Cannabinoid Receptor

1.1 Cannabinoid receptors

Cannabinoid receptors obtained their name as a result of their response to cannabinoids, for example, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) from Cannabis sativa (marijuana) and synthetic analogs. Cannabinoid receptors are members of the endocannabinoid system and are key mediators of many psychological processes (Wotjak, 2005). These receptors belong to the rhodopsin family of G protein-coupled receptors (GPCRs). Two subtypes of receptors have been identified: type 1 (CB₁) and type 2 (CB₂). The CB₁ receptor abounds in the brain and central nervous system (CNS), whereas the CB₂ is found mainly in the immune system (Howlett et al., 2002).

As deduced by complementary DNA encoding (Matsuda et al., 1990) and molecular cloning (Gerard et al., 1991), human CB₁ receptors are encoded by an amino sequence of 472 residues. As for other proteins belonging to the rhodopsin family, the CB₁ receptor has seven transmembrane helical (TMH) domains of which the TMH-4 and -5 domains form the high-affinity ligand binding site (Shire et al., 1996).

Studies of the physiological roles of the CB₁ receptor have revealed strong correlations with inhibition of adenylyl cyclase, pain control, regulation of ion channels, modulation of energy intake, and various other signal transduction pathways (Howlett, 2004; Pagotto et al., 2006). Among these functions, arguably the most promising feature of this receptor's regulatory profile from a therapeutic-potential standpoint, is (or at least was, see below) its role in energy regulation and metabolism. In particular, animal models revealed that CB₁ knockout mice were leaner than wild-type species (Di Marzo et al., 2001). Blocking the CB₁ receptor potentially therefore provides a therapeutic strategy for treatment of obesity and metabolic syndrome as well as drug additions and addiction to smoking (Di Marzo et al., 2001).

4 Receptors of cannabinoids

Cannabinoid receptors are transmembrane proteins that mediate the action of CBs. Two common cannabinoid receptors are CB₁ and CB₂ that belong to the class A family of G-protein coupled receptors. Both receptors contain a glycosylated amino-terminal (extracellular) and carboxy-terminal (intracellular) domain connected by 7 transmembrane domains, 3 extracellular loops and 3 intracellular loops [3,65].

Cannabinoid receptors are found in both central and peripheral tissues of the body [66]. CB₁ is predominant in the regions of brain such as cerebral cortex and cerebellum. CB₂ is mostly located in peripheral tissues including lungs, leukocytes, liver, and spleen [66]. CB₁ and CB₂ receptors along with endogenous CBs and endocannabinoid metabolic enzymes [monoacylglycerol lipase (MAG lipase) and fatty acid amide hydrolase (FAAH)] constitute ECS of the body. Natural and synthetic CBs play important roles in different pathophysiological conditions, including cancer, by modulating ECS [67].

The ability of CBs to modulate tumorigenic properties depends on various factors including the type of cancer, binding affinity between the ligand and the pharmacological target (e.g., cannabinoid receptor) and the site of the tumour [10,68,69]. For instance, binding affinity of WIN-55,212−2 with cannabinoid receptors is higher compared to its natural analogue, THC [10]. However, WIN-55,212−2 is less efficient in promoting cell death as compared to THC [10]. WIN-55,212−2 is a schedule I controlled substance and illegal to use. The anti-tumorigenic action of CBs is attributed to the modulation of various signalling pathways crucial for cancer pathogenesis (Fig. 8) [10,69–71].

Fig. 8. Key signaling pathways modulated by cannabinoids in cancer cells.

CBs exert their apoptotic action by binding to CB₁ and CB₂ receptors. For instance, cannabinoid receptor agonists when bind to receptors enhance pro-apoptotic molecules such as sphingolipid ceramide which in-turn modulates various signalling pathways involved in the pathogenesis of cancer. In leukemic cells, CBs act through ceramide which induces apoptosis by modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. In glioma cells, CBs induces apoptosis by upregulating endoplasmic reticulum stress-related genes [72]. Furthermore, in lung cancer, CBD is reported to up-regulate the expression of cyclooxygenase-2 (COX-2) and pro-apoptotic prostaglandin E-2 (PGE2) which contribute to cellular apoptosis [73].

