Anxiety Modulation by Cannabinoids-The Role of Stress Responses and Coping.

Endocannabinoids were implicated in a variety of pathological conditions including anxiety and are considered promising new targets for anxiolytic drug development. The optimism concerning the potentials of this system for anxiolysis is probably justified. However, the complexity of the mechanisms affected by endocannabinoids, and discrepant findings obtained with various experimental approaches makes the interpretation of research results difficult. Here, we review the anxiety-related effects of the three main interventions used to study the endocannabinoid system: pharmacological agents active at endocannabinoid-binding sites present on both the cell membrane and in the cytoplasm, genetic manipulations targeting cannabinoid receptors, and function-enhancers represented by inhibitors of endocannabinoid degradation and transport. Binding-site ligands provide inconsistent findings probably because they activate a multitude of mechanisms concomitantly. More robust findings were obtained with genetic manipulations and particularly with function enhancers, which heighten ongoing endocannabinoid activation rather than affecting all mechanisms indiscriminately. The enhancement of ongoing activity appears to ameliorate stress-induced anxiety without consistent effects on anxiety in general. Limited evidence suggests that this effect is achieved by promoting active coping styles in critical situations. These findings suggest that the functional enhancement of endocannabinoid signaling is a promising drug development target for stress-related anxiety disorders.

The neuropharmacology of cannabinoid receptor ligands in central signaling pathways.

The endocannabinoid system is a complex neuronal system involved in a number of biological functions, like attention, anxiety, mood, memory, appetite, reward, and immune responses. It is at the centre of scientific interest, which is driven by therapeutic promise of certain cannabinoid ligands and the changing legalization of herbal cannabis in many countries. The endocannabinoid system is a modulatory system, with endocannabinoids as retrograde neurotransmitters rather than direct neurotransmitters. Neuropharmacology of cannabinoid ligands in the brain can therefore be understood in terms of their modulatory actions through other neurotransmitter systems. The CB1 receptor is chiefly responsible for effects of endocannabinoids and analogous ligands in the brain. An overview of the neuropharmacology of several cannabinoid receptor ligands, including endocannabinoids, herbal cannabis and synthetic cannabinoid receptor ligands is given in this review. Their mechanism of action at the endocannabinoid system is described, mainly in the brain. In addition, effects of cannabinoid ligands on other neurotransmitter systems will also be described, such as dopamine, serotonin, glutamate, noradrenaline, opioid, and GABA. In light of this, therapeutic potential and adverse effects of cannabinoid receptor ligands will also be discussed.

Exploration of Multiverse Activities of Endocannabinoids in Biological Systems.

Over the last 25 years, the human endocannabinoid system (ECS) has come into the limelight as an imperative neuro-modulatory system. It is mainly comprised of endogenous cannabinoid (endocannabinoid), cannabinoid receptors and the associated enzymes accountable for its synthesis and deterioration. The ECS plays a proven role in the management of several neurological, cardiovascular, immunological, and other relevant chronic conditions. Endocannabinoid or endogenous cannabinoid are endogenous lipid molecules which connect with cannabinoid receptors and impose a fashionable impact on the behavior and physiological processes of the individual. Arachidonoyl ethanolamide or Anandamide and 2-arachidonoyl glycerol or 2-AG were the endocannabinoid molecules that were first characterized and discovered. The presence of lipid membranes in the precursor molecules is the characteristic feature of endocannabinoids. The endocannabinoids are released upon rapid enzymatic reactions into the extracellular space via activation through G-protein coupled receptors, which is contradictory to other neurotransmitter that are synthesized beforehand, and stock up into the synaptic vesicles. The current review highlights the functioning, synthesis, and degradation of endocannabinoid, and explains its functioning in biological systems.

TRPV1 Activation by Anandamide via a Unique Lipid Pathway.

The capsaicin receptor, transient receptor potential vanilloid type 1 (TRPV1), is a polymodal channel that has been implicated in the perception of pain and can be modulated by a variety of cannabinoid ligands. Here we report TRPV1 channel activation by the endocannabinoid, anandamide (AEA), in a unique, peripheral binding site via extended MD simulations. These results aim to expand the understanding of TRPV1 and assist in the development of new TRPV1 modulators.

Cannabis, the Endocannabinoid System and Immunity-the Journey from the Bedside to the Bench and Back.

