CB1 and CB2 receptor-mediated signalling: a focus ... - Science Direct

Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 66(2&3),161^171 & 2002 Elsevier Science Ltd. All rights reserved. doi:10.1054/plef.2001.0344, available online at http://www.idealibrary.com on

CB1 and CB2 receptor-mediated signalling: a focus on endocannabinoids Sean D. McAllister,1 Michelle Glass2,* 1

Forbes Norris ALS Research Center, California Pacific Medical Centre, 2351 Clay Street, Suite 416, San Francisco, CA 94115, USA Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand

2

Summary The discovery that the major psychoactive component of marijuana activated two G-protein coupled receptors prompted the search for the endogenous cannabinoid ligands now termed endocannabinoids.To date three putative ligands have been isolated, all consisting of arachidonic acid linked to a polar head group. Both synthetic and endogenous cannabinoids have been the focus of extensive study over the past few years.The signalling events produced by endocannabinoids as compared with D9 -THC and synthetic cannabinoids contain many similarities. However, as research focuses more on endogenous ligands the divergence between these classes of compounds grows.This review focuses upon the developments in endocannabinoid signal transduction from receptor-mediated activation of common G-protein linked effector pathways through downstream regulation of gene transcription. & 2002 Elsevier Science Ltd. All rights reserved.

INTRODUCTION While cannabinoid pharmacology derived its beginnings from the study of the marijuana plant component D9tetrahydrocannabinol (D9-THC) the isolation of three putative endogenous cannabinoids has stimulated an emerging focus on endocannabinoid pharmacology. The arachidonic acid derivativesFanandamide,1 2-arachidonyl-glycerol (2AG)2,3 and 2-arachidonyl-glyceryl ether4 are exciting not just for their addition to cannabinoid research, but because they also represent a novel class of neurotransmitters derived from fatty acids that may yet prove to be important in other fields. Endocannabinoids are known to bind to and activate at least two cloned receptorsFCB1 and CB2.5,6 Attention is also currently focused on the search for additional members of this family.7 Much of the research on the signalling aspects of these G-protein coupled receptors has taken advantage of synthetic cannabinoids and D9THC.8 This review will specifically focus on endocannaReceived 30 October 2001 Accepted 10 November 2001 *Correspondence to: M. Glass, Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand.Tel.: +64-9-373-7599x6247; Fax: +64-9373-7556

& 2002 Elsevier Science Ltd. All rights reserved.

binoid signal transduction, except to highlight those situations in which differences have arisen between endocannabinoid and synthetic cannabinoid signalling. Furthermore, as no signal transduction data are available for the recently described 2-arachidonyl-glyceryl ether, the discussion is limited to anandamide and 2AG. Endocannabinoid activation of CB1 and CB2 cannabinoid receptors results in the modulation of multiple intracellular signal transduction pathways via the activation of G-proteins. This review will therefore first examine the endocannabinoid activation of G-proteins and their immediate effectors (adenylate cyclase and ion channels) before examining more downstream or G-protein independent pathways.

ENDOCANNABINOID-MEDIATED ACTIVATION OF G-PROTEINS Both anandamide and 2AG bind to and activate each of the cannabinoid receptors (CB1 and CB2). While both these receptors are clearly coupled to pertussis toxin sensitive G-proteins9 studies have demonstrated that CB1 and CB2 receptors do not couple identically to signal transduction pathways.10 For example, the CB2 receptor is unable to, or interacts poorly with ion channels, whereas

Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 66(2&3), 161^171

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McAllister and Glass

the CB1 receptor couples to a variety of ion channels.9,11,12 One explanation of these findings may be that the receptors couple differentially to G-protein subtypes. Recent studies have demonstrated that this is indeed the caseFwhile both receptors display a high affinity for Gi, the affinity of the CB1 receptor for Go is ten fold higher than that of the CB2 receptor.13 Furthermore, as described in the following two sections, an additional level of selectivity of action comes from the determination that the endocannabinoids differ in their ability to activate G-proteins via the CB1 and CB2 receptors.

