Abstract
Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB1) receptors, as well as peripheral CB1 and CB2 receptors. CB2-selective agonists have the benefit of lacking CB1 receptor-mediated CNS side effects. Anandamide and 2-arachidonoylglycerol (2-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosyn.
The putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed.
Keywords
Cannabinoids; Endocannabinoids: anandamide; Neuropeptides: orexin; Pain; Receptors: metabotropic glutamate;
1. Cannabinoids
Marijuana has been used for relieving pain for more than four centuries, but its analgesic mechanism was only understood during the past two decades, after cannabinoid receptors were discovered. The main active component of marijuana is Δ9-tetrahydrocannabinol (Δ9-THC),1 which exerts several pharmacological actions, including analgesia, immobilization, heightened sensory awareness, euphoria, hypothermia, impairment of short-term memory, and suppression of immune responses.2 Compounds mimicking the effects of Δ9-THC through activating cannabinoid receptors are termed cannabinoids with three categories. Phytocannabinoids are the constituents isolated from marijuana.3 Endocannabinoids are endogenous ligands of cannabinoid receptors.4 Synthetic cannabinoids are compounds developed for potential medical uses, based on the concept that they would mimic the therapeutic effects of phytocannabinoids while having no psychoactivity.
2. CB1 and CB2 receptors
The first cannabinoid receptor, CB1, was cloned by Matsuda et al5 from a rat brain cDNA library. Thereafter, Munro's group6 cloned another cannabinoid receptor, CB2, from an HL-60 cell cDNA library. Both CB1 and CB2 receptors are seven-transmembrane G-protein-coupled receptors, mainly coupled to Gi/o proteins,7 and share 44% overall identity (68% identity for the transmembrane domains).
CB1 receptors are enriched in the nervous systems and moderately expressed in peripheral tissues.5, 8, 9 In both rat and human brains, CB1 receptors are densely distributed in the frontal cerebral cortex, basal ganglia, cerebellum, hippocampus, hypothalamus, and anterior cingulate cortex, but rarely in the brainstem nuclei.10 The latter finding may account for the low toxicity of cannabinoids when given in accidental overdose.
At the cellular level, CB1 receptors are mainly localized to axons and nerve terminals and are largely absent from neuronal somas or dendrites.11 This ultra-structural finding, suggesting a predominantly presynaptic localization of CB1 receptors, is consistent with the functional finding that activation of CB1 receptors inhibits calcium channels and activates potassium channels, leading to inhibition of neurotransmitter release.12, 13
The first cannabinoid receptor, CB1, was cloned by Matsuda et al5 from a rat brain cDNA library. Thereafter, Munro's group6 cloned another cannabinoid receptor, CB2, from an HL-60 cell cDNA library. Both CB1 and CB2 receptors are seven-transmembrane G-protein-coupled receptors, mainly coupled to Gi/o proteins,7 and share 44% overall identity (68% identity for the transmembrane domains).
CB1 receptors are enriched in the nervous systems and moderately expressed in peripheral tissues.5, 8, 9 In both rat and human brains, CB1 receptors are densely distributed in the frontal cerebral cortex, basal ganglia, cerebellum, hippocampus, hypothalamus, and anterior cingulate cortex, but rarely in the brainstem nuclei.10 The latter finding may account for the low toxicity of cannabinoids when given in accidental overdose.
At the cellular level, CB1 receptors are mainly localized to axons and nerve terminals and are largely absent from neuronal somas or dendrites.11 This ultra-structural finding, suggesting a predominantly presynaptic localization of CB1 receptors, is consistent with the functional finding that activation of CB1 receptors inhibits calcium channels and activates potassium channels, leading to inhibition of neurotransmitter release.12, 13
CB2 receptors are mainly distributed in immune cells.6, 14 Although originally thought absent from the CNS, CB2 receptors were later found to be expressed in microglia, dorsal root ganglion, spinal cord, and sparsely in several brain regions, such as the cerebellum, cortex, and brainstem.15, 16Nevertheless, CB2 receptors in the CNS can be strongly induced in sensory neurons and spinal cord in neuropathic and inflammatory pain models, as well as in spinal microglia and macrophages in human postmortem spinal cord specimens of multiple sclerosis and amyotrophic lateral sclerosis.17
3. Endocannabinoids
The finding of cannabinoid receptors in the brain motivated the search for their endogenous ligands to elucidate their functional roles. Indeed, several endogenous arachidonic acid derivatives have been identified to have cannabimimetic actions and are termed endocannabinoids; they include anandamide (N-arachidonoylethanolamine),18 2-arachidonoylglycerol (2-AG),19, 20 noladin ether (2-arachidonoylglyceryl ether),21 virodhamine (O-arachidonoylethanolamine),22 and N-arachidonoyldopamine.23 These endocannabinoids, like other lipid mediators, such as prostaglandins, are synthesized and then released locally on demand in activity-dependent and receptor-regulated manners.24 Released endocannabinoids are rapidly inactivated by uptake and enzymatic degradation. Agents that interfere with the inactivation of endocannabinoids may provide a pharmacological means to modify cannabinoid-mediated functions.25, 26 Anandamide and 2-AG are the two best studied endocannabinoids. Despite similar chemical structures, they have distinct pathways for biosynthesis and degradation.