A pathway by which Δ⁹-THC promotes apoptosis is via upregulation of Tribbles homolog 3 (TRB3) protein. Upregulation of TRB3 leads to cellular autophagy and inhibition of protein kinase B (PKB) leading to increased synthesis of Bcl2-associated agonist of cell death (BAD), which is a pro-apoptotic protein [73,74]. Autophagy either acts as an alternate to apoptosis causing cell death or acts together with the apoptotic pathway leading to cell death. For instance, Δ⁹-THC and JWH-015, when bind to CB₂ receptors, induced cellular autophagy and reduced cellular viability in HepG2 and HuH-7 hepatocarcinoma cell lines and tumour models [75]. Here, JWH-015 and Δ⁹-THC upregulated TRB3.This resulted in the suppression of the PKB/mammalian target of rapamycin Complex1 (mTORC1) axis and stimulation of adenosine monophosphate-activated kinase (AMPK). Further, calmodulin-dependent protein kinase kinase-beta (CAMKK-β) induced AMPK phosphorylation leads to cellular autophagy [75].

Arachidonoyl cyclopropamide (ACPA) and GW405833 (GW), upon binding to CB₁ and CB₂ receptors respectively, release reactive oxygen species (ROS) which inhibit glycolysis, Krebs’ cycle and oxidative phosphorylation in pancreatic cancer cells (Fig. 9) [76,77]. Additionally, inhibition of oxidative phosphorylation changes the Adenosine Triphosphate/Adenosine Monophosphate ratio of cells and induces AMPK, the enzyme responsible for maintaining the intracellular homeostasis and energy regulation of cells. Activation of AMPK leads to inhibition of mTORC1, a key regulator of protein synthesis and cell growth. All these events eventually trigger the cell towards autophagy. mTORC1 inhibition also downregulates its downstream targets, hypoxia-inducible factor-1 alpha (HIF-1α) and pyruvate dehydrogenase kinase (PDHK) resulting in cell death (Fig. 9) [76].

Highlights

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Cannabinoid receptors are pleiotropically-coupled GPCRs.

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Few studies quantify cannabinoid bias using Black and Leff's operational model.

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Correlations between cannabinoid bias in vitro and in vivo are just being measured.

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Understanding cannabinoid bias could yield effective cannabinoid-based drugs.

1 Introduction

1.2 Antagonists and inverse agonists of CB₁ receptors

A 1,5-diarylpyrazole-based ligand, SR141716A, later known as rimonabant (acomplia), was released in 1994 by Sanofi-Aventis to target the CB₁ receptor. It displayed nanomolar affinity for this receptor and just micromolar affinity for the CB₂ receptor making it the first efficient, competitive agent for selective blocking of CB₁ receptors (Rinaldi-Carmona et al., 1994). Rimonabant was first categorized as a CB₁ receptor antagonist; however, after comprehensive pharmacological studies taking into account the intrinsic activity of the endocannabinoid system, it was recategorized as an inverse agonist (Pertwee, 2005). An analog, AM251, which has an iodine-substituent in place of a chlorine atom in the 5-phenyl ring of rimonabant, has similar inhibitory activity and selectivity for CB₁ whilst also providing an opportunity to introduce a radioactive-iodine isotope (¹²³I or ¹²⁴I) to allow visualization of the CNS by position emission tomography (PET) or single photon emission computed tomography (SPECT; Howlett, 2004; Pagotto et al., 2006). A ring-constrained analog NESS0327 was reported as having femtomolar affinity for the CB₁ receptor in vitro; however, no inhibition was shown when administered orally (Murineddu et al., 2005; Stoit et al., 2002). The poor biodistribution of this compound in vivo doubtless contributed to this outcome. Extensive studies have also been reported toward the development of new CB₁ ligands by changing the central core of rimonabant to imidazole, triazole, and phenyl units (e.g., O-1803) and by de novo design to give novel scaffolds such as found in CP-945598 (Pfizer), MK-0364 (taranabant, Merck), and AVE-1625 (Sanofi-Aventis; Scheme 27.1).

Scheme 27.1. “Drug-like” potential CB₁ inhibitors.

Clinical trials of rimonabant for treatment of obesity suggested that it was a promising and effective medicine and so the compound was approved by the European Medicines Agency for sale in Europe as acomplia in 2006. Although only minor side-effects were reported during the clinic trials, several serious psychiatric disorder cases emerged once the drug was available on prescription. As a consequence, this drug was withdrawn from the market in 2008 and many other ongoing projects targeting the CB₁ receptor as a strategy for developing antiobesity drugs were also apparently terminated (Jones, 2008).