G-protein coupling of CB1 receptors Many studies have examined the ability of cannabinoid agonists to activate G-proteins by analysing the activation of [35S]GTPgS in brain homogenates.14,15 While these studies have predominantly focused on synthetic cannabinoids, some information on endocannabinoids has been gained. These studies have demonstrated anandamide to be either ineffective,16 or a partial agonist in the stimulation of GTPgS binding compared with synthetic cannabinoids.17–19 While this may seem in conflict with the previous observation that anandamide is a full agonist in the inhibition of cAMP accumulation in brain membranes, it likely reflects the mixed G-protein population present in the brain. The dominant neuronal Gprotein is Go, furthermore this G-protein shows a more rapid rate of GDP–GTP exchange than Gi, thus the bulk of the signal observed in these assays is likely to reflect activation of Go by CB1. Consistent with this observation, anandamide has been demonstrated to be a partial agonist in the activation of Go in reconstitution assays.13 In this same study, anandamide was a full agonist in the activation of Gi , consistent with its observed efficacy in the inhibition of adenylate cyclase. Few studies have examined the ability of 2AG to stimulate G-proteins via CB1. A recent study demonstrated maximal activation of Gai-1 by 2AG in an in situ reconstitution model.20 This finding is consistent with previous data suggesting that 2AG is a full agonist in the inhibition of cAMP accumulation.21,22 In contrast to anandamide 2AG produced a maximal receptor-mediated activation of Gao in this study. This finding suggests that the two endocannabinoids exhibit differential sensitivity for the activation of Gi and Go. Thus, it appears that one ligand can induce a receptor conformation that is maximally active in stimulating one G-protein, while only partially active in its ability to activate a different Gprotein. Thus, 2AG produced a receptor conformation that conferred maximal stimulation of both Gi and Go. In contrast anandamide was able to produce full activation of Gi, but only partial activation of Go. The

finding of differential regulation of G-proteins by endocannabinoids provides a mechanism by which endocannabinoids may produce different effects within the same cell population.

G-protein coupling of CB2 receptors Reports in the literature demonstrated 2AG could produce maximal inhibition of cAMP accumulation23 and maximal release of intracellular calcium24 in cells expressing CB2 receptors while anandamide is only partially efficacious in these systems. These findings may be explained by recent data. Glass and Northup20 found anandamide was a partial agonist in the activation of Gi by the CB2 receptor, whereas 2AG proved fully efficacious. The critical influence of the ratios of receptors to G-proteins in studies on transfected cell lines was clearly indicated in the study by Gonsiorek et al.23 This study found that both endocannabinoids were full agonists in activating GTPgS in a cell line expressing 10–14 pmol/mg of CB2 receptor, while anandamide was a partial agonist compared with 2AG in cells expressing half this level of receptor. Endogenous agonists have a multitude of ways to produce different signals in different cells; these include receptor subtypes, splice variants of receptors, and differential co-localization of the receptors with signalling molecules. The work on selective G-protein activation provides evidence of an additional level of specificityFthat multiple endogenous agonists might produce divergent effects through their ability to differentially activate G-proteins.

ENDOCANNABINOID MODULATION OF ADENYLATE CYCLASE As described above both CB1 and CB2 cannabinoid receptors can couple to pertussis toxin sensitive Gproteins (Gi/o) to inhibit adenylate cyclase.9 Consistent with its binding affinities, anandamide is approximately 3 times more potent in its inhibition of cAMP accumulation via the CB1 receptor compared with the CB2 receptor.9 2AG but not anandamide was recently demonstrated to be a full agonist for the inhibition of cAMP via the CB2 receptor.23 At the CB1 receptor both anandamide and 2AG appear to be full (or nearly full) agonists in the inhibition of cAMP accumulation.9,22 In addition to inhibiting cAMP accumulation, CB1 but not CB2 receptors can stimulate cAMP formation under certain conditions, consistent with a putative Gs linkage of this receptor.25–27 Interestingly, anandamide appears to be a weak partial agonist on this stimulatory pathway.27