3.1. Anandamide
3.1.1. Anandamide synthesis
Anandamide is an N-acylethanolamine and can be formed by a two-step biosynthesis scheme catalyzed by N-acetyltransferase and N-arachidonoyl phosphatidylethanolamine-phospholipase D (NAPE-PLD).27, 28 Anandamide can also be generated from NAPE through an α/β-hydrolase-4 (ABHD4) and glycerophosphodiesterase 1 (GDE1) enzymatic pathway.29, 30, 31 However, basal levels of brain anandamide in mice lacking the putative synthetic enzymes, either NAPE-PLD32 or GDE1,33 or both GDE1 and NAPE-PLD,33were not different from those in wild type mice. Thus, additional biosynthetic pathways are suggested. Because basal levels of anandamide were measured in these studies, it is not known if activity-dependent anandamide levels differ between the genotypes.
3.1.2. Anandamide degradation
In contrast to its obscure synthetic pathway, anandamide is known to be rapidly inactivated by the degradation enzyme, fatty acid amide hydrolase (FAAH). It is a 579 amino acid serine amidohydrolase, widely distributed in the brain.34, 35 FAAH is mainly located postsynaptically, complimentary to the presynaptically located CB1 receptors, in many, but not all, brain regions.35, 36, 37
3.1.3. Anandamide uptake
In addition to the degradation enzyme, a membrane transporter has been proposed to play a role in the uptake of anandamide. However, its existence remains controversial38, 39 because several putative anandamide transporter inhibitors, such as AM404, VDM11, and LY218240, which enhance the effects of anandamide,40, 41 also inhibit FAAH.42, 43, 44
3.1.4. Anandamide and type 1 transient receptor potential vanilloid channels
Unlike 2-AG, which primarily acts at cannabinoid receptors, anandamide activates both cannabinoid receptors and type 1 transient receptor potential vanilloid (TRPV1) channels.45 The TRPV1 channel is a voltage-gated cation channel which is distributed in both the peripheral and the central nervous system. Capsaicin, the constituent of chili pepper, is a potent agonist. Anandamide and capsaicin have similar binding affinities in displacing [3H]-resiniferatoxin from TRPV1.46 Hence, activation of TRPV1 by anandamide may complicate the interpretation of the effects of experiments using FAAH or anandamide transporter inhibitors.
3.2. 2-AG
3.2.1. 2-AG synthesis
2-AG is a monoacylglycerol (MAG) and is mainly formed by the hydrolysis of diacylglycerol (DAG) through DAG lipases (DAGLs). DAG can be generated through postsynaptic depolarization-induced Ca2+ influx and/or activation of Gq-protein-coupled receptors (GqPCRs) through phospholipase C (PLC).47, 48DAGLs are postsynaptic integral membrane proteins and exist as two isoforms, α and β.49, 50 DAGLα is the major synthetic enzyme for 2-AG in the brain.51
3.2.2. 2-AG degradation
2-AG is mainly degraded by a cytosolic serine hydrolase, MAG lipase (MAGL),52, 53 which is localized in presynaptic terminals, where CB1receptors are also expressed.52 FAAH, the main degradation enzyme of anandamide, metabolizes 2-AG in vitro54, 55 but not efficiently in vivo.56
4. Cannabinoids are antinociceptive
After the identification of CB1 and CB2 receptors, subtype-selective agonists and antagonists, as well as knockout mice, were actively developed. With these tools, the analgesic mechanisms of cannabinoids were explored. Systemic administration of cannabinoids can produce an analgesic effect with an efficacy comparable to opioids in acute pain animal models,57 and was even more effective than opioids in some chronic pain models.58, 59 These analgesic effects are mainly mediated by CB1 receptors in the CNS,59, 60, 61and by both CB162 and CB263, 64 receptors in the periphery.
