In the previous article of our Understanding Cannabinoids series, we presented a review of the scientific literature related to the study of certain cannabinoids, such as, CBD, CBN, and CBG for their potential therapeutic effects. We also introduced the endocannabinoid network (popularly known as the Endocannabinoid System or ECS).
However, considering "ECS plays a very important role in the human body for our survival," (See, Sallaberry and Astern, JYn this articleI, June 2018, Vol. 34, Issue 6, p 48-55), we delve into the molecular pathways involved in the regulation of the ECS in this article. “This is due to its ability to play a critical role in maintaining the homeostasis of the human body, which encompasses the brain, endocrine, and immune system, to name a few. ECS is a unique system in multiple dimensions.”
To begin with, it is a retrograde system functioning post- to pre-synapse, allowing it to be a “master regulator” in the body.
Secondly, it has a very wide scope of influence due to an abundance of cannabinoid receptors located anywhere from immune cells to neurons.
Finally, cannabinoids are rapidly synthesized and degraded, so they do not stay in the body for very long in high amounts, possibly enabling cannabinoid therapy to be a safer alternative to opioids or benzodiazepines.
Since the discovery of the ECS, there has been a major rise in publications on medical cannabis and cannabinoid-based therapeutics, in addition to cannabinoid receptor pharmacology. These studies have accelerated greatly in untangling the ECS, both in the molecular and the physiological realms. For a detailed account on cannabinoid and endocannabinoid history, please refer to these outstanding reviews: Roger G. Pertwee, Br. J. of Pharm. (2006) 147, S163-S171 and Antonio Waldo Zuradi, Rev. Bras. Psiquiatr. 2006; 28(2):153-7. The identification of biosynthetic and degradative pathways of endocannabinoids in the following years, make up the classical ECS. In our previous article we discussed that the ECS is made up of three components: (i) receptors, (ii) endocannabinoids, and (iii) enzymes. Here we take the opportunity to elaborate on each of the components of the ECS.
The first report of the existence of the brain cannabinoid receptor, termed the cannabinoid receptor type 1 (CB1R), was reported in the late 1980s. See, Devane et al., Mol. Pharmacol. 1988, 34, 605–613. The discovery of the CB1R was followed by the identification of the second cannabinoid receptor, termed the peripheral cannabinoid receptor (due to the lack of expression in brain) or cannabinoid receptor type 2 (CB2R) (See Abstract, Munro et al., Nature 365, 61–65 (1993)) and their two endogenous ligands, anandamide and 2-arachidonoylglycerol. See Abstract, Devane et al., Science, 18 Dec 1992. Vol. 258, Issue 5090, pp. 1946-1949; Mechoulam et al., Biochem. Pharmacol. 1995, 50, 83–90; and Abstract, Sugiura et al., Biochem. Biophys. Res. Commun. 1995, 215, 89–97.
The CB1R and CB2R belongs to the Class A G-protein coupled receptor (GPCR) subfamily, often regarded as one the most diverse subfamilies of GPCRs in humans. See, Liu et al., Genes, Brain and Behavior (2009) 8: 519–530. Similar to the other GPCRs in this family, the CB1R and CB2R comprise of a seven-transmembrane helical domain, extracellular and intracellular loops, an extracellular N terminus and an intracellular carboxy terminal tail and are activated by endogenously produced ligands.
The CB1R is highly expressed in most regions of the central nervous system (CNS), including in cells of central and the peripheral nervous system, with densities that rival other neurotransmitter and neuromodulatory receptors. See, Herkenham et al., Journal of Neuroscience, February 1991, 11 (2) 563-583. Moderate to high expression of CB1Rs have been observed in the cerebral cortex (cingulate gyrus, prefrontal cortex, and hippocampus), basal ganglia (globus pallidus, substantia nigra), periaqueductal gray, hypothalamus, amygdala, and cerebellum. The CB1R is highly expressed in brainstem medullary nuclei, such as the nucleus of the solitary tract and area postrema, serving as the primary integrative centers for the cardiovascular system and emesis, respectively. Moderate CB1R expression was also confirmed in the spinal cord (dorsal horn and lamina I, III, and X). More recently, dense CB1R positive fibers were identified in the ventral horn. Apart from the CNS, CB1R expression was reported in the somatic, sympathetic, parasympathetic, and the enteric nervous systems. See, Gomez et al., The Journal of Neuroscience, November 1, 2002, 22(21):9612–9617; Abstract, Coutts and Izzo, Current Opinion In Pharmacology. Volume 4, Issue 6, December 2004, Pages 572-579;
Croci et al., British Journal of Pharmacology (1998) 125, 1393-1395. The CB1R has also been reported in the peripheral organ systems of the body, albeit at lower, but functional levels, See, Szekeres et al., The Journal of Biological Chemistry Vol. 287, No. 37, pp. 31540 –31550, September 7, 2012. Functional CB1R have been reported in the liver, muscle, adipose tissue, vasculature, heart, pancreatic beta cells, reproductive organs, and alveolar cells.