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ENDOCANNABINOID MODULATION OF ION CHANNELS

Calcium channels Before the discovery of endocannabinoids, it was apparent that activating the CB1 receptor led to the modulation of ion channels.28 The first experiments demonstrating that endocannabinoids could alter ion channel activity utilized the NG108-15 neuroblastoma-glioma cell line that naturally expresses CB1 receptors to demonstrate that anandamide could inhibit N-type voltage-dependent calcium channels.29 This inhibition was blocked by pertussis toxin but was independent of the cAMP pathway29 suggesting that it was directly mediated via Gao.30 This group subsequently demonstrated that anandamide could also inhibit Q-type Ca2+ currents in a pituitary cell line transfected with the CB1 receptor.31 Differences in the efficacy of anandamide were observed between these studies. Anandamide was a partial agonist in the inhibition of calcium channels in N18 neuroblastoma cells,29 in contrast it produced a fully efficacious response in AtT20 cells that express a higher number of receptors.31 These findings, therefore, likely reflect differences in the receptor to G-protein ratios between these two cell models. There is an extensive body of research studying inhibition of pre-synaptic voltage-dependent calcium channels by synthetic cannabinoids and these findings can be correlated with cannabinoid pharmacology in the central and peripheral nervous system.10 It has been demonstrated that the activation of CB1 receptors and subsequent inhibition of calcium channels, on presynaptic terminals containing acetylcholine, GABA, glutamate, or noradrenaline, lead to a decrease in the release of these transmitters.32–36 The physiological outcome of this inhibition clearly depends upon which cell populations are being activated. For example in the hippocampus, the pre-synaptic inhibition of calcium channels on axonal terminals synapsing on pyramidal neurons has been correlated with the ability of endocannabinoids to inhibit long-term potentiation.22,37,38 This mechanism may be one way in which cannabinoids alter learning and memory. The distribution of endocannabinoid synthesis, metabolism and transport machinery suggested that endocannabinoids might act as retrograde signalling molecules that are released from post-synaptic cells to inhibit presynaptic transmitter release.39–41 Recently, this mechanism was revealed in the CNS.42 In both the hippocampus and cerebellum it was shown that activation of postsynaptic neurons resulted in the release of endocannabinoid(s) from these neurons.43–45 The endogenous ligand(s) then acted as retrograde signalling molecules to inhibit pre-synaptic calcium influx in axonal terminals & 2002 Elsevier Science Ltd. All rights reserved.

and subsequently reduced the release of neurotransmitter. In these studies the presence of endocannabinoids was inferred using specific synthetic CB1 receptor agonists and antagonists as well as an endocannabinoid transport inhibitor. In tissue and often in cell lines, direct application of high concentrations of endocannabinoids leads to only subtle effects. This may be due to many factors, including the innate instability of the compounds at physiological temperatures and in the presence of light, the lipophilicity of the molecules, and their susceptibility to degradation by tissue esterases and uptake by transporters.39,46,47 Hence, future studies that require direct exogenous administration of endocannabinoids will present a challenge. In cerebral arterial smooth muscle cells anandamide activation of CB1 receptors has been shown to result in the inhibition of L-type Ca2+ channel currents.48 This inhibition appeared to be related to the ability of anandamide to relax pre-constricted cerebral vessels in a similar preparation. In retinal slices from larval tiger salamander, activation of the CB1 receptor by synthetic cannabinoids led to the inhibition of voltage-dependent L-type calcium channels of bipolar cells.49 In this same study an analysis of the cannabinoid lipids in the rat retina indicated the presence of 2-AG, palmitoyl-ethanolamide, and oleylethanolamide but not anandamide. Although, direct application of endocannabinoids was not carried out it was inferred that the endogenous ligands could alter channel activity on bipolar cell terminals. These two specific findings, taken together with early studies on inhibition of calcium currents,29,31 demonstrate that CB1 receptors can couple to many subtypes of voltage-dependent calcium channels.

NMDA receptor associated ion channels Anandamide has been shown to both inhibit and enhance the effects of NMDA-receptor stimulated responses in the CNS.50,51 In rat brain slices, anandamide and D9-THC inhibited calcium influx is brought about by the addition of NMDA.50 This effect was receptor mediated as assessed by blockade with SR141716A and pertussis toxin, and appeared to be related to the interaction of CB1 with P/Q-type calcium channels. Interestingly, when the CB1 receptor component was blocked, anandamide but not D9-THC produced a stimulatory effect on NMDA-induced calcium responses. Thus, in addition to its inhibitory action via the CB1 receptor anandamide may directly interact with the NMDA channel to enhance calcium influx. Since arachidonic acid, the product of anandamide hydrolysis, can enhance NMDA receptor activity52 the investigators repeated experiments with a hydrolysis-resistant analogue, methanandamide. Similar results were observed with