1 Introduction

Cannabinoid receptors are considered to be key regulators of nausea, obesity (Bellocchio et al., 2006; DiMarzo and Deprés, 2009), pain (Manzanares et al., 2006; Sagar et al., 2009), anxiety, depression, substance use disorders (Mackie, 2006) and neurodegenerative disorders such as Alzheimer's disease (Aso and Ferrer, 2014), and Parkinson's disease (Brotchie, 2003). Despite exhaustive research, few cannabis-based therapeutics have reached clinical use, although nabilone (Cesamet) (Frank et al., 2008), dronabinol (Marinol) (Pertwee, 2006) and a Δ⁹-tetrahydrocannabinol (THC)/cannabidiol blend (Sativex) (Blake, 2006) are approved for the treatment of spasticity, nausea and pain in various countries. Conventional drug design has primarily targeted the orthosteric site of cannabinoid receptors, creating ligands that compete with the endogenous cannabinoid, anandamide and 2-arachidonoyl-glycerol (2-AG), for binding to this site. However, psychoactive side effects are frequent and often preclude clinical usefulness for ligands that target these sites on the CB₁ cannabinoid receptor (CB₁) which is expressed in high numbers in many of the regions of the brain (Mackie, 2006). It is now well appreciated that allosteric sites exist on many G-protein coupled receptors (GPCRs), including CB₁ (Kenakin, 2012; Conn et al., 2014). Targeting these sites offers in principle several advantages such as greater subtype-selectivity (Conn et al., 2009), maintenance of spatial and temporal aspects of receptor activation and consequent attenuation of side effects (Conn et al., 2009; Burford et al., 2013). This manuscript focuses on the allosteric modulation of the CB₁ receptor and gives a structural and functional review of its known allosteric modulators and their outcome.

2.2 Cannabinoid receptors

The cannabinoid receptors (CB1 and CB2), as well as a putative cannabinoid receptor GPR55, are seven transmembrane G protein-coupled receptors (GPCRs) that signal predominately through inhibitory Gα_i/o G proteins. The CB1 receptor was cloned in 1990 (Matsuda, Lolait, Brownstein, Young, & Bonner, 1990) based on its binding affinity for the natural ligand (delta9-tetrahydrocannabinol, THC) and a synthetic analogue with potent analgesic properties (CP-55,940). This new receptor inhibited forskolin-stimulated adenylyl cyclase activity in a G protein-dependent manner, a hallmark of cannabinoid compounds isolated from cannabis. This opened the door for development of synthetic compounds, both agonists and antagonists, that bind CB1 receptors (Herkenham et al., 1990). CB1 receptors are the most abundant GPCRs in the central nervous system (Busquets-Garcia, Bains, & Marsicano, 2018) and are expressed in neurons throughout the central nervous system (Busquets-Garcia et al., 2018; Herkenham et al., 1990; Stella, 2010; Turcotte, Blanchet, Laviolette, & Flamand, 2016) where they are primarily expressed in presynaptic terminals and act to inhibit neurotransmitter release (Chevaleyre, Takahashi, & Castillo, 2006; Freund & Hajos, 2003; Freund, Katona, & Piomelli, 2003; Hajos et al., 2000; Huang et al., 2001; Kano, Ohno-Shosaku, Hashimotodani, Uchigashima, & Watanabe, 2009; Katona et al., 1999; Katona et al., 2001; Katona et al., 2006; Mackie, 2005; Morisset & Urban, 2001). More recently, postsynaptic actions of CB1 receptors have been described (Maroso et al., 2016), but appear to be rare compared to the ubiquitous expression of presynaptic CB1 receptors (for review, see Busquets-Garcia et al., 2018).

