The neurobiology and evolution of cannabinoid signalling

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1. Two putative endocannabinoid ligands, arachidonylethanolamide ('anandamide') and 2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of 'classical' neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues, indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.     

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.     

Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.     

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.     

Localization of cannabinoid receptors CB1, CB2, GPR55, and PPARα in the canine gastrointestinal tract

The endocannabinoid system (ECS) is composed of cannabinoid receptors, their endogenous ligands, and the enzymes involved in endocannabinoid turnover. Modulating the activity of the ECS may influence a variety of physiological and pathophysiological processes. A growing body of evidence indicates that activation of cannabinoid receptors by endogenous, plant-derived, or synthetic cannabinoids may exert beneficial effects on gastrointestinal inflammation and visceral pain. The present ex vivo study aimed to investigate immunohistochemically the distribution of cannabinoid receptors CB1, CB2, G protein-coupled receptor 55 (GPR55), and peroxisome proliferation activation receptor alpha (PPARα) in the canine gastrointestinal tract. CB1 receptor immunoreactivity was observed in the lamina propria and epithelial cells. CB2 receptor immunoreactivity was expressed by lamina propria mast cells and immunocytes, blood vessels, and smooth muscle cells. Faint CB2 receptor immunoreactivity was also observed in neurons and glial cells of the submucosal plexus. GPR55 receptor immunoreactivity was expressed by lamina propria macrophages and smooth muscle cells. PPARα receptor immunoreactivity was expressed by blood vessels, smooth muscle cells, and glial cells of the myenteric plexus. Cannabinoid receptors showed a wide distribution in the gastrointestinal tract of the dog. Since cannabinoid receptors have a protective role in inflammatory bowel disease, the present research provides an anatomical basis supporting the therapeutic use of cannabinoid receptor agonists in relieving motility disorders and visceral hypersensitivity in canine acute or chronic enteropathies.     

Biased agonism at the cannabinoid receptors – Evidence from synthetic cannabinoid receptor agonists

The type 1 and type 2 cannabinoid receptors are G protein-coupled receptors implicated in a variety of physiological processes and diseases. Synthetic cannabinoid receptor agonists (SCRAs) were originally developed to explore the therapeutic benefits of cannabinoid receptor activation, although more recently, these compounds have been diverted to the recreational drug market and are increasingly associated with incidences of toxicity. A prominent concept in contemporary pharmacology is functional selectivity or biased agonism, which describes the ability of ligands to elicit differential activation of signalling pathways through stabilisation of distinct receptor conformations. Biased agonists may maximise drug effectiveness by reducing on-target adverse effects if they are mediated by signalling pathways distinct from those that drive the therapeutic effects. For the cannabinoid receptors, it remains unclear as to which signalling pathways mediate desirable and adverse effects. However, given their structural diversity and potential to induce a plethora of signalling effects, SCRAs provide the most promising prospect for detecting and studying bias at the cannabinoid receptors. This review summarises the emerging evidence of SCRA bias at the cannabinoid receptors.     

An Overview on the Biochemistry of the Cannabinoid System

About 40 years ago, cannabinoids were considered as the substances responsible for the psychoactive properties of marijuana and other derivatives of Cannabis sativa, whereas their medicinal use remained unexplored. However, with the discovery of the endocannabinoid system 20 years later, the compounds able to modify this system are being reconsidered for their therapeutic potential. Thus, the term "cannabinoid" includes now much more compounds than those present in C. sativa derivatives, for instance, numerous synthetic cannabinoids obtained by modifications from plant-derived cannabinoids or from the compounds that behave as endogenous ligands for the different cannabinoid receptor types. The term "cannabinoid" should also refer to some prototypes of selective antagonists for these receptors. The explanation for this exponential growth in cannabinoid pharmacology is the discovery and characterization of the endocannabinoid signaling system (receptors, ligands, and inactivation system) which plays a modulatory role mainly in the brain but also in the periphery. The objective of the present review article was to give an overview of the present state-of-the-art of biochemistry of the endocannabinoid system. Other authors in this volume will review their functions in the brain, their alterations in a variety of neurological and psychiatric pathologies, and the proposed therapeutic benefits in these diseases of new cannabinoid-related compounds that improve the pharmacological properties of classic cannabinoids.     