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methanandamide and further studies in Xenopus oocytes expressing NMDA receptors were also used to confirm the direct potentiating effects of anandamide on these receptors.50 Methanandamide has been demonstrated to enhance NMDA-evoked calcium release in primary cerebellar cultures and cerebellar slices.51 In contrast to a previous study50 this effect was demonstrated to be due to activation of the CB1 receptor. The data suggested that the enhancement of NMDA-evoked calcium release was due to CB1 receptor activation of the phospholipase C (PLC) pathway leading to the downstream release of calcium from intracellular stores. Importantly, it was noted that blockade of the phospholipase C pathway unmasked a CB1-mediated inhibition of the NMDAevoked calcium release consistent with earlier findings.50 Netzeband et al.,51 hypothesized that under conditions of strong cellular stimulation, an inhibitory effect on voltage-gated calcium channels by endocannabinoids might be more apparent whereas under periods of lower stimulation the PLC-mediated pathway may dominate.

Potassium channels Anandamide has been demonstrated to enhance the activity of G-protein coupled inwardly rectifying potassium channels (GIRK/Kir) in AtT20 cells transfected with the CB1 receptor31 and in oocytes expressing the CB1 receptor and GIRK 1 and 4.12 Recent evidence for CB1 modulation of GIRK channels in native tissue comes from a study of glutamatergic signalling in the mouse nucleus accumbens.53 In this study, the inhibition of pre-synaptic transmitter release was due to CB1 receptor-mediated inhibition of pre-synaptic potassium currents. In cerebellar granule and COS cells the acid-sensitive background K+-channel, TASK-1, is directly inhibited by anandamide independent of the CB1 receptor.54 TASK-1 is a member of a family of leak or background K+-selective channels that set resting membrane potential and input resistance55 and may be a mechanism behind the neuronal effects of inhalation anaesthetics.56 Taking into account the localization of TASK-1 in areas of motor control, such as motor neurons and cerebellar granular cells, Maingret et al.54 suggested that anandamide might influence motor behaviours through its interaction with TASK-1 but further studies are needed to support this hypothesis. A recent study demonstrated that methanandamide, acting through CB1 receptors, could decrease postsynaptic K+ M-current in hippocampal CA1 neurons.57 The author suggested that CB1-mediated stimulation of intracellular calcium stores, leading to increase in intracellular calcium, could be one of the mechanisms behind the IM inhibition. In the CNS, a majority of

literature points to a pre-synaptic locus for CB1 receptors and activation of these receptors leads to a decrease in transmitter release.41 However, indirectly stimulating a post-synaptic neuron by decreasing the pre-synaptic release of inhibitory transmitters and directly stimulating the same cell may be complementary processes produced by CB1 receptors.57 It should also be noted that the data presented by Netzeband et al.51 (see above) suggested that cell excitation produced by endocannabinoids was a process mediated by CB1 receptors on post-synaptic cells. It is clear that future studies are needed to determine how these particular pre- and post-synaptic CB1 receptor mechanisms are related. In the central nervous system pre-synaptic inhibition of ion channels by cannabinoids has been primarily attributed to the inhibition of N- or P/Q-type voltagedependent calcium channels or activation of cAMPdependent A-type potassium currents.8 Comparison of past studies with more recent literature suggests we should be cautious in generalizing a pre- and postsynaptic endocannabinoid receptor/channel signalling mechanism in the brain. It appears that at the pre- and post-synaptic level, depending on the cell population studied, endocannabinoids may influence a variety of ion channels.

