Agonist AM251 Research Articles

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen‐related receptor α protein

AM251 is an inverse agonist of the cannabinoid 1 receptor (CB(1)R) that can exert 'off-target' effects in vitro and in CB(1)R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function. The various biological functions of AM251 were measured in CB(1)R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα. AM251 up-regulates EGFR expression and signalling via a novel non-CB(1)R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines.

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

BackgroundK2 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 FindingsJWH-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 Ki 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/SignificanceUnlike 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.

Rimonabant Redux and Strategies to Improve the Future Outlook of CB1 Receptor Neutral‐Antagonist/Inverse‐Agonist Therapies

Rimonabant Redux and Strategies to Improve the Future Outlook of CB1 Receptor Neutral‐Antagonist/Inverse‐Agonist Therapies

The cannabinoid receptor CB1 inverse agonist AM251 potentiates the anxiogenic activity of urocortin I in the basolateral amygdala

The basolateral amygdala is reported to play an important role in the neural bases of emotional processing. Previous studies have shown that injections of urocortin I (UcnI) into the basolateral amygdala (BLA) elicit anxiety-like behaviors in animal models. The present study examined the anxiogenic effects of UcnI administered directly into the BLA of male Sprague-Dawley rats. UcnI was administered at doses of 0.1–10.0 pmol and rats were then placed in an elevated plus maze for 10 min. UcnI reliably decreased the percent time spent in the open arms of the elevated plus maze (EPM) as well as open arm entries. This effect was observed across all doses tested, indicating the induction of anxiety-like behavior. In separate groups of rats, the CB1 inverse agonist AM251 was administered systemically (0.03–3.0 mg/kg IP) or directly into the BLA (0.25–25.0 pmol) and EPM performance assessed. Both routes of AM251 administration produced a reduction in open arm entries and in time spent in the open arms. Moreover, when rats were pretreated with AM251 either systemically or directly into the BLA, the anxiogenic effect of UcnI was potentiated. That is, co-administration of AM251 and UcnI produced a greater suppression of percent time spent in the open arms and open arm entries as compared to UcnI alone. Based on these findings, we propose that urocortin and endocannabinoid signaling are part of an integrated neural axis modulating anxiety states within the basolateral amygdala.This article is part of a Special Issue entitled ‘Anxiety and Depression’.

The antinociceptive potency of N-arachidonoyl-dopamine (NADA) and its interaction with endomorphin-1 at the spinal level

The endogenous N-arachidonoyl-dopamine (NADA) activates both transient receptor potential vanilloid1 (TRPV1) and cannabinoid-1 (CB1) receptors. The goal of this study was to characterize the antinociceptive potential of NADA on inflammatory thermal hyperalgesia in rats at spinal level, and to determine its interaction with endomorphin-1 (EM) at the spinal level.The effects of NADA and EM on thermal hyperalgesia were evaluated in rats with a unilateral hind paw carrageenan-induced inflammation. Intrathecal injection of either EM (0.03–10μg) or NADA (1.5–50μg) caused dose-dependent antihyperalgesia, but NADA was 5.4 times less potent than EM. The antihyperalgesia caused by 15μg NADA was inhibited by the TRPV1 antagonist AMG9810, but not by CB1 antagonist/inverse agonist AM 251, whereas the effect of 50μg NADA was decreased by both drugs. Co-administration of EM with NADA in 1:15 and 1:50 ratios produced a short-lasting potentiation, but isobolographic analysis for the whole investigated period revealed additive interaction between the two endogenous ligands.The results show that both TRPV1 and CB1 receptor activation play a substantial role in the antinociceptive effects of NADA at spinal level, while co-administration of NADA with EM did not show potentiation.

New fat and new neurons: endocannabinoids control neurogenesis in obesity (Commentary on Rivera et al.)

