The role of central CB2 cannabinoid receptors on food intake in neonatal chicks

The endocannabinoids (ECBs) have diverse physiological functions including the regulation of food intake and metabolism. In mammals, ECBs regulate feeding primarily through the CB1 receptors within the brain whereas the CB2 receptors are primarily involved in the regulation of immune function by direct action on peripheral immune cells and central glia. The central effect of ECBs on feeding behavior has not been studied in non-mammalian species. Therefore, the present study investigated the effect of CB65, a selective CB2 receptors agonist, on food intake in the neonatal chicks. In addition, the effect of astressin, a CRF receptor antagonist, on CB65-induced food intake was also investigated. Intracerebroventricular injection of the CB65 (1.25 μg) increased the food intake at 30- and 60-min post-injection significantly as compared to the control group. Pretreatment with a selective CB2 receptor antagonist, AM630, but not astressin, significantly attenuated the CB65-induced food intake. These results suggested that CB2 receptor agonists act on the brain to induce food intake.     

Tetrazole-biarylpyrazole derivatives as cannabinoid CB1 receptor antagonists

Cannabinoid CB1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed a new series of tetrazole-biarylpyrazoles. The various analogues were efficiently prepared and bio-assayed for binding to cannabinoid CB1 receptor. Six of the new compounds which displayed high in vitro CB1 binding affinities were assayed for binding to CB2 receptor. Noticeably, cyclopentyl-tetrazole (9a) demonstrated good binding affinity and selectivity for CB1 receptor (IC(50)=11.6nM and CB2/CB1=366).     

New natural noncannabinoid ligands for cannabinoid type-2 (CB2) receptors

Since the discovery that Delta 9-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB2) receptors. Cannabinoid type-1 (CB1) and CB2 receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB2 receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB2-directed drugs.     

UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis

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.     

Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors

Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.     

Molecular biology of cannabinoid receptors

During the last decade, research on the molecular biology and genetics of cannabinoid receptors has led to a remarkable progress in understanding of the endogenous cannabinoid system, which functions in a plethora of physiological processes in the animal. At present, two types of cannabinoid receptors have been cloned from many vertebrates, and three endogenous ligands (the endocannabinoids arachidonoyl ethanolamide, 2-arachidonoyl glycerol and 2-arachidonoyl-glycerol ether) have been characterized. Cannabinoid receptor type 1 (CB(1)) is expressed predominantly in the central and peripheral nervous system, while cannabinoid receptor type 2 (CB(2)) is present almost exclusively in immune cells. Cannabinoid receptors have not yet been cloned from invertebrates, but binding proteins for endocannabinoids, endocannabinoids and metabolic enzyme activity have been described in a variety of invertebrates except for molting invertebrates such as Caenorhabditis elegans and Drosophila. In the central nervous system of mammals, there is strong evidence emerging that the CB(1) and its ligands comprise a neuromodulatory system functionally interacting with other neurotransmitter systems. Furthermore, the presynaptic localization of CB(1) together with the results obtained from electrophysiological experiments strengthen the notion that in cerebellum and hippocampus and possibly in other regions of the central nervous system, endocannabinoids may act as retrograde messengers to suppress neurotransmitter release at the presynaptic site. Many recent studies using genetically modified mouse lines which lack CB(1) and/or CB(2) finally could show the importance of cannabinoid receptors in animal physiology and will contribute to unravel the full complexity of the cannabinoid system.     

Coevolution between cannabinoid receptors and endocannabinoid ligands

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.     