Calcium mobilization and the dual effects of endocannabinoids Early studies in CB1 and CB2 transfected cell lines demonstrated that while anandamide could mobilize intracellular calcium this was not the result of receptor activation of the PLC pathway.9,21 These investigations were in agreement with studies in other tissues and transfected cells lines that failed to see CB1-mediated mobilization of intracellular calcium.58,59 However, other investigations have demonstrated CB1 receptor-mediated activation of intracellular calcium stores, suggesting that the cellular background may be imperative in the investigation of receptor-dependent calcium interactions. Many of the studies indicating intracellular calcium control by cannabinoid receptors have emerged from the research of Sugiura and colleagues who have utilized NG108-15 cells that contain an endogenous CB1 receptor. These studies have suggested a cannabinoid-mediated increase in transient Ca2+ release that is both CB1 and Gi/Go mediated.60–63 Furthermore, their studies have suggested different efficacy profiles of cannabinoid ligands compared with other assays, with 2AG as the most efficacious and anandamide and all synthetic agonists tested acting as partial agonists.63 Interestingly, when increases in [Ca2+]i were induced by depolarizing neuroblastoma cells with high K+, a treatment paradigm that can activate voltage-gated ions channels, 2AG




actually acted as a inhibitor in the micromolar range.60 Under this protocol the high K+ caused a sustained rise in the [Ca2+]i. When endocannabinoids were added this effect was inhibited. It could be suggested that this observation was due to CB1 receptor-mediated inhibition of voltage-gated channels but the involvement of CB1 receptors could not be confirmed because SR141716A alone also inhibited the sustained calcium increases. As discussed in the previous sections, other studies also suggested a cannabinoid receptor-mediated pathway that activates intracellular Ca2+ pools.51,57 CB1 receptors may activate PLC through Gi bg subunits as these subunits have previously been demonstrated to stimulate PLCb in NG108-15 cells.64 Taken together these studies suggest dual effects of endocannabinoids on calcium mobilization in neuronal cells with the outcome dependent on the pathway evaluated. CB1 receptor activation of the PLC pathway results in stimulation of [Ca2+]i, whereas a cell paradigm focusing on ion channel activity favours CB1 receptor-dependent inhibition of [Ca2+]i.51,60 Similar discrepancies exist for CB2 receptors. Results from CHO cells transfected with the CB2 receptor indicated that these receptors do not activate phospholipases A2, C or D, or mobilize intracellular calcium.9,65 However, a recent paper by Sugiura et al.24 demonstrated rapid transient increases in intracellular free Ca2+ concentrations in response to 2AG in HL-60 cells that naturally express the CB2 receptor. This effect was blocked by the CB2 receptor antagonist SR144528, and was pertussis toxin sensitive. As for the CB1 receptor, this group found that 2AG was the most potent agonist tested for this response, while anandamide was only a weak partial agonist. These findings suggest that as for CB1, the cellular background may be critical in evaluating the ability of CB2 to couple to intracellular calcium pools.

ENDOCANNABINOID REGULATION OF SIGNAL TRANSDUCTION PATHWAYS RELATED TO CELL FATE A new and exciting area of cannabinoid research has been investigations into the role of cannabinoids in cell fate i.e. proliferation and cell death. The potential role of cannabinoids in this area, while still developing, has recently been extensively reviewed,66 and appears to involve many novel signalling pathways. Endocannabinoids have been demonstrated to produce numerous down stream events following the activation of mitogen-activated protein kinases (MAPK). The MAPK family includes the extracellular signal regulated kinases (ERK), which are activated in response to growth factors and involved in cell growth and differentiation, and the two stress-related kinases p38-MAPK and c-jun-N-term& 2002 Elsevier Science Ltd. All rights reserved.