Historically, CB2 receptors were thought to be expressed exclusively in the periphery, primarily on immune cells, but functional and anatomical evidence now indicates that these receptors are also expressed in the central nervous system (Atwood & Mackie, 2010). Basally, CB2 receptors are expressed at lower levels than CB1 receptors in the midbrain and brainstem (Gong et al., 2006), although localization studies using putative CB2 receptor antibodies should be interpreted with caution due to issues with specificity (Brownjohn & Ashton, 2012; Cecyre, Thomas, Ptito, Casanova, & Bouchard, 2014; Marchalant, Brownjohn, Bonnet, Kleffmann, & Ashton, 2014). Functional studies using multiple CB2-selective agonists and antagonists provide convincing evidence for CB2-dependent effects in the rostral ventromedial medulla (RVM) (Deng et al., 2015; Li, Suchland, & Ingram, 2017) and spinal cord (Beltramo et al., 2006; Burston et al., 2013; Guindon & Hohmann, 2008a). Interestingly, CB2 receptor expression appears to be highly dynamic and dependent on the environment as CB2 expression is induced by inflammation and neuropathic pain (Hsieh et al., 2011; Li et al., 2017). CB2 receptor expression has been observed on microglia (Stella, 2010) and is upregulated in inflammation (Maresz, Carrier, Ponomarev, Hillard, & Dittel, 2005).

While CB1 and CB2 receptors are the best studied receptors in the cannabinoid system, both endocannabinoids and exogenous cannabinoids can target other receptors. GPR55 is an orphan GPCR that is stimulated by AEA and some lipophilic derivatives of endocannabinoids, as well as the CB1 receptor antagonist AM251 and inverse agonist SR141716A (rimonabant) (Kapur et al., 2009; Yang, Zhou, & Lehmann, 2016). GPR55 is expressed on neurons in the dorsal root ganglion (Lauckner et al., 2008), on adipose tissue (Tuduri, Lopez, Dieguez, Nadal, & Nogueiras, 2017) and microvascular endothelial cells (Leo et al., 2019) suggesting myriad functions of the endocannabinoid system that are largely unexplored.

Another binding site for AEA is the transient receptor potential channel TRPV1 (Di Marzo & De Petrocellis, 2010; Di Marzo, De Petrocellis, Fezza, Ligresti, & Bisogno, 2002). AEA is a full agonist at TRPV1 channels expressed on nociceptive primary afferents, as well as on many central neurons comprising ascending pain circuits. TRPV1 channels are non-selective cation channels gated by capsaicin, protons and heat that promote neuronal excitability. AEA is pro-nociceptive in some situations, promoting responses to painful stimuli (Dinis et al., 2004) but AEA activation of TRPV1 channels is also antinociceptive, especially in the presence of inflammation and neuropathic pain (Guindon, Lai, Takacs, Bradshaw, & Hohmann, 2013; Horvath, Kekesi, Nagy, & Benedek, 2008). Taken together, the actions of endocannabinoids depend both on expression of the target receptors on specific cells and on adaptations within specific brain areas that are induced in different pain states.

It should also be noted that there are documented variations in cannabinoid receptor function across species. The CB1 receptor appears to be well conserved with 98.7% amino acid sequence homology between guinea-pig and human CB1 receptors, 99.2% homology between guinea pig and rat or mouse (Kurz, Gottschalk, Schlicker, & Kathmann, 2008), and even 70% homology between pufferfish and human CB1 receptor amino acid sequence (Yamaguchi, Macrae, & Brenner, 1996). In contrast, the CB2 receptor is not as well conserved across species. CB2 receptor mRNA splicing and expression vary between mice and rats, which impacts CB2 receptor-dependent effects on cocaine self-administration between the species (Zhang et al., 2015). Rat and human CB2 receptors share 81% amino acid homology (Mukherjee et al., 2004) with sequence divergence in the carboxy terminus of mammalian CB2 receptors that could differentially impact receptor regulation, including desensitization and internalization (Brown, Wager-Miller, & Mackie, 2002). Appropriate caution should be employed when comparing CB2 receptor function across species.

Cannabinoid receptors: what they do

The cannabinoid receptors as part of the endocannabinoid system have been shown to play important roles in a variety of physiological conditions, including neuronal development, neuronal plasticity, food intake and energy balance, perception process, immune modulation, cell apoptosis, cardiovascular and reproductive functions (Marzo, Bifulco, & De Petrocellis, 2004; Rodriguez de Fonseca et al., 2005). The multifaceted functions of cannabinoid receptors are rooted by their cellular physiology and complex signaling pathways in transducing the binding of cannabinoid ligands into biological responses.