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups     

Cannabinoid receptors and their ligands

There are at least two types of cannabinoid receptors, CB(1) and CB(2), both coupled to G proteins. CB(1) receptors exist primarily on central and peripheral neurons, one of their functions being to modulate neurotransmitter release. CB(2) receptors are present mainly on immune cells. Their roles are proving more difficult to establish but seem to include the modulation of cytokine release. Endogenous agonists for cannabinoid receptors (endocannabinoids) have also been discovered, the most important being arachidonoyl ethanolamide (anandamide), 2-arachidonoyl glycerol and 2-arachidonyl glyceryl ether. Other endocannabinoids and cannabinoid receptor types may also exist. Although anandamide can act through CB(1) and CB(2) receptors, it is also a vanilloid receptor agonist and some of its metabolites may possess yet other important modes of action. The discovery of the system of cannabinoid receptors and endocannabinoids that constitutes the "endocannabinoid system" has prompted the development of CB(1)- and CB(2)-selective agonists and antagonists/inverse agonists. CB(1)/CB(2) agonists are already used clinically, as anti-emetics or to stimulate appetite. Potential therapeutic uses of cannabinoid receptor agonists include the management of multiple sclerosis/spinal cord injury, pain, inflammatory disorders, glaucoma, bronchial asthma, vasodilation that accompanies advanced cirrhosis, and cancer. Following their release onto cannabinoid receptors, endocannabinoids are removed from the extracellular space by membrane transport and then degraded by intracellular enzymic hydrolysis. Inhibitors of both these processes have been developed. Such inhibitors have therapeutic potential as animal data suggest that released endocannabinoids mediate reductions both in inflammatory pain and in the spasticity and tremor of multiple sclerosis. So too have CB(1) receptor antagonists, for example for the suppression of appetite and the management of cognitive dysfunction or schizophrenia.     

Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes

To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.     

Cannabinoid signalling

After their discovery, the two known cannabinoid receptors, CB(1) and CB(2), have been the focus of research into the cellular signalling mechanisms of cannabinoids. The initial assessment, mainly derived from expression studies, was that cannabinoids, via G(i/o) proteins, negatively modulate cyclic AMP levels, and activate inward rectifying K(+) channels. Recent findings have complicated this assessment on different levels: (1) cannabinoids include a wide range of compounds with varying profiles of affinity and efficacy at the known CB receptors, and these profiles do not necessarily match their biological activity; (2) CB receptors appear to be intrinsically active and possibly coupled to more than one type of G protein; (3) CB receptor signalling mechanisms are diverse and dependent on the system studied; (4) cannabinoids have other targets than CB receptors. The aim of this mini review is to discuss the current literature regarding CB receptor signalling pathways. These include regulation of adenylyl cyclase, MAP kinase, intracellular Ca(2+), and ion channels. In addition, actions of cannabinoids that are not mediated by CB(1) or CB(2) receptors are discussed.     

Controlled downregulation of the cannabinoid CB1 receptor provides a promising approach for the treatment of obesity and obesity-derived type 2 diabetes

Increased activity of the endocannabinoid system has emerged as a pathogenic factor in visceral obesity, which is a risk factor for type 2 diabetes mellitus (T2DM). The endocannabinoid system is composed of at least two G-protein-coupled receptors (GPCRs), the cannabinoid receptor type 1 (CB1), and the cannabinoid receptor type 2 (CB2). Downregulation of CB1 activity in rodents and humans has proven efficacious to reduce food intake, abdominal adiposity, fasting glucose levels, and cardiometabolic risk factors. Unfortunately, downregulation of CB1 activity by universally active CB1 inverse agonists has been found to elicit psychiatric side effects, which led to the termination of using globally active CB1 inverse agonists to treat diet-induced obesity. Interestingly, preclinical studies have shown that downregulation of CB1 activity by CB1 neutral antagonists or peripherally restricted CB1 inverse agonists provided similar anorectic effects and metabolic benefits without psychiatric side effects seen in globally active CB1 inverse agonists. Furthermore, downregulation of CB1 activity may ease endoplasmic reticulum and mitochondrial stress which are contributors to obesity-induced insulin resistance and type 2 diabetes. This suggests new approaches for cannabinoid-based therapy in the management of obesity and obesity-related metabolic disorders including type 2 diabetes.     