inal kinase ( JNK). The most studied of these in terms of cannabinoid interactions are the ERKs. Synthetic cannabinoids can increase the activity of ERK by means of a G-protein via both CB1 and CB2 receptors.67,68 Anandamide has also been shown to stimulate ERK activation in several cell types.69,70 The pathways for activation of ERK may be different for each receptor as CB2, but not CB1, receptor stimulation of ERK can be attenuated by a PKC inhibitor, suggesting that it is PKC dependent.68 ERK activation by CB1 has recently been demonstrated to lead to the activation of the Na+/ H+-exchanger NHE-1, a electroneutral transmembrane transporter involved in multiple cellular functions such as intracellular pH regulation and control of cell volume.71 Furthermore, it is possible that ERK activation is an intermediate step in the cannabinoid receptor-mediated induction of multiple transcription factors. These include activation of krox 24, increased AP-1 DNA-binding activity and increased Fos-related-antigen (FRA) activity.72–74 In addition to krox 24, in vivo activation of the transcription factors c-fos, and c-jun have been reported in rat cortical and striatal regions following intra-peritoneal administration of D9-THC.75 Intriguingly, the CB1 inverse agonist SR141716A has been demonstrated to block insulin receptor mediated stimulation of ERK.76 As both insulin- and cannabinoidmediated stimulations of ERK were blocked by the phosphotidylinositol 3-kinase (PI3K) inhibitor wortmannin, this was interpreted to suggest that insulin stimulated ERK through the G-protein sensitive PI3K1b. Thus, the authors suggested that antagonism of this pathway by SR141716A was due to inverse agonist induced internalization of the receptors and associated G-proteins. The insulin and cannabinoid pathways appeared to converge at the stimulation of ERK. Furthermore, another study has demonstrated that while activation of the Gprotein dependent PI3K isoenzyme mediates cannabinoid activation of Protein Kinase B (PKB), insulin-mediated activation of PKB is mediated through a non-G-protein sensitive PI3K77 suggesting subsequent divergence of these pathways. Recent studies have investigated the action of endocannabinoids on stress-related kinases. In transfected CHO cells,78 and vascular endothelial cells70 one or both of the endocannabinoids activated p38-MAPK. In contrast, in hippocampal slices p38-MAPK, but not JNK was activated by anandamide and 2AG, suggesting that these effects maybe cell type specific.79 Further evidence of cell type specificity came from investigations into the ability of endocannabinoids to produce apoptosis.78 JNK is important in stress response pathways leading to apoptosis.80 CB1 receptor-mediated activation of JNK is Gprotein, PI3K and p21-RAS dependent.78 Importantly, when a range of cell types were tested induction of


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apoptosis following long-term cannabinoid exposure was only observed in the cell types that displayed robust JNK induction.78 Another intriguing aspect of this study was the implication of platelet-derived growth factor (PDGF) receptor transactivation as a critical component of the signal transduction pathway, as antagonism of this receptor blocked the CB1 receptor stimulation of JNK. Activation of stress-related kinases such as p38-MAPK may account in part for the ability of endocannabinoids to interfere in the formation of LTP22 by blocking synaptic plasticity.81 p38-MAPK has also been implicated in neuroprotection82 and anti-proliferative effects in tumour cells,83 both of which are areas that cannabinoids appear to exert effects.

Sphingomyelin hydrolysis and ceramide D-9 THC can induce apoptosis in C6 glioma cells42a and in vivo.83 Since ceramide, a product of sphingomyelin hydrolysis in the plasma membrane has been demonstrated to be an important second messenger involved in apoptotis,84 this signalling pathway was investigated. In these studies D9-THC stimulated sphingomyelin hydrolysis resulting in increased ceramide levels, furthermore ceramide analogues could mimic the ability of D9-THC to promote cell death.85 Initially, the effects of THC were thought to be receptor independent but a more recent study demonstrated that apoptosis could be prevented by simultaneous blockade of both CB1 and CB2. Furthermore, apoptosis was also produced by a range of synthetic cannabinoids consistent with a receptor-mediated mechanism.83 While D9-THC and ceramide analogues could induce cell death in some other tumour cell lines, this phenomenon was not observed in primary astrocytes and neuronal cultures.85 Interestingly, while not leading to apoptosis, cannabinoids did cause sphingomyelin hydrolysis and this correlated with increases in ceramide levels and the upregulation of the protein FAN (factor associated with neutral sphingomyelinase activation) in primary astrocytes.86 A possible explanation for these differences between cell lines has emerged from studies comparing cell death in two differentiated C6 glioma clones, C6.9 that is sensitive to D9-THC mediated apoptosis and C6.4 that is D9-THC insensitive.83 Both of these cell lines contain both CB1 and CB2 receptors, and both demonstrate acutely increased ceramide and ERK induction following THC exposure, however, major differences in their signalling pathways are observed in the chronic effects of D9-THC. After 5 days (the time point where cell viability was compromised in the C6.9 cells) considerable accumulation of ceramide and activation of ERK had occurred in the C6.9 but not C6.4 cells. Interestingly, the ceramide analogue C2-ceramide also produced sustained activation of ERK in C6.9 but not

C6.2 cells. These studies suggest that cannabinoidmediated cell death required sustained ceramide accumulation and ERK activation. Furthermore, this pathway appeared to involve Raf1, but not Ras.83 Clearly, there is much to be learned about this signalling pathway and how it relates to cannabinoid pharmacology.