4.1. CB1 receptor-mediated central site of action
In the CNS, the analgesic effect of cannabinoids is mainly mediated through CB1 receptors located in structures that mediate nociceptive neurotransmission, including the spinal dorsal horn, periaqueductal gray (PAG),65 dorsal raphe nuclei,66 and thalamic ventroposterolateral nucleus.67 At the spinal level, electrophysiological and c-fos expression studies support that cannabinoids inhibit spinal dorsal neuronal activity to produce analgesia.59, 61, 62, 68 Among the supraspinal areas involved in the modulation of nociception, the midbrain PAG has been extensively studied.
4.2. The midbrain PAG is a supraspinal site of action
Activation of the PAG produces analgesia,69 through activating the downstream rostroventral medulla (RVM), which sends inhibitory projections to the dorsal horn of the spinal cord.70 This PAG-RVM-spinal dorsal horn circuit constitutes a key endogenous descending pain inhibitory pathway. Studies using microinjection techniques in pain models suggest that the PAG is the site for cannabinoids producing CB1-mediated analgesic effects.66, 71Immunohistochemical72 and electrophysiological73 studies support that cannabinoids produce analgesic effects in the PAG via a disinhibition (inhibition of GABAergic transmission) mechanism mediated by CB1receptors.
4.3. CB2 receptor-mediated peripheral site of action
In the periphery, the antinociceptive effect of cannabinoids can be mediated both by CB162 and CB2 receptors. Peripherally mediated CB2 antinociception has been an especially attractive therapeutic target, as compounds that selectively activate CB2 receptors have the merit of avoiding CB1-mediated CNS side effects, such as hypomotility, catalepsy, hypothermia, and cognitive impairment. Intraplantar (i.pl.) administration of the CB2-preferring agonists, AM1241 or GW405833, effectively reduced nociceptive responses in several inflammatory pain models.74, 75, 76 These antinociceptive effects were blocked by i.pl. injection of a CB2, but not a CB1 antagonist74 and were absent in CB2 receptor-knockout mice.76, 77 However, activation of CB2 receptors may also have a proinflammatory effect.78 In addition, CB2 receptors were strongly induced in sensory neurons and the spinal cord in neuropathic and inflamed rats.79, 80 This high inducibility of CB2 receptors renders them an attractive potential therapeutic target for pain treatment. A small scale, proof-of-concept clinical trial (32 patients, single site, double-blind, two-way crossover) using a CB2 receptor agonist, LY2828360 (80 mg/kg), for the treatment of osteoarthritis was conducted by Eli Lilly (Indianapolis, IN, USA). However, it failed to meet the primary endpoint.81
5. The role of endocannabinoids in pain regulation
Our understanding of the role of endocannabinoids in pain regulation comes from studies using both pharmacological and genetic approaches by blocking or silencing CB1 and CB2 receptors, or by inhibiting or silencing degradation or synthetic enzymes of anandamide and 2-AG. Table 1 summarizes the effects of inactivating the endocannabinoid system on nociceptive responses in various pain models.
5.1. CB1 receptor-mediated
5.1.1. CB1 antagonists
Systemic administration of SR141716A, the first CB1 antagonist,82 was initially found to have no effect in several pain models (Table 1). Later, it was reported to increase nociceptive responses when given by intrathecal (i.t.) or intraperitoneal (i.p.), but not i.pl., routes of administration, although replication of these findings has been variable (Table 1). Because SR141716A also acts as an inverse agonist of CB1 receptors,83 this might lead to pronociceptive effects in the above studies. The finding that systemic administration of AM4113,84 a neutral CB1 antagonist, produced no changes in nociception suggests there is little tonic endocannabinoid modulation of pain thresholds. However, i.t. injection of CB1 receptor antisense nucleotide caused hyperalgesia in the hot-plate test in mice,85, 86 suggesting an analgesic tone produced by endocannabinoids via CB1 receptors, at least at the spinal level.