The Cannabis plant contains numerous components, including cannabinoids and other active molecules. The phyto-cannabinoid activity is mediated by the endocannabinoid system. Cannabinoids affect the nervous system and play significant roles in the regulation of the immune system. While Cannabis is not yet registered as a drug, the potential of cannabinoid-based medicines for the treatment of various conditions has led many countries to authorize their clinical use. However, the data from basic and medical research dedicated to medical Cannabis is currently limited. A variety of pathological conditions involve dysregulation of the immune system. For example, in cancer, immune surveillance and cancer immuno-editing result in immune tolerance. On the other hand, in autoimmune diseases increased immune activity causes tissue damage. Immuno-modulating therapies can regulate the immune system and therefore the immune-regulatory properties of cannabinoids, suggest their use in the therapy of immune related disorders. In this contemporary review, we discuss the roles of the endocannabinoid system in immunity and explore the emerging data about the effects of cannabinoids on the immune response in different pathologies. In addition, we discuss the complexities of using cannabinoid-based treatments in each of these conditions.

Endocannabinoid system in neurodegenerative disorders.

Most neurodegenerative disorders (NDDs) are characterized by cognitive impairment and other neurological defects. The definite cause of and pathways underlying the progression of these NDDs are not well-defined. Several mechanisms have been proposed to contribute to the development of NDDs. These mechanisms may proceed concurrently or successively, and they differ among cell types at different developmental stages in distinct brain regions. The endocannabinoid system, which involves cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), endogenous cannabinoids and the enzymes that catabolize these compounds, has been shown to contribute to the development of NDDs in several animal models and human studies. In this review, we discuss the functions of the endocannabinoid system in NDDs and converse the therapeutic efficacy of targeting the endocannabinoid system to rescue NDDs.

Endocannabinoid system in sexual motivational processes: Is it a novel therapeutic horizon?

The endocannabinoid system (ECS), which is composed of the cannabinoid receptors types 1 and 2 (CB1 and CB2) for marijuana's psychoactive ingredient Δ9-tetrahydrocannabinol (Δ9-THC), the endogenous ligands (AEA and 2-AG) and the enzymatic systems involved in their biosynthesis and degradation, recently emerged as important modulator of emotional and non-emotional behaviors. For centuries, in addition to its recreational actions, several contradictory claims regarding the effects of Cannabis use in sexual functioning and behavior (e.g. aphrodisiac vs anti-aphrodisiac) of both sexes have been accumulated. The identification of Δ9-THC and later on, the discovery of the ECS have opened a potential therapeutic target for sexual dysfunctions, given the partial efficacy of current pharmacological treatment. In agreement with the bidirectional modulation induced by cannabinoids on several behavioral responses, the endogenous cannabinoid AEA elicited biphasic effects on sexual behavior as well. The present article reviews current available knowledge on herbal, synthetic and endogenous cannabinoids with respect to the modulation of several aspects of sexuality in preclinical and human studies, highlighting their therapeutic potential.

Dietary DHA reduces downstream endocannabinoid and inflammatory gene expression and epididymal fat mass while improving aspects of glucose use in muscle in C57BL/6J mice.

OBJECTIVES: Endocannabinoid system (ECS) overactivation is associated with increased adiposity and likely contributes to type 2 diabetes risk. Elevated tissue cannabinoid receptor 1 (CB1) and circulating endocannabinoids (ECs) derived from the n-6 polyunsaturated acid (PUFA) arachidonic acid (AA) occur in obese and diabetic patients. Here we investigate whether the n-3 PUFA docosahexaenoic acid (DHA) in the diet can reduce ECS overactivation (that is, action of ligands, receptors and enzymes of EC synthesis and degradation) to influence glycemic control. This study targets the ECS tonal regulation of circulating glucose uptake by skeletal muscle as its primary end point. DESIGN: Male C57BL/6J mice were fed a semipurified diet containing DHA or the control lipid. Serum, skeletal muscle, epididymal fat pads and liver were collected after 62 and 118 days of feeding. Metabolites, genes and gene products associated with the ECS, glucose uptake and metabolism and inflammatory status were measured. RESULTS: Dietary DHA enrichment reduced epididymal fat pad mass and increased ECS-related genes, whereas it reduced downstream ECS activation markers, indicating that ECS activation was diminished. The mRNA of glucose-related genes and proteins elevated in mice fed the DHA diet with increases in DHA-derived and reductions in AA-derived EC and EC-like compounds. In addition, DHA feeding reduced plasma levels of various inflammatory cytokines, 5-lipoxygenase-dependent inflammatory mediators and the vasoconstrictive 20-HETE. CONCLUSIONS: This study provides evidence that DHA feeding altered ECS gene expression to reduce CB1 activation and reduce fat accretion. Furthermore, the DHA diet led to higher expression of genes associated with glucose use by muscle in mice, and reduced those associated with systemic inflammatory status.

Metabolism of endocannabinoids.