Prolactin and trk NGF Both anandamide and 2AG have been shown to inhibit hormone-induced breast cancer cell proliferation by down-regulating the receptor for prolactin.87 These same compounds have also been shown to reduce nerve growth factor (NGF)-induced breast cancer cell proliferation by down regulating the levels of trk NGF receptor.88 These down stream signalling events are related to CB1 receptor-mediated inhibition of cAMP/PKA pathways, Raf1 translocation and the subsequent stimulation of ERKs.89

Superoxide and caspase As was observed with breast cancer cells, and C6 gliomas, anandamide has also been shown to inhibit the proliferation of rat pheochromocytoma (PC-12) cells90 via an apoptotic pathway. The investigators demonstrated that anandamide treatment of PC-12 cells caused generation of superoxide anion formation, an important signalling event in apoptosis. Indeed the anti-oxidant N-acetyl cysteine, was able to rescue the PC-12 cells from anandamide-induced apoptosis.90 This study also reported that levels of caspase-3, a cysteine protease implicated in apoptosis signalling, was increased, while cell lines lacking caspase-3 (MCF-7) did not undergo anandamide-induced apoptosis. Many of the pathways involved in anandamide control of cell fate pathways may require reinterpretation in light of the recent finding that anandamide is an agonist at vanilloid receptors.91 This is particularly relevant to cell fate studies as anandamide has recently been demonstrated to produce apoptosis via the vanilloid receptor in a range of human neuroblastoma and lymphoma human cell lines.92 Intriguingly, in this study cannabinoid receptors exhibited a protective role against apoptosis induced by anandamide via vanilloid receptors, perhaps accounting for some of the discrepancies observed in the studies described in this review. THE ROLE OF ENDOCANNABINOIDS IN IMMUNE CELL SIGNAL TRANSDUCTION The role of cannabinoids in immune function will be covered in detail by Parolaro et al., in this issue. However, study in this area has resulted in the discovery of




numerous interesting and novel signal transduction pathways for the endocannabinoids that will be discussed briefly here. Cannabinoids have been known for some time to be able to inhibit macrophage activity.93,94 Macrophages play an essential role in local host defence against invading micro-organisms. In response to foreign invaders and/or chemical signals generated by the affected tissues, macrophages recognize, phagocytose, and destroy the foreign agent. During the activation process, such as by treatment with the bacterial endotoxin, lipopolysaccharide (LPS), macrophages are a major source releasing various inflammatory mediators which contribute to the local inflammatory response, including nitric oxide (NO), tumour necrosis factor-a, eicosanoid prostaglandin E2 (PGE2) and interleukin-6 (IL6). Anandamide and 2AG are synthesized by macrophages,95–98 which have been demonstrated to express CB2 receptors and low levels of CB1 receptor.99,100 The role of endocannabinoids on macrophage signal transduction is still largely being determined. THC produces a marked inhibition of iNOS transcription and nitric oxide production in response to LPC.101 More recently, Chang et al.102 confirmed this finding for THC and demonstrated that anandamide also caused inhibition of iNOS-related NO production, while 2AG produced a slight enhancement. While this effect is thought to be at least in part cannabinoid receptor-mediated, there may also be nonreceptor mediated components.102,103 In addition to modulating NO levels, anandamide also mediates the inhibition of LPS-induced PGE2 and IL-6 release in J774 macrophages through a potential CB2 pathway.102 In contrast, 2AG appeared to serve more as an arachidonic acid precursor, whose metabolism by COX and generation of PGE2 participates in the positive modulation of iNOS and COX-2 induction by this compound.102 In contrast to the inhibitory actions of cannabinoids on macrophages, a detailed set of studies conclude that activation of the CB2 receptor enhances immune function. A microarray study on the CB2 expressing promyelocytic HL-60 cell has demonstrated the up-regulation of multiple chemokines (MCP-1, IL-8 and MIP-1b) and the cytokine TNF-a by the synthetic agonist CP55,940104 however, neither anandamide nor 2AG were able to replicate these responses, so in the absence of additional endocannabinoid ligands, the physiological relevance of these findings is unclear. SIGNALLING THROUGH CB(X) AND VANILLOID RECEPTORS Since the discovery of the CB1 and CB2 receptors, the hunt for CB(x) has been a continual endeavour. Although not often documented, academic and private labs have made efforts to discover new cannabinoid proteins. The & 2002 Elsevier Science Ltd. All rights reserved.

