenous cannabinoids have been implicated in the control of a large number of physiological processes, and most of their effects appear to be mediated by CB 1 or CB 2 receptors. In view of the documented promiscuity of receptor interactions by lipid messengers (Lim & Dey, 2002), it may not be surprising that some cannabinoid effects resist classification as either CB 1 -orcb 2 -mediated. While some of these effects may not be mediated by specific receptors, there is sufficient evidence to suggest the involvement of additional receptors, which include TRPV 1 receptors and at least 2 GPCRs so far defined only pharmacologically. Further studies are needed to reveal the molecular identity of these latter sites, which could then be used to map out their tissue distribution and to fully explore their physiological functions. 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Cannabinoid antagonist SR inhibits endotoxic hypotension by a cardiac mechanism not involving CB1 or CB2 receptors. Am J Physiol Heart Circ Physiol 287, H595 H600. Begg, M., Mo, F. M., Offertáler, L., Bátkai, S., Pacher, P., Razdan, R. K., et al. (2003). G protein-coupled endothelial receptor for atypical cannabinoid ligands modulates a Ca 2+ -dependent K + current. J Biol Chem 278, Bisogno, T., Melsck, D., Bobrov, M. Y., Gretskaya, N. M., Bezuglov, V. V., De Petrocellis, L., et al. (2000). N-acyl-dopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo. Biochem J 351, Bonz, A., Laser, M., Kullmer, S., Kniesch, S., Babin-Ebell, J., Popp, V., et al. (2003). Cannabinoids acting on CB1 receptors decrease