Endocannabinoids belong to a group of ester, ether and amide derivatives of fatty acids, which are endogenous ligands of receptors CB1, CB2, TRPV1 and GPR55 that are included in the endocannabinoid system of the animal organism. The best known endocannabinoids are: N-arachidonylethanolamide called anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They occur in all organisms, and their highest level is observed in the brain. In this review the mechanisms of synthesis and degradation of both AEA and 2-AG are shown. Endocannabinoids are synthesized from phospholipids (mainly phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) located in the cell membrane. As a result of arachidonic acid transfer from phosphatidylcholine to phosphatidylethanolamine, N-arachidonoyl phosphatidylethanolamine is formed, which is hydrolyzed to AEA by phospholipase D, C and A2. However, 2-AG is formed during the hydrolysis of phosphatidylinositol catalyzed mainly by DAGL. The primary role of endocannabinoids is the activation of cannabinoid receptors. Both AEA and 2-AG are primarily agonists of the CB1 receptor and to a lower degree CB2 and TRPV1r eceptors, but 2-AG has stronger affinity for these receptors. Through activation of receptors, endocannabinoids affect cellular metabolism and participate in the metabolic processes by receptor-independent pathways. Endocannabinoids which are not bound to the receptors are degraded. The main enzymes responsible for the hydrolysis of AEA and 2-AG are FAAH and MAGL, respectively. Apart from hydrolytic degradation, endocannabinoids may also be oxidized by cyclooxygenase-2, lipoxygenases, and cytochrome P450. It has been shown that the metabolites of both endocannabinoids also have biological significance.

Effects of activation of endocannabinoid system on myocardial metabolism.

Endocannabinoids exert their effect on the regulation of energy homeostasis via activation of specific receptors. They control food intake, secretion of insulin, lipids and glucose metabolism, lipid storage. Long chain fatty acids are the main myocardial energy substrate. However, the heart exerts enormous metabolic flexibility emphasized by its ability to utilzation not only fatty acids, but also glucose, lactate and ketone bodies. Endocannabinoids can directly act on the cardiomyocytes through the CB1 and CB2 receptors present in cardiomyocytes. It appears that direct activation of CB1 receptors promotes increased lipogenesis, pericardial steatosis and bioelectrical dysfunction of the heart. In contrast, stimulation of CB2 receptors exhibits cardioprotective properties, helping to maintain appropriate amount of ATP in cardiomyocytes. Furthermore, the effects of endocannabinoids at both the central nervous system and peripheral tissues, such as liver, pancreas, or adipose tissue, resulting indirectly in plasma availability of energy substrates and affects myocardial metabolism. To date, there is little evidence that describes effects of activation of the endocannabinoid system in the cardiovascular system under physiological conditions. In the present paper the impact of metabolic diseases, i. e. obesity and diabetes, as well as the cardiovascular diseases - hypertension, myocardial ischemia and myocardial infarction on the deregulation of the endocannabinoid system and its effect on the metabolism are described.

Blockade of monoacylglycerol lipase inhibits oligodendrocyte excitotoxicity and prevents demyelination in vivo.

The endocannabinoids 2-araquidonoylglycerol (2-AG) and anandamide (AEA) are bioactive lipids crucially involved in the regulation of brain function in basal and pathological conditions. Blockade of endocannabinoid metabolism has emerged as a promising therapeutic strategy for inflammatory diseases of the central nervous system, including myelin disorders such as multiple sclerosis. Nevertheless, the biological actions of endocannabinoid degradation inhibitors in oligodendrocytes and white matter tracts are still ill defined. Here we show that the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 suppressed cell death by mild activation of AMPA receptors in oligodendrocytes in vitro, an effect that was mimicked by MAGL substrate 2-AG and by the second major endocannabinoid AEA, in a concentration-dependent manner, whereas inhibition of the AEA metabolizing enzyme fatty acid amide hydrolase with URB597 was devoid of effect. Pharmacological experiments suggested that oligodendrocyte protection from excitotoxicity resulting from MAGL blockade involved the activation of cannabinoid CB1 receptors and the reduction of AMPA-induced cytosolic calcium overload, mitochondrial membrane depolarization, and production of reactive oxygen species. Administration of JZL184 under a therapeutic regimen decreased clinical severity, prevented demyelination, and reduced inflammation in chronic experimental autoimmune encephalomyelitis. Furthermore, MAGL inactivation robustly preserved myelin integrity and suppressed microglial activation in the cuprizone-induced model of T-cell-independent demyelination. These findings suggest that MAGL blockade may be a useful strategy for the treatment of immune-dependent and -independent damage to the white matter.

The endocannabinoid anandamide affects the synthesis of human syncytiotrophoblast-related proteins.