Neurogenesis in the adult brain is a special form of structural plasticity whose function spans from protective responses to brain injury to modulation of memory and emotional processes (Abrous et al., 2005; Holtmaat & Svoboda, 2009). Adult neurogenesis is specific to discrete brain regions, such as the subventricular zone (SVZ) and the subgranular zone of the dentate gyrus (SGZ) of the hippocampus, although recent evidence also suggests that it might be observed in the hypothalamus, and could causally participate in the regulation of energy balance (Kokoeva et al., 2005). Neurogenesis in the adult brain is affected by a plethora of factors, including nutrients derived from the diet and physical exercise (Abrous et al., 2005; Bouret, 2010). In particular, consumption of a high-fat diet and development of obesity might negatively affect neurogenesis, mostly in the hippocampus (Stranahan et al., 2008; Park et al., 2010), thus potentially impacting on mental health. The endocannabinoid system (ECS) is known to regulate energy balance by engaging both central and peripheral mechanisms, and has been investigated as a potential target for the pharmacotherapy of obesity (Pagotto et al., 2006). Moreover, endocannabinoid signaling is known to modulate adult neurogenesis in both physiological and pathophysiological conditions (Aguado et al., 2005, 2007). In the present issue of EJN, Rivera et al., (2011) merged these aspects of the ECS, and investigated, in rats, whether subchronic treatment with the cannabinoid type 1 inverse agonist AM251, which is known to cause a reduction in food intake and body weight, could also lead to changes in cell proliferation in structures such as the SVZ, SGZ, and hypothalamus. Interestingly, although the drug reliably decreased food intake and body weight in both low-fat-fed and high-fat-fed animals, the effects on brain cell proliferation were seen only during exposure to the high-fat diet. This suggests that the AM251-induced modulation of cell proliferation depends upon the types of nutrient (e.g. increased dietary fat) ingested, and not on caloric intake or adiposity changes. Neuroinflammation, which is known to impair neurogenesis (Monje et al., 2003), develops independently of increased adiposity when animals consume a high-fat diet (Posey et al., 2009). Although neuroinflammation was not investigated in the present study, its potential modulation through pharmacological blockade of CB1 could have participated in the described changes. Another possibility is that the treatment impacted differently on hypothalamic–pituitary–adrenal axis activity and consequently glucocorticoid levels, the latter having important effects on cell proliferation and neurogenesis (Stranahan et al., 2008). Interestingly, the authors described a differential response to the treatment in the different areas studied. Whereas AM251 increased cell proliferation in the SGZ, it reduced the number of bromodeoxyuridine-positive cells in the SVZ and hypothalamus of high-fat-fed rats. This could be attributable to different neurogenic cellular pools being present in these areas. However, it could also be the result of an off-target effect of AM251 on vanilloid type 1 receptors, for instance, which has been implicated in the reduction in cell proliferation observed in the SVZ after AM251 administration (Goncalves et al., 2008). Although precursor cell proliferation does not necessarily lead to functional neurogenesis, the findings by Rivera et al. (2011) point to the existence of a functional link between the ECS, high-fat diet exposure, and modulation of brain structural plasticity, adding a novel level of complexity to the role played by the ECS in the modulation of brain function.

The Type 2 Cannabinoid Receptor Regulates Bone Mass and Ovariectomy-Induced Bone Loss by Affecting Osteoblast Differentiation and Bone Formation

The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass and bone turnover but the mechanisms responsible are incompletely understood. In this study we investigated the role that the CB2 pathway plays in bone metabolism using a combination of genetic and pharmacological approaches. Bone mass and turnover were normal in young mice with targeted inactivation of CB2 receptor (CB2(-/-)), but by 12 months of age, they had developed high-turnover osteoporosis with relative uncoupling of bone resorption from bone formation. Primary osteoblasts from CB2(-/-) mice had a reduced capacity to form bone nodules in vitro when compared with cells from wild-type littermates and also had impaired PTH-induced alkaline phosphatase (ALP) activity. The CB2-selective agonist HU308 stimulated bone nodule formation in wild-type osteoblasts but had no effect in CB2(-/-) osteoblasts. Further studies in MC3T3-E1 osteoblast like cells showed that HU308 promoted cell migration and activated ERK phosphorylation, and these effects were blocked by the CB2 selective inverse agonist AM630. Finally, HU308 partially protected against ovariectomy induced bone loss in wild-type mice in vivo, primarily by stimulating bone formation, whereas no protective effects were observed in ovariectomized CB2(-/-) mice. These studies indicate that the CB2 regulates osteoblast differentiation in vitro and bone formation in vivo.