Cannabinoid pharmacology in the cardiovascular system: potential protective mechanisms through lipid signalling

Cannabinoids include not only plant-derived compounds (of which delta9-tetrahydrocannabinol is the primary psychoactive ingredient of cannabis), but also synthetic agents and endogenous substances termed endocannabinoids which include anandamide (2-arachidonoylethanolamide) and 2-arachidonoylglycerol. Cannabinoids act on specific, G-protein-coupled, receptors which are currently divided into two types, CB1 and CB2. Relatively selective agonists and antagonists for these receptors have been developed, although one agent (SR141716A) widely used as an antagonist at CB1 receptors has non-cannabinoid receptor-mediated effects at concentrations which are often used to define the presence of the CB1 receptor. Both cannabinoid receptors are primarily coupled to Gi/o proteins and act to inhibit adenylyl cyclase. Stimulation of CB1 receptors also modulates the activity of K+ and Ca2+ channels and of protein kinase pathways including protein kinase B (Akt) which might mediate effects on apoptosis. CB, receptors may activate the extracellular signal-regulated kinase cascade through ceramide signalling. Cannabinoid actions on the cardiovascular system have been widely interpreted as being mediated by CB1 receptors although there are a growing number of observations, particularly in isolated heart and blood vessel preparations, that suggest that other cannabinoid receptors may exist. Interestingly, the currently identified cannabinoid receptors appear to be related to a wider family of lipid receptor, those for the lysophospholipids, which are also linked to Gi/o protein signalling. Anandamide also activates vanilloid VR1 receptors on sensory nerves and releases the vasoactive peptide, calcitonin gene-related peptide (CGRP), which brings about vasodilatation through its action on CGRP receptors. Current evidence suggests that endocannabinoids have important protective roles in pathophysiological conditions such as shock and myocardial infarction. Therefore, their cardiovascular effects and the receptors mediating them are the subject of increasing investigative interest.     

Astrocytes in endocannabinoid signalling

Astrocytes are emerging as integral functional components of synapses, responding to synaptically released neurotransmitters and regulating synaptic transmission and plasticity. Thus, they functionally interact with neurons establishing tripartite synapses: a functional concept that refers to the existence of communication between astrocytes and neurons and its crucial role in synaptic function. Here, we discuss recent evidence showing that astrocytes are involved in the endocannabinoid (ECB) system, responding to exogenous cannabinoids as well as ECBs through activation of type 1 cannabinoid receptors, which increase intracellular calcium and stimulate the release of glutamate that modulates synaptic transmission and plasticity. We also discuss the consequences of ECB signalling in tripartite synapses on the astrocyte-mediated regulation of synaptic function, which reveal novel properties of synaptic regulation by ECBs, such as the spatially controlled dual effect on synaptic strength and the lateral potentiation of synaptic efficacy. Finally, we discuss the potential implications of ECB signalling for astrocytes in brain pathology and animal behaviour.     

New metabolically stable fatty acid amide ligands of cannabinoid receptors: Synthesis and receptor affinity studies

We investigated the structure-activity relationships for the interactions of fatty acid amide analogs of the endocannabinoid anandamide with human recombinant cannabinoid receptors. Thirty-five novel fatty acid amides were synthesized using five different types of acyl chains and 11 different aromatic amine 'heads.' Although none of the new compounds was a more potent ligand than anandamide, we identified three amine groups capable of improving the metabolic stability of arachidonoylamides and their CB(1)/CB(2) selectivity ratio to over 20-fold, and several aromatic amines capable of improving the affinity of short chain or monosaturated fatty acids for cannabinoid CB(1) receptors. For the first time a tertiary amide of arachidonic acid was found to possess moderate affinity (K(i)=300 nM) for cannabinoid CB(1), but not CB(2), receptors.     

Formation and fate of extranuclear chromatin bodies in Tetrahymena.

The fate of extranuclear chromatin bodies (ECBs) formed by exclusion of macronuclear material at the time of karyokinesis was followed quantitatively in Tetrahymena pyriformis strain GL-I. In a logarithmic growth phase culture, 51% of the dividing cells produced one (43%) or more (8%) ECBs. Most of these gradually disappear before the next cell division, but approximately 13% are retained and carried into subsequent cell cycles. The random distribution of ECBs into anterior or posterior daughter cells, their staining and morphological characteristics, and their rapid loss in cells in starvation medium, all indicate that ECBs play no more of a role in cellular activity than that of an internally produced food vacuole.     

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2–OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2–OT column can be used to determine the binding affinities (K_i values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2–OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.     

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.