The human syncytiotrophoblast (hST) has a major role in the production of important placental hormones. Several molecules regulate hST endocrine function but the role of endocannabinoids in this process is still unknown. Here, we report that the endocannabinoid anandamide (AEA) decreased cAMP levels, impaired human chorionic gonadotropin secretion, placental alkaline phosphatase activity and decreased aromatase mRNA levels and protein expression, through cannabinoid (CB) receptor activation. AEA also downregulated leptin and placental protein 13 transcription, though via a CB receptor-independent mechanism. All this evidence suggests AEA is a novel modulator of hormone synthesis by the syncytiotrophoblast, supporting the importance of the endocannabinoid signalling in placental function.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression.

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

New vistas for treatment of obesity and diabetes? Endocannabinoid signalling and metabolism in the modulation of energy balance.

Growing evidence suggests that pathological overactivation of the endocannabinoid system (ECS) is associated with dyslipidemia, obesity and diabetes. Indeed, this signalling system acting through cannabinoid receptors has been shown to function both centrally and peripherally to regulate feeding behaviour as well as energy expenditure and metabolism. Consequently, modulation of these receptors can promote significant alterations in body weight and associated metabolic profile. Importantly, blocking cannabinoid receptor type 1 function has been found to prevent obesity and metabolic dysfunction in various murine models and in humans. Here we provide a detailed account of the known physiological role of the ECS in energy balance, and explore how recent studies have delivered novel insights into the potential targeting of this system as a therapeutic means for treating obesity and related metabolic disorders.

Unique inhibitory synapse with particularly rich endocannabinoid signaling machinery on pyramidal neurons in basal amygdaloid nucleus.

2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB(1) receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB(1) is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGLα, CB(1), and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGLα was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB(1), MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGLα and CB(1) were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGLα-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons.

Endocannabinoid signal in the gut controls dietary fat intake.

Oral sensory signals drive dietary fat intake, but the neural mechanisms underlying this process are largely unknown. The endocannabinoid system has gained recent attention for its central and peripheral roles in regulating food intake, energy balance, and reward. Here, we used a sham-feeding paradigm, which isolates orosensory from postingestive influences of foods, to examine whether endocannabinoid signaling participates in the positive feedback control of fat intake. Sham feeding a lipid-based meal stimulated endocannabinoid mobilization in the rat proximal small intestine by altering enzymatic activities that control endocannabinoid metabolism. This effect was abolished by surgical transection of the vagus nerve and was not observed in other peripheral organs or in brain regions that control feeding. Sham feeding of a nutritionally complete liquid meal produced a similar response to that of fat, whereas protein or carbohydrate alone had no such effect. Local infusion of the CB(1)-cannabinoid receptor antagonist, rimonabant, into the duodenum markedly reduced fat sham feeding. Similarly to rimonabant, systemic administration of the peripherally restricted CB(1)-receptor antagonist, URB 447, attenuated sham feeding of lipid. Collectively, the results suggest that the endocannabinoid system in the gut exerts a powerful regulatory control over fat intake and might be a target for antiobesity drugs.

The highs and lows of cannabinoid receptor expression in disease: mechanisms and their therapeutic implications.

Alterations in the endogenous cannabinoid system have been described in almost every category of disease. These changes can alternatively be protective or maladaptive, such as producing antinociception in neuropathic pain or fibrogenesis in liver disease, making the system an attractive therapeutic target. However, the challenge remains to selectively target the site of disease while sparing other areas, particularly mood and cognitive centers of the brain. Identifying regional changes in cannabinoid receptor-1 and -2 (CB(1)R and CB(2)R) expression is particularly important when considering endocannabinoid system-based therapies, because regional increases in cannabinoid receptor expression have been shown to increase potency and efficacy of exogenous agonists at sites of disease. Although there have been extensive descriptive studies of cannabinoid receptor expression changes in disease, the underlying mechanisms are only just beginning to unfold. Understanding these mechanisms is important and potentially relevant to therapeutics. In diseases for which cannabinoid receptors are protective, knowledge of the mechanisms of receptor up-regulation could be used to design therapies to regionally increase receptor expression and thus increase efficacy of an agonist. Alternatively, inhibition of harmful cannabinoid up-regulation could be an attractive alternative to global antagonism of the system. Here we review current findings on the mechanisms of cannabinoid receptor regulation in disease and discuss their therapeutic implications.

Cannabinoids provoke alcoholic steatosis through a conspiracy of neighbors.

Cannabinoid signaling by CB1 receptors drives fibrogenesis and fat accumulation in liver. A report in this issue of Cell Metabolism (Jeong et al., 2008) now links hepatic stellate cells, a resident liver fibrogenic cell type, to the generation of steatosis through production of the endocannabinoid 2-arachidonoylglycerol (2-AG) after ethanol feeding, leading to paracrine stimulation of hepatocyte CB1 receptors.

Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver.

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.