Pre-training anandamide infusion within the basolateral amygdala impairs plus-maze discriminative avoidance task in rats

Endocannabinoids (eCBs) modulate a variety of brain functions via activation of the widely expressed CB1 receptor. One site of high density of this receptor is the basolateral amygdala (BLA), a structure involved in the formation of aversive memories. The activation and blockade of CB1 receptors by systemic or hippocampal drug administrations have been shown to modify memory processing. However, little is known about the role of the BLA endocannabinoid system in aversive memories. Additionally, BLA endocannabinoid transmission seems to be related to emotional states, but the relevance of these effects to memory formation is still unknown. In this study we investigated the effects of the eCB anandamide (AEA) and the CB1 antagonist/inverse agonist AM251 infused into the BLA on the acquisition of an aversive memory task, concomitantly evaluating basal anxiety levels in rats. Male rats received pre-training micro-injection of AEA, AM251 or vehicle bilaterally into the BLA, and were studied with the plus-maze discriminative avoidance task (a paradigm that allows concomitant and independent evaluation of anxiety-like behavior and the memory of an aversive task). Our results showed that AEA into the BLA before training prevented memory retrieval 24h later, as evaluated by exploration of the aversive arm of the maze, while AM251 into the BLA did not interfere with animals’ performance. In addition, AEA had no effect on anxiety-like behavior (as evaluated by open arm exploration and risk assessment), while AM251 induced an anxiogenic effect. Our data indicate an important role of BLA CB1 receptors in aversive memory formation, and suggest that this involvement is not necessarily related to a possible modulation of anxiety states.

Obesity-dependent cannabinoid modulation of proliferation in adult neurogenic regions

Endocannabinoid signalling participates in the control of neurogenesis, especially after brain insults. Obesity may explain alterations in physiology affecting neurogenesis, although it is unclear whether cannabinoid signalling may modulate neural proliferation in obese animals. Here we analyse the impact of obesity by using two approaches, a high-fat diet (HFD, 60% fat) and a standard/low-fat diet (STD, 10% fat), and the response to a subchronic treatment with the cannabinoid receptor type 1 (CB1) inverse agonist AM251 (3 mg/kg) on cell proliferation of two relevant neurogenic regions, namely the subventricular zone in the striatal wall of the lateral ventricle (SVZ) and the subgranular zone of the dentate gyrus (SGZ), and also in the hypothalamus given its role in energy metabolism. We found evidence of an interaction between diet-induced obesity and CB1 signalling in the regulation of cell proliferation. AM251 reduced caloric intake and body weight in obese rats, as well as corrected plasma levels of cholesterol and triglycerides. AM251 is shown, for the first time, to modulate cell proliferation in HFD-obese rats only. We observed an increase in the number of 5-bromo-2-deoxyuridine-labelled (BrdU+) cells in the SGZ, but a decrease in the number of BrdU+ cells in the SVZ and the hypothalamus of AM251-treated HFD rats. These BrdU+ cells expressed the neuron-specific βIII-tubulin. These results suggest that obesity may impact cell proliferation in the brain selectively, and provide support for a role of CB1 signalling regulation of neurogenesis in response to obesity.

Dynamic Mass Redistribution as a Means to Measure and Differentiate Signaling via Opioid and Cannabinoid Receptors

