The endocannabinoid system consists of the endogenous cannabinoids (endocannabinoids), cannabinoid receptors and the enzymes that synthesise and degrade endocannabinoids. Many of the effects of cannabinoids and endocannabinoids are mediated by two G protein-coupled receptors (GPCRs), CB(1) and CB(2), although additional receptors may be involved. CB(1) receptors are present in very high levels in several brain regions and in lower amounts in a more widespread fashion. These receptors mediate many of the psychoactive effects of cannabinoids. CB(2) receptors have a more restricted distribution, being found in a number of immune cells and in a few neurones. Both CB(1) and CB(2) couple primarily to inhibitory G proteins and are subject to the same pharmacological influences as other GPCRs. Thus, partial agonism, functional selectivity and inverse agonism all play important roles in determining the cellular response to specific cannabinoid receptor ligands.
Cannabinoids used in cancer are best-known for their palliative effects, including reducing nausea and vomiting, alleviating cancer pain, and stimulating appetite [178,179]. It has been argued that cannabinoids can exert anti-tumor effects directly through the inhibition of cell proliferation and induction of apoptosis, or indirectly through the inhibition of angiogenesis, invasion and metastasis . Numerous studies using synthetic/endo-/phyto-cannabinoids and endocannabinoid system regulators in various cancer cell lines support this notion . The antitumor effects of cannabinoids have also been observed in various animal tumor models . In general, an enhanced endocannabinoid system is seen in tumor tissues [179,182,183]. However, the role of upregulated endocannabinoid system activity is still controversial as contrasting results have been reported supporting a proliferative as well as an anti-proliferative role of cannabinoids on cancer cells [180,181]. Interestingly, a bimodal effect of cannabinoids on cancer cell growth has also been observed, with low concentrations being proliferative and high concentrations being pro-apoptotic .
Given the widespread distribution of CB1Rs in the human body, it is reasonable for one to speculate a broad spectrum of physiological roles of the CB1R [3,9,63,126]. Indeed, the CB1R and the endocannabinoid system are largely involved in various aspects of central neural activities and disorders, including appetite, learning and memory, anxiety, depression, schizophrenia, stroke, multiple sclerosis, neurodegeneration, epilepsy, and addiction [3,9,126,127]. The CB1R is also involved in physiological and pathological conditions in the PNS and peripheral tissues, including pain, energy metabolism, cardiovascular and reproductive functions, inflammation, glaucoma, cancer, and liver and musculoskeletal disorders . The expression of CB1R remarkably fluctuates in many pathological conditions, underscoring its critical role in a wide spectrum of biological activities . Interestingly, in some cases, both positive and negative alterations in CB1R expression and functionality have been reported . Moreover, the administration of CB1R agonists exert biphasic effects in several conditions . On the other hand, the widespread presence of the CB1R limits the therapeutic application of CB1R ligands due to various side effects. These facts underscore the significance of understanding and manipulating the endocannabinoid system in a condition-specific manner.
Simplified scheme representing endocannabinoid retrograde signaling mediated synaptic transmission. Endocannabinoids are produced from postsynaptic terminals upon neuronal activation. As the two major endocannabinoids shown in the scheme, 2-arachidonolglycerol (2-AG) is biosynthesized from diacylglycerol (DAG) by diacylglycerol lipase-α (DAGLα), and anandamide (AEA) is synthesized from N-acyl-phosphatidylethanolamine (NAPE) by NAPE-specific phospholipase D (NAPE-PLD). As lipids, endocannabinoids, mainly 2-AG, readily cross the membrane and travel in a retrograde fashion to activate CB1Rs located in the presynaptic terminals. Activated CB1Rs will then inhibit neurotransmitter (NT) release through the suppression of calcium influx. 2-AG is also able to activate CB1Rs located in astrocytes, leading to the release of glutamate. Extra 2-AG in the synaptic cleft is taken up into the presynaptic terminals, via a yet unclear mechanism, and degraded to arachidonic acid (AA) and glycerol by monoacylglycerol lipase (MAGL). On the other hand, AEA, synthesized in postsynaptic terminal, activates intracellular CB1R and other non-CBR targets, such as the transient receptor potential cation channel subfamily V member 1 (TRPV1). Although endocannabinoid retrograde signaling is mainly mediated by 2-AG, AEA can activate presynaptic CB1Rs as well. Fatty acid amide hydrolase (FAAH) is primarily found in postsynaptic terminals and is responsible for degrading AEA to AA and ethanolamine (EtNH2). Although NAPE-PLD is expressed in presynaptic terminals in several brain regions, it is not clear yet whether AEA is responsible for anterograde signaling in the endocannabinoid system. Note that alternative routes exist for the metabolism of endocannabinoids, depending on the brain region and physiological conditions. Thin arrows indicate enzymatic process; thick arrows indicate translocation; blunted arrow indicates inhibition.
CB1R has been found to inhibit GABA and glutamate release from presynaptic terminals, which confers the CB1R with the ability to modulate neurotransmission [60,129]. This has been proposed as a plausible underlying mechanism of CB1R-mediated neuroprotection against excitotoxicity, a prominent pathological process of many neurological disorders, including epilepsy and neurodegenerative diseases [34,130,131]. To date, numerous studies have shown that the CB1R plays a neuroprotective role against excitotoxicity induced by various stimuli [131,132,133,134]. It has been demonstrated recently that in mouse brain, the neuroprotective effect exerted by CB1R against excitotoxicity is restricted to the CB1R population located on glutamatergic terminals . In addition to the prominent inhibitory effects on Ca 2+ influx and glutamate release, CB1R-mediated neuroprotection also involves inhibition of nitric oxide (NO) production, reduction of zinc mobilization, and increase of BDNF expression [134,135,136]. Recent studies have implicated a direct physical interaction between CB1Rs and NMDARs in the presence of histidine triad nucleotide-binding protein 1, which allows CB1Rs to negatively regulate NMDAR activity and protects neural cells from excitotoxicity [136,137].