The CB2R, on the other hand is expressed in high levels in immune cells and in lymphoid tissues. See, Li et al., Cell 176, 459–467, January 24, 2019. Cells that participate in both innate and adaptive immune response, such as the spleen, thymus, and peripheral blood mononuclear cells, are known to express high levels of CB2R. See, Galiègue et al., Eur. J. Biochem. 232, 54-61 (1995). Under pathological conditions, various peripheral cell types have also been shown to express detectable levels of the CB2R. This includes activated hepatic stellate cells (See Abstract, Julien et al., Volume 128, Issue 3, March 2005, Pages 742-755) and renal cells from fibrotic kidney (See Abstract, Zhou et al., Kidney International, Volume 94, Issue 4, October 2018, Pages 756-772), However, there have been reports of detectable levels of the CB2R under normal physiological conditions in pancreatic acinar cells (See, Michler et al., Am. J. Physiol. Gastrointest. Liver Physiol. 304: G181–G192, 2013), adipocytes (See Abstract, Roche et al., Histochemistry and Cell Biology, Volume 126, pages177–187(2006)), skeletal muscle cells (See Abstract, Yu et al., Intl. J. of Legal Medicine, Volume 124, pages 397–404 (2010)), cardiomyocytes, and endothelial cells. See Abstract, Lépicier et al., Life Sciences, Volume 81, Issues 17–18, 13 October 2007, Pages 1373-1380. Additionally, both CB1R and CB2R have been detected in connective tissue such as fascial fibroblasts and osteoclast cells. See, Fede et al., Eur. J. Histochem. 2016 Apr 11; 60(2): 2643. An overview of tissue distribution of the CB1R and the CB2R in healthy conditions is shown in the figure below:
“Not only do both the CB1R and the CB2R have complimentary roles in various pathological conditions, but they also exhibit contrasting/unique roles in several disease states.” See, Hapsula and Clark, Int. J. Mol. Sci. 2020 Oct. 17;21(20):7693. “The ubiquitous nature of cannabinoid receptors lends itself to regulate a variety of cellular and physiological processes. From regulation of cellular functions, such as neuromodulation, to orchestrating complex metabolic and immune responses, cannabinoid receptors serve essential role in both physiological and pathological conditions. Since the endocannabinoid system is vital for homeostasis of several biological processes, several pathological conditions pertaining to cardiovascular, neurological, metabolic and immunological diseases, are often associated with alterations of endocannabinoid tone.”
Strategies to alter endocannabinoid tone in pathological conditions, with minimal-to-no adverse effect profiles, are highly advantageous. This is especially true, since targeting central CB1R has been associated with severe neurological adverse effects. This must be achieved by either employing a direct strategy, which involves either activating or inactivating cannabinoid receptors using mono- or combination therapies, or indirectly, by inhibiting degradative enzymes of endocannabinoids resulting in enhanced cannabinoid receptor activation. See, Roger G. Pertwee, British Journal of Pharmacology (2009), 156, 397–411; Abstract, Rizzo et al., Neuroscience Letters, Volume 462, Issue 2, 22 September 2009, Pages 135-139; Abstract Sallaberry and Astern, JYn this articleI, June 2018, Vol. 34, Issue 6, p 48-55); and McPartland et al., PLOS ONE, published March 12, 2014, Vol. 9 (3).
There are multiple known endocannabinoids that play a role in the ECS. All of them seem to play a role in anti-proliferative, anti-inflammatory, and anti-metastatic effects (See, Madia & Daeninck, Current Oncology, Vol. 23, No. 6, December 2016). Additionally, it appears that they have a role in neurotransmitter, immune system, and mitochondrial function. There are two main endocannabinoids: anandamide and 2-archidonyl glycerol (2-AG). See, Sallaberry and Astern, JYI, June 2018, Vol. 34, Issue 6, p 48-55.