Classically, G protein-coupled receptor activation by a ligand has been viewed as producing a defined response such as activation of a G protein, activation or inhibition of adenylyl cyclase, or stimulation of phospholipase C and/or alteration in calcium flux. Newer concepts of ligand-directed signaling recognize that different ligands, ostensibly acting at the same receptors, may induce different downstream effects, complicating the selection of a screening assay. Dynamic mass redistribution (DMR), a label-free technology that uses light to measure ligand-induced changes in the mass of cells proximate to the biosensor, provides an integrated cellular response comprising multiple pathways and cellular events. Using DMR, signals induced by opioid or cannabinoid agonists in cells transfected with these receptors were blocked by pharmacologically appropriate receptor antagonists as well as by pertussis toxin. Differences among compounds in relative potencies at DMR versus ligand-stimulated GTPγS or receptor binding endpoints, suggesting functional selectivity, were observed. Preliminary evidence indicates that inhibitors of intermediate steps in the cell signaling cascade, such as receptor recycling inhibitors, mitogen-activated protein kinase kinase/p38 mitogen-activated protein kinase inhibitors, or cytoskeletal disruptors, altered or attenuated the cannabinoid-induced response. Notable is the finding that mitogen-activated protein kinase kinase 1/2 inhibitors attenuated signaling induced by the cannabinoid type 2 receptor inverse agonist AM630 but not that stimulated by the agonist CP 55,940. Thus, DMR has the potential to not only identify ligands that activate a given G protein-coupled receptor, but also ascertain the signaling pathways engaged by a specific ligand, making DMR a useful tool in the identification of biased ligands, which may ultimately exhibit improved therapeutic profiles.

Cannabinoid‐induced delayed gastric emptying is selectively increased upon intermittent administration in the rat: role of CB1 receptors

Cannabinoids acutely administered depress central, cardiovascular and gastrointestinal functions. These effects might be modified upon repeated administration. Compared to the effects induced by daily administration, those induced by intermittent administration are less known. The effect of intermittent treatment with the CB1/CB2 cannabinoid agonist WIN55,212-2 (WIN) was studied in the rat. Male rats received saline, vehicle or WIN at 0.5 (low-WIN) or 5 (high-WIN) mg kg(-1) week(-1) for 4 weeks. WIN effects on the central nervous system (cannabinoid tetrad tests), cardiovascular function and gastrointestinal motor function were evaluated after the first and last doses, and, where appropriate, 1 week after the last dose. To determine the involvement of CB1 receptors in the chronic effect of WIN, the CB1 receptor antagonist/inverse agonist AM251 (1 mg kg(-1)) was used. High- (but not low-) WIN induced the four signs of the cannabinoid tetrad, and reduced gastrointestinal motility, but did not alter cardiovascular parameters. Upon chronic intermittent administration, tolerance did not clearly develop to WIN effects. Quite the opposite, depression of gastric emptying was intensified. No effect was long-lasting. Repeated administration of AM251 was more efficacious than single administration to block WIN chronic central effects, but the opposite occurred regarding lower intestinal motility. Upon intermittent administration, hypersensitization may develop to some effects (particularly delayed gastric emptying) induced by cannabinoid agonists. CB1 antagonists/inverse agonists may show different efficacy upon repeated or single administration to block cannabinoid-induced central and gastrointestinal effects. Thus, cannabinoid effects are dependent on the pattern of drug administration.

Estradiol Decreases Cortical Reactive Astrogliosis after Brain Injury by a Mechanism Involving Cannabinoid Receptors

The neuroactive steroid estradiol reduces reactive astroglia after brain injury by mechanisms similar to those involved in the regulation of reactive gliosis by endocannabinoids. In this study, we have explored whether cannabinoid receptors are involved in the effects of estradiol on reactive astroglia. To test this hypothesis, the effects of estradiol, the cannabinoid CB1 antagonist/inverse agonist AM251, and the cannabinoid CB2 antagonist/inverse agonist AM630 were assessed in the cerebral cortex of male rats after a stab wound brain injury. Estradiol reduced the number of vimentin immunoreactive astrocytes and the number of glial fibrillary acidic protein immunoreactive astrocytes in the proximity of the wound. The effect of estradiol was significantly inhibited by the administration of either CB1 or CB2 receptor antagonists. The effect of estradiol may be in part mediated by alterations in endocannabinoid signaling because the hormone increased in the injured cerebral cortex the messenger RNA levels of CB2 receptors and of some of the enzymes involved in the synthesis and metabolism of endocannabinoids. These findings suggest that estradiol may decrease reactive astroglia in the injured brain by regulating the activity of the endocannabinoid system.