5.1.2. CB1 knockout mice
Pain sensitivity has been examined in three lines of CB1 knockout mice of distinct genetic backgrounds, C57BL/6J,87 CD1,88 and 129/SvJ.89 Knockout of CB1 in C57BL/6J mice resulted in lower pain sensitivity in the hot-plate and formalin tests, but not the tail-flick test. However, the antinociceptive responses in these mutants may be confounded by their impaired locomotor activity.87 The other two lines of CB1 knockout mice displayed normal pain sensitivity in several nociceptive tests (Table 1). These results also suggest that tonic levels of endocannabinoids play a minor role in setting pain thresholds.
5.2. CB2 receptor-mediated
5.2.1. CB2 antagonists
SR144528, the first CB2 receptor antagonist, was used to investigate the role of CB2 receptor activation in the effect of endocannabinoids on pain regulation, mainly in inflammatory pain models (Table 1). In the formalin test, it was hyperalgesic when given by Intravenous (i.v.) injection63 but was ineffective by i.p. administration.90, 91 This CB2 antagonist induced hyperalgesia and enhanced edema when given by i.p. injection in the carrageenan model,92 but displayed anti-inflammatory effects by oral administration in the same model.78 Again, the inverse agonist property of SR144528 might also explain its hyperalgesic effects in these studies.
5.2.2. CB2 knockout mice
CB2 knockout mice, as compared with wild type mice, had increased thermal nociceptive responses in the plantar test, while having normal pain sensitivity in the hot-plate and tail-immersion assays.77 In a neuropathic pain model, CB2 knockout mice displayed enhanced nociceptive responses in the contralateral paw compared to control mice, which was also associated with enhanced interferon-γ and microglial expression in the contralateral spinal dorsal horn.93, 94 These results suggest that endocannabinoids play a CB2receptor-mediated tonic analgesic role, but also exert a proinflammatory tone during neuropathic pain state.
5.3. Role of anandamide in pain regulation
5.3.1. FAAH inhibitors
Several lines of evidence suggest that inhibition of FAAH, the degradation enzyme of anandamide, effectively reduced nociceptive responses in various pain models (Table 1). A series of irreversible carbamate FAAH inhibitors, such as URB532 and URB597, has been developed and patented95, 96 for pain treatment. Later, an α-ketooxazole reversible FAAH inhibitor, OL-135, was demonstrated to have high in vivo efficacy for reducing nociceptive responses in tail-immersion, hot-plate, formalin tests97 and neuropathic pain models.98Thereafter, a highly selective urea-based irreversible FAAH inhibitor, PF-3845, was developed by Pfizer (NYC, NY, USA) and shown to be an efficacious analgesic in inflammatory99, 100 and neuropathic101 pain models. These FAAH inhibitors significantly increased anandamide levels in the brain and mainly induced a CB1 receptor-mediated antinociception, suggesting that endogenous anandamide, when protected from degradation, can produce antinociception through CB1 receptors. Chronic systemic treatment with PF-3845 produced a persistent analgesic effect without tolerance in mice.101Interestingly, this compound was also effective when given by i.pl. administration.100 Recently, a peripherally acting FAAH inhibitor, URB937, was shown to be effective in inflammatory pain, neuropathic pain, and arthritis pain models.102 URB597 and URB937 were also found to be effective in the cisplatin (a chemotherapeutic agent) neuropathic pain model.103 An orally bioavailable urea-based irreversible carbamate FAAH inhibitor, PF-04457845, was developed by Pfizer and displayed significant CB1 receptor-dependent reductions in inflammatory and arthritis pain models.104, 105
The promising findings with FAAH inhibitors in reducing nociceptive responses in various animal pain models (Table 1) motivated advancing an FAAH inhibitor to a clinical pain trial. However, in a randomized, placebo-controlled clinical trial, chronic administration of PF-04457845 did not have significant analgesic effects in patients with osteoarthritis, although their plasma levels of endocannabinoids were significantly elevated and no significant side effects were reported.106 Although this Phase II clinical trial was terminated early due to ineffectiveness in an interim analysis, it remains to be further elucidated if FAAH inhibitors would be clinically effective for other pain indications,107 such as migraine, cancer pain or neuropathic pain induced by chemotherapy,103 diabetes, or herpes virus. The possible species difference in the FAAH between human and rodents should also be investigated.106 Interestingly, a noncovalent, reversible and noncompetitive FAAH inhibitor, AZ513, was published by AstraZenica,108 with a higher potency in inhibiting human FAAH than rat FAAH. It is not known if this compound will be more promising if there is any clinical trial in the future.