widespread use of homology screening for this venture suggests that if there is a CB(x) then it shares little structural identity to CB1 or CB2 because this technique has yet to yield results. Nevertheless, recent papers have presented the possibility of novel receptors that recognize endocannabinoids. As reviewed by Hillard105 and Hogestatt and Zygmunt in this issue endocannabinoids have significant effects in the vascular system. In the anesthetized rat administration of anandamide intravenously causes an initial short tachycardia.106 This event is then followed by a prolonged bradychardia. Detailed studies on the hypotensive effects of anandamide administered intravenously have shown these CB1-dependent effects to be related to inhibition of norepinepherine release from sympathetic terminals107 and this mechanism can be related to calcium channel inhibition as discussed previously in this article. In isolated vascular smooth muscle cells the interaction between CB1 receptors and calcium channels has also been implicated in the direct production of vasorelaxation.48 However, in isolated mesenteric arterial beds the effects of anandamide and synthetic cannabinoids suggested the possibility of cannabinoid receptor subtypes. In particular, anandamide can cause vasodilation that is dependent on the presence of endothelial cells in mesenteric arterial beds.108 This effect could not be reproduced by WIN55,212-2 and HU-210 but it could be blocked by nearly micromolar concentrations of SR141716A.108 This finding could also be reproduced in cannabinoid receptor knockout animals further suggesting the presence of an endocannabinoid receptor subtype.109 Using CB1 knockout animals investigators have also provided evidence for CB(x) in the brain.7 Of 24 cannabinoid compounds tested for agonist-stimulated GTPgS binding only anandamide and WIN55,212-2 produced significant stimulation in brain slices from these animals. Important caveats in the search for cannabinoid receptor subtypes came with studies of vanilloid receptors and the emergence of non-specific effects of cannabinoid antagonists. Anandamide acts as a full agonist at vanilloid receptors.91 Activation of these receptors results in vasodilator effects and production of apoptosis in neuronal and immune cells.91,92,110 The contribution of this receptor system must now be ruled out when searching for receptor subtypes using anandamide. Furthermore, it has become evident that SR141716A can produce non-CB1 related effects at concentrations in the micromolar range and in some cases these appear to be antagonism of the original response.7,60,111 These findings suggest investigators should be cautious when claiming CB1-dependent effects only on the premise of blockade by high concentrations of SR141716A. The situation has also been complicated


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by recent in vivo data that have shown that the CB2 antagonist SR144528 can block the effects of systemically administered palmitoyl ethanolamide112,113 despite this ligand clearly not binding to or activating the CB2 cannabinoid receptor.24,114 Regardless of these caveats, with the availability of cannabinoid receptor knockout animals and an ever-increasing array of molecular techniques we may yet see the discovery of a novel endocannabinoid receptor.

CONCLUSION As indicated by this review, endocannabinoids modulate many signal transduction pathways. An additional layer of complexity in this field arises from non-cannabinoid receptor mediated signal transductionFeither through direct endocannabinoid interaction with other signalling moieties (e.g. vanilloid receptors), or indirectly through metabolism of the primary compounds.98,115,116 At the level of CB1 and CB2 receptor/G-protein complex, anandamide and 2AG can produce selective responses through the differential activation of G-protein subtypes. The endocannabinoid receptor CB1 may then in turn drive the activation or inhibition of adenylate cyclase and modulation of ion channels. Further downstream investigations into the role of these compounds in areas such as cell fate, immune cell regulation, and cardiac response have revealed new and exciting signalling events in a complex array of intracellular pathways. Future studies of endocannabinoid signalling will expand our basic knowledge base and reveal a more detailed picture of the interrelationship between specific signalling events and the physiological outcomes produced by the endogenous ligands. It is certain that a better understanding of these pathways will expand the potential for the modulation of endocannabinoid signalling as a therapeutic target.

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