Cannabinoid CB<Subscript>2</Subscript> Receptor-Mediated Anti-nociception in Models of Acute and Chronic Pain

The endocannabinoid system consists of cannabinoid CB₁ and CB₂ receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB₁ receptors. Following the recent evidence for CB₂ receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB₂ receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB₂ receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB₂ receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action. Jhaveri and Sagar joint first author.     

Role of endocannabinoids in brain development.

In addition to those functions that have been extensively addressed in this special issue, such as nociception, motor activity, neuroendocrine regulation, immune function and others, the endogenous cannabinoid system seems to play also a role in neural development. This view is based on a three-fold evidence. A first evidence emerges from neurotoxicological studies that showed that synthetic and plant-derived cannabinoids, when administered to pregnant rats, produced a variety of changes in the maturation of several neurotransmitters and their associated-behaviors in their pups, changes that were evident at different stages of brain development. A second evidence comes from studies that demonstrated the early appearance of elements of the endogenous cannabinoid system (receptors and ligands) during the brain development. The atypical location of these elements during fetal and early postnatal periods favours the notion that this system may play a role in specific molecular events related to neural development. Finally, a third evidence derives from studies using cultures of fetal glial or neuronal cells. Cannabinoid receptors are present in some of these cultured cells and their activation produced a set of cellular effects consistent with a role of this system in the process of neural development. All this likely supports that endocannabinoids, early synthesized in nervous cells, play a role in events related to development, by acting through the activation of second messenger-coupled cannabinoid receptors.     

The neurobiology of cannabinoid analgesia.

The discovery of cannabinoid receptors and their putative endogenous ligands raises questions as to the nature of the effects produced by cannabinoids on neural circuits that mediate pain and whether endogenous cannabinoids produced by the brain or in the periphery serve naturally to modulate pain. A sizable body of previous work showed that cannabinoid agonists suppress pain behavior in a variety of models of acute and chronic pain. However, at appropriate doses, cannabinoids also profoundly suppress motor behavior (see Sa?udo-Pe?a et al., this volume), which complicates the interpretation of behavioral analgesia since a motor response is the endpoint of virtually all such studies. Studies conducted in this laboratory used biochemical and neurophysiological measures to determine whether cannabinoids suppress nociceptive neurotransmission. The results showed that cannabinoids suppress nociceptive neurotransmission at the level of the spinal cord and the thalamus. These effects are reversible, receptor mediated, selective for painful as opposed to nonpainful somatic stimuli, and track the behavioral analgesia both in time course and potency.     

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.     

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.     

Effects of cannabinoids on energy metabolism.

The present review summarizes the recent work carried out by our group on the link between signal transduction pathways and metabolic regulation systems as affected by cannabinoids. In cells such as astrocytes and lymphocytes, which express cannabinoid receptors, physiologically relevant doses of cannabinoids induce a remarkable metabolic stimulation as determined e.g. by enhanced glucose utilization. Studies performed in astrocytes show that the cannabinoid-evoked stimulation of glucose metabolism is independent of adenylyl cyclase inhibition, and seems to rely on the cascade CB1 cannabinoid receptor --> Sphingomyelin breakdown --> Ceramide --> Raf-1 --> Mitogen-activated protein kinase (MAPK) --> Glucose utilization. A role for phosphoinositide 3'-kinase in the stimulation of glucose utilization by cannabinoids is also put forward. In addition, ceramide generated upon CB1 cannabinoid receptor activation may enhance ketone body production by astrocytes independently of MAPK. Anandamide has also been shown to exert metabolic effects in hepatocytes, cells that do not express cannabinoid receptors. The biological role of cannabinoids as modulators of metabolism is as yet unclear.     