A Pilot Study into the Effects of the CB1 Cannabinoid Receptor Agonist WIN55,212-2 or the Antagonist/Inverse Agonist AM251 on Sleep in Rats

The plant cannabinoid Δ9-tetrahydrocannabinol and the endocannabinoid anandamide increase the amount of sleep via a CB1 receptor mediated mechanism. Here, we explored the use of a novel electroencephalogram (EEG) recording device based on wireless EEG microchip technology (Neurologger) in freely-moving rats, and its utility in experiments of cannabinoids-induced alterations of EEG/vigilance stages. EEG was recorded through epidural electrodes placed above pre-frontal and parietal cortex (overlaying the dorsal hippocampus). As cannabinoids, we acutely administered the full synthetic CB1 receptor agonist, WIN55,212-2 (1 mg/kg), and the antagonist/inverse agonist, AM251 (2 mg/kg), either alone or together through the intraperitoneal route. WIN55,212-2 increased the total amount of NREM sleep and the length of each NREM bout, but this was unlikely due to CB1 receptor activation since it was not prevented by AM251. However, WIN55,212-2 also lowered overall EEG spectral power especially in theta and alpha frequency bands during wakefulness and NREM sleep, and this effect was reversed by AM251. The antagonist/inverse agonist caused no sleep alterations by itself and moderately increased spectral power in Theta, alpha and beta frequency bands during NREM sleep when administered on its own. Implications of endocannabinoid modulation of the sleep-wake cycle and its possible interactions with other transmitter systems are considered.

Expression of the cannabinoid system in muscle: effects of a high-fat diet and CB1 receptor blockade

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) β and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.

Residual effects of focal brain ischaemia upon cannabinoid CB1 receptor density and functionality in female rats

Ischaemic insult results in short-term changes in cannabinoid-1 (CB1) receptor expression in the brain, but it is not known whether long-term changes occur, which could potentially mean a change in the intrinsic ability of the brain to withstand new ischaemic episodes. In this study, we have investigated the expression and functionality of CB1 receptors in coronal brain slices obtained from ovariectomised female rats 46days after middle cerebral artery occlusion (MCAO). The animals were treated with either 17ß-oestradiol or placebo pellets 6h after MCAO and thereafter housed either in isolated or enriched environments. [3H]CP55,940 autoradiography indicated no significant effect of 17ß-oestradiol treatment or housing environment upon CB1 receptor densities. There was, however, a modest but significant decrease in the CB1 receptor density on the ipsilateral side relative to the contralateral side in the frontal cortex, parietal cortex, CA1–CA3 regions of the hippocampus, thalamus and hypothalamus. CB1 receptor functionality was assessed by measurement of basal and CP55,940-stimulated [35S]GTPγS autoradiography. In the frontal cortex, parietal cortex, CA1–CA3 regions of the hippocampus and dentate gyrus, a robust stimulation, blocked by the CB1 receptor inverse agonist AM251, was seen. There were no significant changes in the response to CP55,940 with respect either to the 17ß-oestradiol treatment, housing environment or MCAO. Our results reveal that although there are modest long-term decreases in ipsilateral CB1 receptor densities following MCAO in female rats, these decreases do not result in a functional CB1 receptor deficit.

Effects of cannabinoids on neuropeptide Y and β-endorphin expression in the rat hypothalamic arcuate nucleus