2 Cannabinoid receptors in the respiratory system

Cannabinoid receptors belong to a family of receptors associated with G-proteins. There are two types of cannabinoid receptors: cannabinoid receptor type 1 (CB₁) and type 2 (CB₂). Cannabinoid receptors belong to a family of receptors associated with G-proteins. There are two types of cannabinoid receptors: cannabinoid receptor type 1 (CB₁) and type 2 (CB₂). CB₁ receptors bind a various types of G proteins. Interaction with the G_i/o protein causes inhibition of adenylate cyclase, cyclic AMP-dependent signaling and voltage-gated calcium channels. Simultaneously, activation of mitogen-activated protein kinases (MAPKs), activation of K⁺ currents, and modulation of nitric oxide-dependent signaling are found. In addition to G_i/o, CB₁ receptors can bind other proteins such as G_s and G_q/11 but their role in signal transduction has not been clearly established (Krishna Kumar et al., 2019; Walsh and Andersen, 2020). CB₂ receptors are conjugated to the protein Gi/o. However, interaction with G_i/o induces adenylate cyclase inhibition but does not influences on ion channels activity. Other types of G proteins which coupled to CB₂ receptors include G_β/γ protein (Ibsen et al., 2017).

Most of the CB₁ receptors are expressed in areas of the central nervous system such as the hippocampus, hypothalamus, and cerebral cortex. The interaction between cannabinoids and central CB₁ receptors is responsible for the psychoactive properties some of these compounds (e.g., Δ⁹-THC). In rodents, activation of the central CB₁ receptors causes symptoms known as the cannabinoid tetrad which includes hypothermia, antinociception, catalepsy, and a decrease in physical activity (Walsh and Andersen, 2020).

CB₁ receptors are located peripherally in the cardiovascular system, digestive system, reproductive system, kidneys, tonsils, thymus, skin, bones, skeletal muscles, leukocytes, and adipose tissue (Kaur et al., 2016; Zou and Kumar, 2018; An et al., 2020). The central and peripheral CB₁ receptors control various physiological functions in the body. More importantly, overactivity or overstimulation of peripheral CB₁ receptors is linked to several pathological processes such as fibrosis (liver, heart, kidney, and skin), inflammation, oxidative/nitrosative stress, and insulin resistance (An et al., 2020; Marquart et al., 2010; Mukhopadhyay et al., 2010; Rajesh et al., 2012; Lecru et al., 2015; Jourdan et al., 2017; Dao et al., 2019; Tan et al., 2020).

In contrast to CB₁ receptors, the density of CB₂ receptors in the central nervous system is relatively small. The largest number of peripheral CB₂ receptors are expressed within the cells of the immune system. Cannabinoid activation of CB₂ receptors act to modulate the immune system through the induction of apoptosis, inhibition of cell proliferation and pro-inflammatory cytokines synthesis, increased production of anti-inflammatory cytokines and antibodies. The activation of peripheral CB₂ receptors produces anti-inflammatory, immunosuppressive, and anti-fibrotic effects (Tahamtan et al., 2016; Kaur et al., 2016; Jang et al., 2020).

Studies examining cannabinoid receptor expression and function in the respiratory system are limited and unclear. Both CB₁ and CB₂ receptors are expressed in human lung tissue and bronchi, however CB₁ receptor expression is higher (Galiègue et al., 1995; Grassin-Delyle et al., 2014; Turcotte et al., 2016; Staiano et al., 2016). There is no scientific data about the expression of cannabinoid receptors in the human trachea but according to Spicuzza et al. (2000), cannabinoid receptors were not detected in the tracheas of guinea pigs.

Respiratory epithelial cells display both CB₁ and CB₂ receptors (Fantauzzi et al., 2020). The pulmonary arteries also express CB₁ and CB₂ receptors but with a predominance of CB₁ receptors (Karpińska et al., 2017). The expression of CB₂ receptors on fibroblasts (Fu et al., 2017) and CB₁ receptors on alveolar type II cells (Rice et al., 1997) have been confirmed in cultures of rodent cells. Fig. 2 shows the ratios of cannabinoid receptor expression among the individual components of the respiratory system.