5.3.2. FAAH knockout mice
FAAH knockout mice exhibited reduced CB1-mediated pain sensitivity, but locomotor activity and body temperature were similar to those of wild type mice.34 This suggests that FAAH plays an important role in regulating endogenous anandamide-mediated analgesic tone but not in its motor- or thermo-regulation. CB1-mediated nociceptive responses in acute and inflammatory pain models were significantly reduced in FAAH knockout mice, as seen in animals treated with FAAH inhibitors. However, FAAH knockout mice had unchanged sensitivity in neuropathic pain models, as compared with wild type mice (Table 1).
5.4. Role of 2-AG in pain regulation
5.4.1. MAGL inhibitors
URB602, the first MAGL inhibitor, given by i.pl. injection, significantly reduced mechanical and thermal allodynia in a neuropathic pain model.109JZL184, a much more potent and selective MAGL inhibitor,110 was also effective in reducing nociceptive responses in several inflammatory and neuropathic pain models by either i.p., i.t., or i.pl. injection (Table 1). This suggests that acute administration of MAGL inhibitors has an analgesic effect.
5.4.2. MAGL knockout mice
Brain 2-AG levels were elevated more than 10-fold in MAGL knockout mice101, 111 close to the 8-fold elevation induced by acute administration of JZL184.110 However, because of this elevation of 2-AG desensitized CB1receptors in MAGL knockout mice, fewer and less active CB1 receptors existed in the brains of these mutants.101, 111 Similarly, treatment with a high dose of JZL184 for 6 consecutive days also led to desensitization of CB1 receptors, loss of analgesic activity, and cross-tolerance to the antiallodynic effects of CB1agonists and FAAH inhibitors.101, 111 This is significantly different from the finding that CB1 receptor function remained normal in mice lacking FAAH or which were chronically treated with FAAH inhibitors.34, 101, 112 The difference between FAAH and MAGL inhibition may be because 2-AG has a higher efficacy than anandamide at CB1 receptors.113, 114, 115 Thus, after long-term inhibition of its degradation enzyme, the enhanced and prolonged action of 2-AG, but not anandamide, leads to CB1 receptor desensitization. Interestingly, lower doses of MAGL inhibitors produce significant therapeutic benefits in preclinical models, while avoiding desensitization and tolerance of CB1 signaling.116, 117, 118
6. GqPCR activation-initiated 2-AG retrograde disinhibition in the PAG: A novel analgesic mechanism
A major pathway to generate 2-AG is by GqPCR activation. Activation of postsynaptic GqPCRs results in phospholipid hydrolysis by PLCβ to yield DAG. DAG is then hydrolyzed by DAGLα located at postsynaptic membranes to generate 2-AG, which then diffuses retrogradely across the synapse to activate presynaptic CB1 receptors and inhibit transmitter release.119 This neurotransmission inhibitory mechanism mediated by a GqPCR-PLC-DAGL-2-AG retrograde signaling cascade has been reported in several brain regions,119 including the PAG. Stimulation of the type 5 metabotropic glutamate receptor (mGluR5),120, 121, 122, 123, 124, 125 M1/M3 muscarinic acetylcholine receptor (M1/M3 mAChR),126 and orexin 1 receptor (OX1R),127has been reported to initiate the GqPCR-PLC-DAGL-2-AG signaling-mediated retrograde inhibition of GABAergic transmission (disinhibition) in PAG slices. This 2-AG-mediated disinhibition mechanism may contribute to the analgesic effects induced by activation of these GqPCRs.
6.1. mGluR5 activation
There are eight subtypes of mGluRs classified into three groups according to their coupling signaling pathways: Group I (mGluR1/5), Group II (mGluR2/3), and Group III (mGluR4/6-8).128, 129 Only Group I mGluRs belong to the GqPCR family.130 Drew and Vaughan131 found that activation of all three groups of mGluRs inhibits PAG GABAergic transmission, but only the effect of activating Group I mGluRs, specifically mGluR5, is mediated through endocannabinoids.123
6.2. Inhibiting glutamate transport
As postsynaptic mGluR5s are mainly located perisynaptically,132 they are primarily activated by spillover glutamate, such as when glutamate transporters are inhibited or overwhelmed, subsequently leading to disinhibition of the PAG through the mGluR5-PLC-DAGL-2-AG signaling.123
6.3. mGluR5 agonists
Recently, Gregg and colleagues,120 found that microinjection of an mGluR5 agonist into the dorsolateral PAG (dlPAG) triggered the release of 2-AG, but not anandamide. This effect was reversed by intra-dorsolateral PAG (intra-dlPAG) injection of a CB1 antagonist and DAGL inhibitor, or by expressing siRNA to silence DAGLα in the dlPAG. Immunohistochemical staining showed that mGluR5s were colocalized with DAGLα in the postsynaptic dendritic site, which is juxtaposed to the presynaptic localization of CB1receptors. These data support the fact that postsynaptic mGluR5-DAGLα cascade triggers retrograde 2-AG signaling in the PAG in vivo.
6.3.1. Activating NK1, NTS1/2, and CCK1 receptors
In addition to inhibiting glutamate transporter or directly applying mGluR agonists, there are several ways to release glutamate in sufficient amounts to activate perisynaptic mGluR5s, leading to disinhibition of the PAG through the mGluR5-PLC-DAGL-2-AG signaling. Indeed, Vaughan and colleagues proved that this 2-AG-mediated disinhibition signaling triggered by mGluR5 activation in ventrolateral PAG (vlPAG) slices can be induced by several analgesic neuropeptides.122, 124, 125 Substance P,122 neurotensin,125 and cholecystokinin (CCK),124 activated neurokinin 1, neurotensin 1/2, cholecystokinin 1 receptors, respectively, on glutamatergic somas to release glutamate, which then activated perisynaptic mGluR5s, and produced disinhibition in the PAG through 2-AG. It remains to be elucidated by in vivostudies if this PAG disinhibition mechanism mediated by the mGluR5-PLC-DAGL-2-AG signaling contributes to the antinociceptive effects of intra-PAG injection of substance P,133 neurotensin,134 and CCK.135
6.3.2. TRPV1 activation
Capsaicin, when injected into the PAG, induced antinociception, in contrast to its pronociceptive effect in the periphery.136, 137 In a study examining the analgesic mechanism of capsaicin at the supraspinal level, we recently found that capsaicin can also induce substantial glutamate release in the vlPAG to activate the mGluR5-PLC-DAGL-2-AG disinhibition mechanism, leading to analgesia.121 We found that capsaicin markedly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs), but decreased evoked inhibitory postsynaptic currents (eIPSCs) in PAG slices. This IPSC inhibitory effect of capsaicin was antagonized by TRPV1, mGluR5, and CB1 antagonists and by a DAGL inhibitor. These results suggest that capsaicin activates the TRPV1 channels on glutamatergic terminals to release massive amounts of glutamate to activate postsynaptic mGluR5s, leading to disinhibition in the PAG through the mGluR5-PLC-DAGL-2-AG signaling. Finally, we proved that this mechanism contributes to the analgesic effect induced by intra-ventrolateral PAG (intra-vlPAG) injection of capsaicin,121 because this analgesic effect was reversed by intra-vlPAG injection of AM251 and 2-methyl-6-(phenylethynyl)pyridine, an mGluR5 antagonist. In agreement with our findings, Starowicz et al137 also found that intra-vlPAG capsaicin induced analgesia by releasing glutamate from the PAG to activate the OFF neurons in the RVM.
6.4. M1/M3 mAChR activation
The PAG receives dense cholinergic projections from the pontine tegmentum.138 Microinjection of cholinergic agonists into the PAG produces analgesia and related behaviors.139, 140 In PAG slices, Lau and Vaughan126found that carbachol suppressed IPSCs in PAG slices. This effect was mimicked by inhibiting acetylcholinesterase, occluded by a CB1 agonist, and reduced by blocking M1/M3 mAChRs and CB1 receptors, and inhibiting DAGL. These results suggest that activation of postsynaptic M1/M3 mAChR, like other GqPCRs, initiates the GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG.