8. Future Directions of Cannabinoid-Based Drug Discovery
The first conclusive evidence supporting retrograde endocannabinoid signaling came from the observation of depolarization-induced suppression of inhibition (DSI)/excitation (DSE) [9,33,40]. Later, it was discovered that the endocannabinoid system is involved not only in short-term depression, but also in long-term depression (LTD) at both excitatory and inhibitory synapses [9,33]. Since then, the endocannabinoid system has become the most-studied retrograde signaling system in the brain.
Major localization sites and associated functions of the CB1R in the human body. The majority of CB1Rs expressed in human body is found in the brain, where it is involved in various neurological activities. CB1Rs on the peripheral sites, although to a lesser extent, participates in the regulation of local tissue functions.
Specifically, altered expression of the CB1R and other elements of the endocannabinoid system have been observed in various neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) . The upregulation of the CB1R and endocannabinoid system activity has been observed in the basal ganglia of experimental models of PD, which could be a mechanism to compensate the degenerated dopaminergic neurons of the substantia nigra, or a pathological process that contributes to the worsening of the disease . Interestingly, decreased endocannabinoid system activity has also been reported in PD models . Moreover, both the FAAH inhibitors and CB1R antagonists have been shown to alleviate the motor symptoms in PD models . Similarly, although changes of CB1R expression in AD patients or animal models are still controversial, the activation of the CB1R has been shown to prevent amyloid β-induced neurotoxicity in several cell models [139,140,141,142,143,144]. In addition, the activation of the CB1R has been reported to be beneficial in AD animal models with memory deficits and cognitive disorders [145,146,147]. On the other hand, studies have emphasized the beneficial potentials of the CB1R in HD pathogenesis. In 1993, decreased expression of the CB1R was first reported in the substantia nigra of HD patients via autoradiography . Further studies revealed a progressive loss of CB1Rs as an early sign of HD, which occurred before the onset of actual neurodegeneration, and hastened the worsening of HD . This observation was confirmed at the mRNA level as well as with CB1R immunoreactivity in several transgenic HD mouse models (reviewed in ). A recent study described downregulation of the CB1R not only in medium spiny projection neurons (MSNs) but also in a subpopulation of interneurons that are selectively preserved in both transgenic HD mice and HD patients . Delayed loss of CB1Rs in HD transgenic mice R6/1 was seen in enriched environment, accompanied by delayed onset of motor disorders and disease progression . Moreover, in HD transgenic mice R6/2, CB1R knockout leads to the worsening of motor performances, increased susceptibility to 3-nitropropionic acid, and exacerbated striatal atrophy and Huntingtin (Htt) aggregates [133,152]. Selective increase in CB1R expression in MSNs improves the survival of excitatory projection neurons, but does not promote the motor performances of HD transgenic R6/2 mice . Administration of THC has been reported to ameliorate motor disorders, striatal atrophy, and Htt aggregates in transgenic mice, although controversy exists [133,154]. Activation of the CB1R inhibits glutamate release while increases BDNF release from presynaptic terminals in mice . Further investigation in HD cell models revealed that CB1R activation can protect striatal cells against excitotoxicity through increased BDNF expression via PI3K/Akt pathway . These observations support a critical and possibly beneficial role of the CB1R in neurodegenerative diseases.
6. Cannabinoid Receptor Signaling
Both the CB1R and CB2R are members of the GPCR family and are coupled to pertussis toxin (PTX)-sensitive Gi/o protein, suppress AC and the formation of cAMP upon receptor activation . However, the CB1R but not the CB2R has been reported to activate other G proteins in certain circumstances in a cell type- and ligand-dependent manner . The CB1R is able to stimulate specific AC isoforms via Gβγ subunits . Also, the CB1R stimulates cAMP via coupling to Gs when the dopamine receptor 2 (D2R) is activated simultaneously in cultured striatal neurons, and when Gi is blocked by PTX in transfected CHO-K1 cells, and in response to a relatively high concentration of WIN 55, 212-2 (WIN) in rat globus pallidus slices [90,91,92]. However, the same concentration of WIN but not other CB1R agonists, increases intracellular Ca 2+ concentration via Gq/11 protein in transfected HEK-293 cells and cultured hippocampal neurons with endogenous receptor expression . Moreover, in mice hippocampal slices, CB1R expressed in astrocytes is coupled to Gq/11, increases intracellular Ca 2+ concentrations and triggers astrocytic release of glutamate that stimulates N-methyl- d -aspartate receptor (NMDAR) on pyramidal neurons, indirectly involved in synaptic transmission .
Due to the lipophilic nature of cannabinoids, it was initially thought that these compounds exert various biological effects by disrupting the cell membrane nonspecifically. However, following the discovery of THC and subsequent emerging of several chemically synthesized cannabinoids, the successful mapping and the pharmacological characterization of cannabinoid binding sites in the brain revealed the existence of a putative CBR and its similarity to GPCR nature, which was matched with the properties of an orphan GPCR that is now known as CB1R [4,5,6,7,18].