Fig. 2. Expression of cannabinoid receptors in respiratory system and recruited cells of immune system. Expression of cannabinoid receptors on structures of respiratory system and cells of immune system recruited in respiratory diseases is found. There are differences in the expression of cannabinoid receptors between individual structures and cells. Additionally, expression of cannabinoid receptors in some structures and cells is a moot point. Methods used for receptors detection: ¹ Ligand binding assay; ² Quantitative reverse transcriptase real-time PCR; ³ Reverse transcription polymerase chain reaction; ⁴ Western blot; ⁵ Microarray analysis techniques; ⁶ Flow cytometry.

Cannabinoid receptors are found on pulmonary macrophages and dendritic cells in the respiratory system and other immune system cells recruited in respiratory diseases (Moldoveanu et al., 2009; Turcotte et al., 2016). Pulmonary macrophages and dendritic cells express both cannabinoid receptors but in macrophages, the level of CB₂ receptors is higher (Turcotte et al., 2016; Staiano et al., 2016; Matias et al., 2002). In monocytes, the presence of CB₁ and CB₂ receptors has been observed with a predominance of CB₂ receptors (Galiègue et al., 1995; Sexton et al., 2013).

The existence of cannabinoid receptors, especially CB₂ receptors, in neutrophils is ambiguous (Small-Howard et al., 2005). Neutrophils are thought to express both CB₁ and CB₂ receptors, with the a dominance of CB₁ receptors but some authors have suggested that neutrophils do not express CB₂ receptors (Oka et al., 2004; Chouinard et al., 2011, 2013) or express them to a small extent (Graham et al., 2010). Eosinophils, mast cells, and basophils express CB₁ and CB₂ receptors but mast cells and basophils express more CB₁ than CB₂ receptors (Small-Howard et al., 2005). While eosinophils express CB₂ receptors in higher quantities than CB₁ receptors (Oka et al., 2004; Small-Howard et al., 2005; Frei et al., 2016; Chouinard et al., 2011, 2013). Cannabinoid receptor expression on lymphocytes is dependent on their type. B lymphocytes and NK cells express CB₁ and CB₂ receptors with a predominance of CB₂ receptors (Galiègue et al., 1995). B lymphocytes have the highest level of CB₂ receptors amongst all mononuclear leukocytes (Carayon et al., 1998). The expression of both cannabinoid receptors is observed in TCD8+ lymphocytes while TCD4+ cells express only CB₂ receptors (Galiègue et al., 1995). The expression of cannabinoid receptors in immune system cells is summarized in Fig. 2.

Interestingly, cannabinoid receptors are also expressed on lung cancer cells. The presence of CB₁ and CB₂ receptors has been confirmed in cancerous tissue obtained from patients suffering from NSCLC (non-small-cell lung carcinoma) (confirmed by immunohistochemical staining) and in human lung cancer cell lines such as A549 and SW-1573 (confirmed by reverse transcription polymerase chain reaction or Western blot analysis) (Preet et al., 2008, 2011; Ravi et al., 2014; Milian et al., 2020). An overexpression of cannabinoid receptors has been associated with longer survival in patients (Milian et al., 2020). However, according to Xu et al. (2019), an increased expression of CB₂ receptors signified a worse prognosis. Determination of cannabinoid receptor expression could be a useful tumor marker but further research is needed to determine the relationship between cannabinoid receptors with the severity, stage, and types of the lung cancer.

Introduction

In the 1990s, a number of surprising discoveries laid a strong, new foundation for both Cannabis and cannabinoid research. The identification and early characterization of the cannabinoid receptors (Console-Bram, Marcu, & Abood, 2012; Matsuda, Lolait, Brownstein, Young, & Bonner, 1990; Pacher, 2006) literally provided substrate for the future of cannabinoid science. The CB1 receptor is now understood to be one of the most abundant proteins in the mammalian brain. The CB2 receptor—sharing significant genetic sequence (homology) with the CB1 receptor—has been found to be expressed predominately in immune tissue and cells of immune origin. Because of this characteristic distribution, cannabinoid receptors provide an attractive therapeutic target. The discovery and subsequent availability of various synthetic cannabinoid analogues have allowed researchers to begin characterizing the various physiological roles of these abundant G-protein-coupled receptors (GPCRs). The sum of all knowledge of how these cannabinoid receptors and their endogenous ligands work together to modulate mammalian biology and behavior is referred to as the endocannabinoid system (ECS).