The classical endogenous cannabinoids or endocannabinoids are 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamide [AEA]), which belong to a class of compounds called eicosanoids that are synthesized on-demand from arachidonic acid-containing phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylethanolamine (PE), respectively. See, Di Marzo and De Petrocellis, Phil. Trans. R. Soc. B (2012) 367, 3216–3228. “2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide) are the best-studied endogenous cannabinoids. Despite similarities in chemical structure, 2-AG and anandamide are synthesized and degraded by distinct enzymatic pathways, which impart fundamentally different physiological and pathophysiological roles to these two endocannabinoids.” See, Lu and Mackie, Biol. Psychiatry. 2016 Apr 1; 79(7): 516–525.
AEA biosynthesis includes enzymatic cleavage of a phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine (NAPE). NAPE is synthesized by the enzymatic transfer of arachidonic acid in the sn-1 position of a phosphatidylcholine to the amide group of a phosphatidylethanolamine under the supervision of the calcium-independent N-acyltransferase (NAT). See, Pacher et al., Pharmacol Rev. 2006 Sep; 58(3): 389–462. NAPE is then hydrolyzed to AEA by a specific phospholipase D (NAPE-PLD) which has recently been cloned and molecularly characterized. See, Okamoto et al., The J. Biol. Chem. Vol. 279, No. 7, Issue of February 13, pp. 5298–5305, 2004.
Anandamide may be a very important cannabinoid to manipulate for controlling pain stimuli. See, Madia & Daeninck, Current Oncology, Vol. 23, No. 6, December 2016. “This is due to an interesting quality of anandamide in which the concentration of anandamide dictates the type and number of receptors activated. Anandamide also has the ability to make or break short-term connections between nerve cells that directly affect memory. There is speculation whether anandamide dulls and removes not only physical pain but psychological discomfort as well. If so, this could be utilized to help individuals with posttraumatic stress disorder (PTSD). This argument has particular merit as repression is a well-known coping mechanism. See, De Petrocellis et al., PNAS July 7, 1998 95 (14) 8375-8380. Furthermore, anandamide has been shown to have anti-proliferative effects in breast cancer. It has also been shown to bind with a strong affinity to the CB1 receptors, which may play a greater role in the analgesic effects of the endocannabinoids.”
2-AG is synthesized in a two-step process. See, Basavarajappa, Protein Pept. Lett. 2007; 14(3): 237–246. First, the 2-AG precursor diacylglycerol (DAG) is formed from enzymatic cleavage of membrane phospholipid precursors by the enzyme phospholipase C (PLC). DAG is subsequently hydrolyzed by a diacylglycerol lipase (DAGL) selective for the sn-1 position to generate 2-AG.
2-AG is the most prevalent endocannabinoid in the human body. Its chemical structure is quite similar to anandamide, having the same carbon backbone but a different R-group, C23H38O4. See Abstract, Gonsiorek et al., Molecular Pharmacology May 2000, 57 (5) 1045-1050. It is considered a full agonist of both the CB1 and CB2 receptors, playing a major role in ECS. Due to its high expression in peripheral immune cells, it seems to play a large role in anti-inflammation through immune suppression. Nonetheless, it also functions as a psychoactive endocannabinoid when it binds to CB1 receptors within brain cells. See, Madia & Daeninck, Current Oncology, Vol. 23, No. 6, December 2016.
As mentioned above, endocannabinoids (including AEA and 2-AG) are not stored in cellular vesicles. However, they are rapidly synthesized following the increase in the intracellular calcium level. See, Hanlon et al., Breast Cancer (Dove Med Press). 2016; 8:59-71. Released from depolarized postsynaptic neurons, endocannabinoids act as retrograde messengers, binding and activating CB1 receptors at presynaptic terminals. See, Lerner and Krietzer, Neuron. 2012 Jan 26; 73(2): 347–359. Their action results in the inhibition of neurotransmitter release, mostly glutamate and γ-aminobutyric acid.
Given the subsequent recognition of the ubiquitous distribution of AEA, AEA-metabolizing enzymes, and their receptors, it is not surprising that AEA has been linked with so many diverse physiologic and pathophysiologic actions and functions including modulatory effects on sensory and autonomic nerve signaling (See Abstract, Ralevic and Kendall, Curr. Vasc. Pharmacol. 2009 Jan;7(1):15-25.), regulation of energy consumption and balance (See Abstract, Bermudez-Silva et al., Pharmacology Biochemistry and Behavior, Volume 95, Issue 4, June 2010, Pages 375-382), and initiation and control of inflammation (See Abstract, Turcotte et al., J. