Cannabinoid Receptors and Their Endogenous Agonist,Anandamide

Cannabinoids are a class of compound found in marijuana which have been known for their therapeutic and psychoactive properties for at least 4000 years. Isolation of the active principle in marijuana, 9-THC, provided the lead structure in the development of highly potent congeners which were used to probe for the mechanism of marijuana action. Cannabinoids were shown to bind to selective binding sites in brain tissue thereby regulating second messenger formation. Such studies led to the cloning of three cannabinoid receptor subtypes, CB1, CB2, and CB1A all of which belong to the superfamily of G protein-coupled plasma membrane receptors. Analogous to the discovery of endogenous opiates, isolation of cannabinoid receptors provided the appropriate tool to isolate an endogenous cannabimimetic eicosanoid, anandamide, from porcine brain. Recent studies indicate that anandamide is a member of a family of fatty acid ethanolamides that may represent a novel class of lipid neurotransmitters. This review discusses recent progress in cannabinoid research with a focus on the receptors for 9-THC, their coupling to second messenger responses, and the endogenous lipid cannabimimetic, anandamide.     

Cannabinoid pharmacology: implications for additional cannabinoid receptor subtypes

Delta(9)-Tetrahydrocannabinol (delta(9)-THC), the primary psychoactive constituent of marijuana (Cannabis sativa), is known to bind to two cannabinoid receptors: CB(1) receptors, located primarily in the brain, and CB(2) receptors, located primarily in the periphery. Recent research has suggested that other cannabinoids, including anandamide and WIN 55212-2, may also act at novel non-CB(1), non-CB(2) cannabinoid receptor(s). Anandamide produces a number of in vivo pharmacological effects in CB(1) knockout mice that are not produced by delta(9)-THC and cannot be explained by anandamide's rapid metabolism. In addition, in vitro anandamide and WIN 55212-2 stimulate [35S]GTPgammaS binding in both CB(1) knockout and wildtype mice while delta(9)-THC stimulates this binding only in wildtype mice. Although anandamide and vanilloid agonists share pharmacological effects, anandamide's actions in CB(1) knockout mice do not appear to be mediated by vanilloid VR(1) receptors. While not yet conclusive, these results suggest the possibility of additional cannabinoid receptors in the brain and periphery.     

Delta9-tetrahydrocannabinol (Delta9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia

Delta(9)-tetrahydrocannabinol (Delta(9)-THC), a primary psychoactive constituent of cannabis, has been reported to act as a neuroprotectant via the cannabinoid CB(1) receptor. In this study, Delta(9)-THC significantly decreased the infarct volume in a 4 h mouse middle cerebral artery occlusion mouse model. The neuroprotective effect of Delta(9)-THC was completely abolished by SR141716, cannabinoid CB(1) receptor antagonist, and by warming the animals to 31 degrees C. Delta(9)-THC significantly decreased the rectal temperature, and the hypothermic effect was also inhibited by SR141716 and by warming to 31 degrees C. At 24 h after cerebral ischemia, Delta(9)-THC significantly increased the expression level of CB(1) receptor in both the striatum and cortex, but not in the hypothalamus. Warming to 31 degrees C during 4 h cerebral ischemia did not increase the expression of CB(1) receptor at the striatum and cortex in MCA-occluded mice. These results show that the neuroprotective effect of Delta(9)-THC is mediated by a temperature-dependent mechanism via the CB(1) receptor. In addition, warming to 31 degrees C might attenuate both the neuroprotective and hypothermic effects of Delta(9)-THC through inhibiting the increase in CB(1) receptor in both the striatum and cortex but not in the hypothalamus, which may suggest a new thermoregulation mechanism of Delta(9)-THC.     

Neuromodulatory role of the endocannabinoid signaling system in alcoholism: an overview

The current review evaluates the evidence that some of the pharmacological and behavioral effects of ethanol (EtOH), including EtOH-preferring behavior, may be mediated through the endocannabinoid signaling system. The recent advances in the understanding of the neurobiological basis of alcoholism suggest that the pharmacological and behavioral effects of EtOH are mediated through its action on neuronal signal transduction pathways and ligand-gated ion channels, receptor systems, and receptors that are coupled to G-proteins. The identification of a G-protein-coupled receptor, namely, the cannabinoid receptor (CB1 receptor) that was activated by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive component of marijuana, led to the discovery of endogenous cannabinoid agonists. To date, two fatty acid derivatives identified to be arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) have been isolated from both nervous and peripheral tissues. Both these compounds have been shown to mimic the pharmacological and behavioral effects of Delta(9)-THC. The involvement of the endocannabinoid signaling system in the development of tolerance to the drugs of abuse including EtOH has not been known until recently. Recent studies from our laboratory have demonstrated for the first time the down-regulation of CB1 receptor function and its signal transduction by chronic EtOH. The observed down-regulation of CB1 receptor binding and its signal transduction results from the persistent stimulation of the receptors by the endogenous CB1 receptor agonists, AEA and 2-AG, the synthesis of which has been found to be increased by chronic EtOH treatment. This enhanced formation of endocannabinoids may subsequently influence the release of neurotransmitters. It was found that the DBA/2 mice, known to avoid EtOH intake, have significantly reduced brain-CB1-receptor function consistent with other studies, where the CB1 receptor antagonist SR141716A has been shown to block voluntary EtOH intake in rodents. Similarly, activation of the CB1 receptor system promoted alcohol craving, suggesting a role for the CB1 receptor gene in excessive EtOH drinking behavior and development of alcoholism. Ongoing investigations may lead to the development of potential therapeutic strategies for the treatment of alcoholism.     

Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.     

Effects of delta-9-tetrahydrocannabinol on human immune function and host defense

This review examines evidence that delta(9)-tetrahydrocannabinol (THC) can regulate and suppress human immune responses. Leukocytes express both cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), and levels of mRNA encoding for them are increased in peripheral blood leukocytes obtained from marijuana smokers, suggesting cannabinoid receptor activation in vivo. Exposure of human T-cells to THC suppresses their proliferation, inhibits the release of interferon-gamma, and skews the balance of T-helper cytokines towards a type 2 response. The majority of these effects are CB2 receptor-dependent. Consistent with an impact of THC on cell-mediated immunity, alveolar macrophages (AMs) recovered from the lungs of marijuana smokers are suppressed in their ability to release pro-inflammatory cytokines and nitric oxide (NO), and kill bacteria. Macrophage function is restored by treatment with interferon-gamma, a type 1 cytokine. Habitual exposure to THC appears capable of impacting on human cell-mediated immunity and host defense.     

Cannabinoid receptors in atherosclerosis

PURPOSE OF REVIEW: Recent findings suggesting that cannabinoid receptors are potential targets for the treatment of atherosclerosis are reviewed. RECENT FINDINGS: Cannabinoids, such as Delta9-tetrahydrocannabinol, the major psychoactive compound of marijuana, their synthetic analogs and endogenous cannabinoid ligands, produce their biological effects by interacting with specific receptors. In the apolipoprotein E knockout mouse model of atherosclerosis, Delta9-tetrahydrocannabinol was shown to inhibit disease progression through pleiotropic effects on inflammatory cells. Blocking of cannabinoid receptor CB2, the main cannabinoid receptor expressed on immune cells, abolished the observed effects. The development of novel cannabinoid receptor ligands that selectively target CB2 receptors or pharmacological modulation of the endocannabinoid system might offer novel therapeutic strategies in the treatment of atherosclerosis. Several reports demonstrating an implication of the endocannabinoid system in different inflammatory conditions support this hypothesis. SUMMARY: The immunomodulatory capacity of cannabinoids is now well established and suggests a broad therapeutic potential of cannabinoids for a variety of conditions, including atherosclerosis. New strategies based on nonpsychotropic cannabinoid receptor ligands or compounds modulating endocannabinoid synthesis or stability might solve the problem of the unwanted side effects associated with cannabinoid administration.     

Delta-9-tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway

In this study, we show that Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, suppresses host immune reactivity against lung cancer. In two different weakly immunogenic murine lung cancer models, intermittent administration of THC (5 mg/kg, four times/wk i.p. for 4 wk) led to accelerated growth of tumor implants compared with treatment with diluent alone. In contrast to our findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing SCID mice. The immune inhibitory cytokines, IL-10 and TGF-beta, were augmented, while IFN-gamma was down-regulated at both the tumor site and in the spleens of THC-treated mice. Administration of either anti-IL-10- or anti-TGF-beta-neutralizing Abs prevented the THC-induced enhancement in tumor growth. Both APC and T cells from THC-treated mice showed limited capacities to generate alloreactivity. Furthermore, lymphocytes from THC-treated mice transferred the effect to normal mice, resulting in accelerated tumor growth similar to that seen in the THC-treated mice. THC decreased tumor immunogenicity, as indicated by the limited capacity for tumor-immunized, THC-treated mice to withstand tumor rechallenge. In vivo administration of a specific antagonist of the CB2 cannabinoid receptor also blocked the effects of THC. Our findings suggest the THC promotes tumor growth by inhibiting antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.     

Distinct pharmacology and metabolism of K2 synthetic cannabinoids compared to Δ-THC: Mechanism underlying greater toxicity?

K2 or Spice products are emerging drugs of abuse that contain synthetic cannabinoids (SCBs). Although assumed by many teens and first time drug users to be a “safe” and “legal” alternative to marijuana, many recent reports indicate that SCBs present in K2 produce toxicity not associated with the primary psychoactive component of marijuana, ?⁹-tetrahydrocannabinol (Δ⁹-THC). This mini-review will summarize recent evidence that use of K2 products poses greater health risks relative to marijuana, and suggest that distinct pharmacological properties and metabolism of SCBs relative to Δ⁹-THC may contribute to the observed toxicity. Studies reviewed will indicate that in contrast to partial agonist properties of Δ⁹-THC typically observed in vitro, SCBs in K2 products act as full cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) agonists in both cellular assays and animal studies. Furthermore, unlike Δ⁹-THC metabolism, several SCB metabolites retain high affinity for, and exhibit a range of intrinsic activities at, CB1 and CB2Rs. Finally, several reports indicate that although quasi-legal SCBs initially evaded detection and legal consequences, these presumed “advantages” have been limited by new legislation and development of product and human testing capabilities. Collectively, evidence reported in this mini-review suggests that K2 products are neither safe nor legal alternatives to marijuana. Instead, enhanced toxicity of K2 products relative to marijuana, perhaps resulting from the combined actions of a complex mixture of different SCBs present and their active metabolites that retain high affinity for CB1 and CB2Rs, highlights the inherent danger that may accompany use of these substances.     

Structural modifications of the cannabinoid side chain towards C3-aryl and 1',1'-cycloalkyl-1'-cyano cannabinoids

The compounds reported in this study are Delta(8)-THC analogues in which the C3 five-carbon linear side chain of Delta(8)-THC was replaced with aryl and 1',1'-cycloalkyl substituents. Of the compounds described here analogues 2d (CB(1), K(i)=11.7 nM. CB(2), K(i)=9.39 nM) and 2f (CB(1), K(i)=8.26 nM. CB(2), K(i)=3.86 nM) exhibited enhanced binding affinities for CB(1) and CB(2), exceeding that of Delta(8)-THC. Efficient procedures for the synthesis of these novel cannabinoid analogues are described.     

Behavioral sensitization to delta 9-tetrahydrocannabinol and cross-sensitization with morphine: differential changes in accumbal shell and core dopamine transmission

Although cannabinoid-induced behavioral sensitization and cross-sensitization with opiates has been recently demonstrated, no information is available on the associated state and responsiveness of dopamine (DA) transmission in the nucleus accumbens (NAc) shell and core. In this study we investigate by means of dual probe microdialysis, the effect of exposure to a sensitizing regimen of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and morphine on the extracellular concentrations of DA under basal conditions and after challenge with Delta(9)-THC and morphine in the NAc shell and core. Different groups of male Sprague-Dawley rats were administered twice daily for 3 days with increasing doses of Delta(9)-THC (2, 4, and 8 mg/kg i.p.), morphine (10, 20, and 40 mg/kg s.c.), and vehicle. After 14-20 days from the last injection, the animals were implanted with two microdialysis probes, one aimed at the NAc shell and the other at the core. The following day animals pre-treated with Delta(9)-THC and vehicle controls were challenged with 150 microg/kg i.v. of Delta(9)-THC or 0.5 mg/kg i.v. of morphine. Animals pre-treated with morphine and their vehicle controls were administered with 150 microg/kg i.v. of Delta(9)-THC. Rats pre-exposed to Delta(9)-THC showed behavioral sensitization associated with a reduced stimulation of DA transmission in the NAc shell and an increased stimulation in the NAc core in response to Delta(9)-THC challenge. Pre-exposure to Delta(9)-THC induced behavioral sensitization to morphine also, but only a reduced stimulation of DA transmission in the NAc shell was observed. Animals pre-treated with morphine showed behavioral sensitization and differential changes of DA in the NAc shell and core in response to Delta(9)-THC challenge with a decreased response in the shell and an increased response in the core. The results show that Delta(9)-THC-induced behavioral sensitization is associated with changes in the responsiveness of DA transmission in the NAc subdivisions that are similar to those observed in the sensitization induced by other drugs of abuse.     

Novel, not adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.     

Presence and functional regulation of cannabinoid receptors in immune cells.

In the last 30 years studies on drug-abusing humans and animals injected with cannabinoids, as well as in vitro models employing immune cell cultures, have demonstrated that marijuana and cannabinoids are immunomodulators. Both types of cannabinoid receptors, CB1 and CB2, have been found in immune cells, suggesting they are important in mediating the effects of cannabinoids on the immune system. This article reviews the data on the function and distribution of cannabinoid receptors in the immune system and their involvement in the immunomodulatory effect of these substances.     

Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity

Background

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ⁹-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2''s high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).

Methods/Principal Findings

JWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [³H]CP-55,940, and then for CB1R intrinsic efficacy using an [³⁵S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting K_i values that were lower than or equivalent to Δ⁹-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ⁹-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ⁹-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.

Conclusions/Significance

Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ⁹-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.     

Activation of brain prostanoid EP3 receptors via arachidonic acid cascade during behavioral suppression induced by Delta8-tetrahydrocannabinol

We have previously shown that behavioral changes induced by cannabinoid were due to an elevation of prostaglandin E2 (PGE2) via the arachidonic acid cascade in the brain. In the present study, we investigated the participation of the prostanoid EP3 receptor, the target of PGE2 in the brain, in behavioral suppression induced by Delta8-tetrahydrocannabinol (Delta8-THC), an isomer of the naturally occurring Delta9-THC, using a one-lever operant task in rats. Intraperitoneal administration of Delta8-THC inhibited the lever-pressing behavior, which was significantly antagonized by both the selective cannabinoid CB1 receptor antagonist SR141716A and the cyclooxygenase inhibitor diclofenac. Furthermore, intracerebroventricular (i.c.v.) administration of PGE2 significantly inhibited the lever-pressing performance similar to Delta8-THC. Prostanoid EP3 receptor antisense-oligodeoxynucleotide (AS-ODN; twice a day for 3 days, i.c.v.) significantly decreased prostanoid EP3 receptor mRNA levels as determined by the RT-PCR analysis in the cerebral cortex, hippocampus and midbrain. AS-ODN also antagonized the PGE2-induced suppression of the lever pressing. In the same way, the suppression of lever-pressing behavior by Delta8-THC was significantly improved by AS-ODN. It is concluded that the suppression of lever-pressing behavior by cannabinoid is due to activation of the prostanoid EP3 receptor through an elevation of PGE2 in the brain.     

Receptor-independent effects of natural cannabinoids in rat peritoneal mast cells in vitro

Cannabinoids can activate CB(1) and CB(2) receptors. Since a CB(2) mRNA has been described in rat peritoneal mast cells (RPMC), we investigated a series of cannabinoids and derivatives for their capacity to stimulate RPMC. Effects of natural cannabinoids Delta(9)-tetrahydrocannabinol (Delta(9)-THC), Delta(8)-THC, endocannabinoids (anandamide, palmitoylethanolamide) and related compounds (N-decanoyl-, N-lauroyl-, N-myristoyl-, N-stearoyl- and N-oleoyl-ethanolamines; N-palmitoyl derivatives (-butylamine, -cyclohexylamine, -isopropylamine); and N-palmitoyl, O-palmitoylethanolamine), and synthetic cannabinoids including WIN 55,212-2, SR141716A and SR144528 were assessed for their capacity to induce histamine release or prime RPMC stimulated by compound 48/80. Only Delta(9)-THC and Delta(8)-THC could induce non-lytic, energy- and concentration-dependent histamine releases from RPMC (respective EC(50) values: 23.5+/-1.2; 53.4+/-20.6 microM, and maxima: 71.2+/-5.5; 55.7+/-2.7% of the total RPMC histamine content). These were not blocked by CB(1) (SR141716A) or CB(2) (SR144528) antagonists, but reduced by pertussis toxin (100 ng/ml). Endocannabinoids and analogues did neither induce histamine secretion, nor prime secretion induced by compound 48/80 (0.2 microg/ml). Delta(9)-THC and Delta(8)-THC induced in vitro histamine secretion from RPMC through CB receptor-independent interactions, partly involving G(i/o) protein activation.     

Marijuana-based drugs: innovative therapeutics or designer drugs of abuse?

The principal psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), activates CB1 cannabinoid receptors (CB1Rs). Unfortunately, pharmacological research into the design of effective THC analogs has been hampered by psychiatric side effects. THC-based drug design of a less academic nature, however, has led to the marketing of "synthetic marijuana," labeled as K2 or "Spice," among other terms, which elicits psychotropic actions via CB1R activation. Because of structural dissimilarity to THC, the active ingredients of K2/Spice preparations are widely unregulated. The K2/Spice "phenomenon" provides a context for considering whether marijuana-based drugs will truly provide innovative therapeutics or merely perpetuate drug abuse.     

花生四烯乙醇胺的研究进展

花生四烯乙醇胺（arachidonoylethanolamide, anandamide，ANA）是近年来确定的大麻素受体的内源性配基，它主要分布在中枢神经系统、免疫系统及子宫等部位，具有大麻的主要活性成分——Δ⁹-四氢大麻酚（Δ⁹-THC）的药理功能.ANA有两种受体，即脑型受体（CB1）和脾型受体（CB2），它们都是与GTP偶联的跨膜受体，是ANA发挥作用的主要途径.脂肪酸酰胺水解酶（fatty acid amide hydrolase，FAAH）是ANA特异性极高的水解酶，它可以迅速调节ANA在体内的含量，从而发挥特异的生理作用.