The control of appetite and satiety is extremely complex and involves a balance between neurotransmitters and neuropeptides to stimulate and/or inhibit feeding behaviour. The effect of cannabinoids on food intake is well established, but little is known about the mechanism of action underlying their activity. In the present report, the effect of pharmacological manipulation of the cannabinoid receptor on the expression of hypothalamic neuropeptides is investigated. We used an immunohistochemical approach to examine the effect of intracerebroventricular administration of the cannabinoid receptor agonist WIN55,212-2 and the inverse agonist AM251 on neuropeptide Y (NPY) and the β-endorphin (β-end) neuronal hypothalamic systems. Double immunohistochemistry (c-fos/β-end) was used to assess the number of β-end neurons activated by the cannabinoid agonist. The present results showed that 1μg WIN 55,212-2 increases β-end immunoreactivity within the arcuate nucleus while no significant changes were noted in the NPY-immunoreactive nerve fibres network in comparison to the control group. Injection of 1μg AM251 decreases both NPY and β-end immunoreactivity within the arcuate nucleus. The number of β-end neurons exhibiting c-fos increased significantly in WIN 55,212-2 compared with the control group. These results suggest that cannabinoids affect the expression of hypothalamic neuropeptides, notably the NPY and β-end systems, which may have implications in the orexigenic action of cannabinoids.

Attenuation of food intake in chicks by an inverse agonist of cannabinoid receptor 1 administered by either injection or ingestion in hydrocolloid carriers

The cannabinoid receptor (CB1) was studied primarily in mammals where it was found to comprise a link between reward processes and addictive behavior such as food consumption. The purpose of this study was twofold: first to characterize the effect of the chicken CB1 receptor inverse agonist AM251 on food intake, and second, to establish a stress-free approach for application of AM251 to birds using hydrocolloid carriers, which can be mixed with food. A single administration of AM251 by intravenous injection (at 0.85 or 5mgkg−1BW) or by ingestion of hydrocolloid carriers entrapping AM251 at a concentration of 5mgkg−1BW led to a transient attenuation of food intake. The consequent reduced cumulative food intake and BW were observed in the treated chicks for at least 7h post-administration, with no gender differences. Circulating levels of AM251, assessed by LC–MS following 48h of continuous feeding with hydrocolloid carriers containing 50mg AM251kg−1BWday−1, were physiologically significant at 186±73pmolml−1. It is concluded that unlike some other factors, which act differently in birds compared to mammals such as ghrelin, CB1 inverse agonists attenuate food intake in chicks similar to its effect in mammals. In addition, the new approach for administration of AM251 to birds in hydrocolloid carriers could provide a simple and stress-free tool for prolonged studies of this control mechanism in birds.

Genome-wide Expression Profiling Revealed Peripheral Effects of Cannabinoid Receptor 1 Inverse Agonists in Improving Insulin Sensitivity and Metabolic Parameters

Inhibition of cannabinoid receptor 1 (CB1) has shown efficacy in reducing body weight and improving metabolic parameters, with the effects correlating with target engagement in the brain. The peripheral effects of inhibiting the CB1 receptor has been appreciated through studies in diet-induced obese and liver-specific CB1 knockout mice. In this article, we systematically investigated gene expression changes in peripheral tissues of diet-induced obese mice treated with the CB1 inverse agonist AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide]. CB1 receptor inhibition led to down-regulation of genes within the de novo fatty acid and cholesterol synthetic pathways, including sterol regulatory element binding proteins 1 and 2 and their downstream targets in both liver and adipose tissue. In addition, genes involved in fatty acid beta-oxidation were up-regulated with AM251 treatment, probably through the activation of peroxisome proliferator-activated receptor alpha (PPARalpha). In adipose tissue, CB1 receptor inhibition led to the down-regulation of genes in the tumor necrosis factor alpha signal transduction pathway and possibly to the activation of PPARgamma, both of which would result in improved insulin sensitivity.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Physical and functional interaction between CB1 cannabinoid receptors and β2‐adrenoceptors

The CB(1) cannabinoid receptor and the beta(2)-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells. Physical interactions between CB(1) receptors and beta(2)-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling. Physical interactions between CB(1) receptors and beta(2)-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of beta(2)-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB(1) receptors. Co-expression altered the signalling properties of CB(1 )receptors, resulting in increased Galpha(i)-dependent ERK phosphorylation, but decreased non-Galpha(i)-mediated CREB phosphorylation. The CB(1) receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated beta(2)-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB(1) receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB(1) receptors and beta(2)-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the beta(2)-adrenoceptor-pERK response. A complex interaction was demonstrated between CB(1) receptors and beta(2)-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.