The cannabinoid system and cytokine network

Many advances have been made in the last few years concerning our understanding of the receptors and ligands composing the cannabinoid system. Likewise, the science surrounding cytokine biology has advanced enabling us to measure these proteins more precisely as well as understand and interpret the meaning of changes in their levels. Scientists wishing to study the health consequences of smoking marijuana as well as understand the possible role of endogenous cannabimimetic ligands in immune regulation have continued to study the influence of these substances on the regulation and development of the cytokine network. Research has shown that two major cannabinoid receptor subtypes exist and that subtype 1 (CB1) is expressed primarily in the brain whereas subtype 2 (CB2) is expressed primarily in the periphery. A variety of ligands for these receptors based on the cannabinoid structure have been synthesized and studied as well as low affinity compounds, noncannabinoid ligands, and endogenous ligands derived from fatty acid eicosanoids. Highly selective receptor antagonists have also been introduced and studied. Synthetic, low affinity ligands such as (+)-HU-211 and DMH-11C have been shown to cause anti-inflammatory effects possibly through inhibiting the production and action of TNF-alpha and other acute phase cytokines. In addition, suppression of TNF and other cytokines such as GM-CSF, IL-6, IFNgamma, and IL-12 has also been seen following exposure to high affinity and psychoactive ligands such as marijuana and THC. However, some of these ligands have also been shown to increase rather than decrease interleukins such as IL-1, IL-4, IL-10, and IL-6, cytokines such as TNF-alpha, and chemokines such as IL-8, MIP-1, and RANTES. The endogenous ligand, anandamide, has been shown in culture to either suppress the proliferation response to prolactin or enhance the response to cytokines such as IL-3 and IL-6. This eicosanoid has also been shown to increase the production of interleukins and other cytokines. Cannabinoid receptors have been shown to be involved in some but not all of these effects. It is clear that psychoactive and nonpsychoactive compounds have demonstrated effects in vivo and in vitro on the production and function of a variety of cytokines. Depending upon the model system, these effects are often conflicting, and the involvement of cannabinoid receptors is unclear. However, enough evidence exists to suggest that the cannabinoid system significantly impacts the functioning of the cytokine network, and this association may provide clues to the mechanisms of certain immune diseases and form the basis for new immunotherapies.     

Cannabinoid-sensitive receptors in cardiac physiology and ischaemia

The classical cannabinoid receptors CB1 and CB2 as well as the cannabinoid-sensitive receptor GPR55 are widely distributed throughout the mammalian body. In the cardiovascular field, CB1 and CB2 crucially impact on diseases characterized by inflammatory processes, such as atherosclerosis and acute myocardial infarction. Both receptors and their endogenous ligands anandamide and 2-arachidonoylglycerol are up-regulated in the ischaemic heart in humans and animal models. Pharmacological and genetic interventions with CB1 and CB2 vitally affect acute ischaemia-induced cardiac inflammation. Herein, CB1 rather aggravates the inflammatory response whereas CB2 mitigates inflammation via directly affecting immune cell attraction, macrophage polarization and lymphocyte clusters in the pericardial adipose tissue. Furthermore, cannabinoids and their receptors affect numerous cardiac risk factors. In this context, cannabis consumption is debated to trigger arrhythmias and even myocardial infarction. Moreover, CB1 activation is linked to impaired lipid and glucose metabolism and therefore obesity and diabetes, while its antagonism leads to the reduction of plasma triglycerides, low-density lipoprotein cholesterol, leptin, insulin and glucose. On the other hand, activation of cannabinoid-sensitive receptors can also counteract unfavourable predictors for cardiovascular diseases. In particular, hypertension can be mitigated via CB1 agonism and impaired adrenoceptor responsiveness prevented by functional GPR55.Taken together, current insights identify the cannabinoid system as promising target not only to therapeutically interfere with the vasculature, but also to affect the heart as target organ. This review discusses current knowledge regarding a direct cardiac role of the cannabinoid system and points out its feasible therapeutic manipulation in the ischaemic myocardium.     

Distinct cannabinoid sensitive receptors regulate hippocampal excitation and inhibition

One of the well-known effects of cannabinoids is the impairment of cognitive processes, including short-term memory formation, by altering hippocampal and neocortical functions reflected in network activity. Acting on presynaptically located G protein-coupled receptors in the hippocampus, cannabinoids modulate the release of neurotransmitter molecules. CB1 cannabinoid receptors, so far the only cloned cannabinoid receptor type in the CNS, are selectively expressed on the axon terminals of a subset of GABAergic inhibitory interneurons containing the neuropeptide cholecystokinin. Activation of CB1 receptors reduces GABA release from presynaptic terminals, thereby increasing the excitability of principal cells. Novel, non-CB1 cannabinoid sensitive receptors are present on the hippocampal excitatory axon terminals, which suppress glutamate release. These cannabinoid receptors have distinct pharmacological features compared to CB1, i.e. WIN 55212-2 is an order of magnitude less potent in reducing glutamatergic transmission than in inhibiting GABAergic postsynaptic currents, and the novel receptor binds vanilloid receptor ligands. Thus, at least two different cannabinoid sensitive presynaptic receptors regulate network activity in the hippocampus, CB1 via the GABAergic interneurons, and a new receptor via a direct action on pyramidal cell axon terminals.