6.5. OX1 receptor activation
Orexin A and orexin B, also named hypocretin 1 and 2, respectively, are a pair of hypothalamic neuropeptides and exert their biological functions through two GqPCRs, OX1 and OX2 receptors.141, 142 Orexin-expressing neurons are localized in the perifornical area and the lateral hypothalamus (LH),141, 142but project widely throughout the brain, including the PAG.143, 144, 145Orexins have been implicated in a myriad of physiological functions, such as sleep, reward, energy homeostasis, autonomic central control, and pain.146, 147 However, the mechanism(s) of how orexins regulate pain, especially at the supraspinal level, remained unclear until our findings in 2011.127 We found that orexin A depressed IPSCs in PAG slices and that this effect was inhibited by OX1 and CB1, but not OX2 antagonists, as well as by PLC and DAGL inhibitors. Moreover, the effect of orexin A was mimicked by a cannabinoid agonist and enhanced by an MAGL inhibitor. These results suggest that activation of postsynaptic OX1 receptors, like mGluR5s and M1/M3 mAChRs, initiates the GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG. We also proved that this mechanism contributes to the antinociceptive effect induced by intra-vlPAG injection of orexin A in the rat hot-plate test.127
Taken together, in the PAG, mGluR5 can be activated directly by an mGluR5 agonist,120 or by endogenous spillover glutamate: (1) when glutamate transport is inhibited;123 (2) when glutamatergic terminals are depolarized by TRPV1 activation by capsaicin121 or anandamide;148, 149 and (3) when glutamatergic cell bodies were excited by substance P via NK1 receptors,122neurotensin via NTS1/2 receptors,125 or CCK via CCK1 receptors.124 After mGluR5 activation, 2-AG was generated through the PLC-DAGL enzymatic cascade to produce retrograde inhibition of GABAergic transmission in the PAG, leading to analgesia.
7. Functional role of endocannabinoid analgesia in the PAG
7.1. Stress-induced analgesia
In 2005, Hohmann et al150 demonstrated that stress-induced analgesia (SIA), a phenomenon believed to represent the evolutionary impetus for the development of central pain inhibition mechanisms in humans and animals,151 is associated with the rapid formation of 2-AG and anandamide in the dlPAG. SIA induced by foot shock stress was blocked by CB1 antagonists and enhanced by the MAGL or FAAH inhibitor given by intra-dlPAG injection, but not affected by the CB2, opioid, or TRPV1 antagonist. Valverde et al152 also reported that forced swim stress-induced analgesia was absent in CB1 knockout mice.
Olango et al153 and Butler et al154 also demonstrated that endocannabinoids are involved in fear-conditioned analgesia (FCA) in rats re-exposed to the context previously associated with foot shock. They found that intra-dlPAG injection of the CB1 antagonist attenuated, and the FAAH inhibitor enhanced, FCA in rats injected with i.pl. formalin. Elevated anandamide in the dlPAG was specifically associated with FCA.153
Together, these results suggest that endocannabinoids, either anandamide or 2-AG, can be induced to release by various stressors, either electrical foot shock, swimming or fear, or during a pain state to activate the CB1 receptors in the PAG, serving as an endogenous analgesic protector. However, how a stress triggers endocannabinoid release to induce SIA remains unclear. The disinhibition mechanism mediated by 2-AG in the PAG through the GqPCR-PLC-DAGL signaling might be such a mechanism.
7.2. mGluR5 involvement
Recently, Gregg and colleagues120 found that intra-dlPAG injection of an mGluR5 agonist increased PAG 2-AG and enhanced SIA. This effect was reversed by intra-dlPAG injection of the CB1 receptor antagonist and DAGL inhibitors, and by silencing DAGLα in the dlPAG.120 These data suggest that the mGluR5-PLC-DAGLα-2-AG disinhibition mechanism in the PAG contributes to SIA.
7.3. Orexin involvement
Two studies have suggested that endogenous orexins play a role in SIA. Watanabe et al155 reported that SIA induced by electrical foot shock was absent in prepro-orexin knockout mice. Xie et al156 found that in mice with hypothalamic orexin neurons degenerated by ataxin-3 expression, restraint stress-induced antinociception was significantly reduced, as compared to wild type controls. The reduction of SIA in orexin/ataxin-3 mice can be reversed by intracerebroventricular (i.c.v.) injection of orexin A.
We recently found that a 30-min restraint stress in mice induced analgesia in the hot-plate test accompanied with increased c-fos expression in LH orexin neurons. This SIA was blocked by intra-vlPAG injection of OX1 and CB1antagonists, respectively. These results suggest that restraint stress activates hypothalamic orexin neurons, releasing orexins to activate OX1 receptors in the vlPAG, initiating the GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG, leading to analgesia.157
7.4. Substance P/neurotensin involvements
Substance P is antinociceptive at the supraspinal level, in contrast to a pronociceptive action in the periphery. The PAG is one of the sites of action because intra-PAG injection of substance P induced antinociception.133Chemical (carbachol) stimulation of LH triggered substance P release in the PAG, leading to analgesia.158 Interestingly, activation of LH by restraint stress also released orexins, which in turn acted in the PAG to induce analgesia through 2-AG mediated disinhibition.157 It is interesting to know if substance P also plays a role in endocannabinoid-mediated SIA. Moreover, both orexin and substance P were released after LH activation, so it will be interesting to examine if there are interactions between these two neuropeptide systems during SIA.
Similarly, neurotensin was also suggested to be involved in SIA.159 It will also be interesting to see if neurotensin plays a role in endocannabinoid-mediated SIA.
7.5. Analgesic mechanism of acetaminophen
Acetaminophen, one of the most popular analgesic drugs, has been used for more than a century. Its mode of analgesic action is still a matter of debate. In 2005–2006, a new look for the analgesic effect of acetaminophen, involving the CB1 receptor, was proposed by two groups simultaneously.160, 161, 162Hogestatt et al162 demonstrated that N-arachidonoylphenolamine, known as AM404, can be formed in the brain after systemic administration of acetaminophen in rats. They suggested that after systemic administration, acetaminophen is deacetylated in the liver into p-aminophenol, which is then conjugated with arachidonic acid to form AM404 in the brain by FAAH, the degradation enzyme of anandamide. Further studies showed that the analgesic effects of acetaminophen were reduced by CB1 antagonists,160, 161 and absent in CB1 knockout mice.161
AM404 has complicated pharmacological properties. Like anandamide, it is a C20 unsaturated fatty acid amide, originally developed as a potent inhibitor for anandamide uptake,40 but it is also an FAAH inhibitor. Furthermore, AM404 is also a TRPV1 agonist, as potent as capsaicin.163 Recently, Mallet et al164 found that the TRPV1 channel in the brain is involved in acetaminophen-induced antinociception. They found that the oral antinociceptive effects in several pain models induced by acetaminophen at a clinically effective dose range observed in wild type mice, were absent in TRPV1 or FAAH knockout mice. The analgesic effect of AM404 (i.c.v.) was also absent in TRPV1 knockout mice.
Together, these studies suggest that the analgesic effects of oral acetaminophen, or its central active metabolite, AM404, are mediated by TRPV1 channels and CB1 receptors. However, TRPV1 activation usually causes neuroexcitation, whereas CB1R activation decreases neurotransmitter release. We recently proved that the mechanism we reported for the analgesic mechanism of capsaicin,121 can explain how acetaminophen can produce a CB1R-mediated analgesic effect via TRPV1 activation.165 We found that oral acetaminophen or intra-PAG microinjection of AM404 produced comparable antinociceptive effects in the rat hot-plate test. Both antinociceptive effects were antagonized by intra-PAG microinjection of antagonists of TRPV1, mGluR5, or CB1R. It is suggested that after oral administration, acetaminophen is deacetylated in the liver into p-aminophenol, which is then converted into AM404 in the brain. AM404 then activates TRPV1, like capsaicin, in the vlPAG, to release a great amount of glutamate, which activates the mGluR5-PLC-DAGLα-2-AG disinhibition mechanism in the PAG, leading to analgesia.
8. Conclusions and perspectives
After the identification of cannabinoid receptors and endocannabinoids, accumulating studies have emerged during the past two decades that have aimed to elucidate the regulatory mechanisms of the cannabinoid system in pain control, with a goal of developing new pain therapies. CB2 selective agonists have the merit of avoiding CB1 receptor-mediated CNS side effects. Inhibitors of anandamide and 2-AG degradation enzymes, FAAH and MAGL, respectively, have the merit of focusing action at generating sites. Despite promising preclinical results, the first clinical trial with an FAAH inhibitor failed. The efficacy of MAGL inhibitors remains to be determined. Because of its synthesis on demand, endogenous 2-AG levels can be elevated in the PAG upon GqPCR activation and lead to analgesia via PAG disinhibition. Several endogenous analgesic neuropeptides may serve as endogenous analgesics during SIA via this GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG. As an effective endogenous pain relief mechanism during evolution, it offers a promising strategy for analgesic drug development.
Acknowledgments
This study was supported by the grants to LCC from the National Science Council, Taipei, Taiwan (NSC 101-2325-B002-048 and NSC 102-2321-B002-066) and National Health Research Institutes, Miaoli, Taiwan (NHRI-EX102-10251NI).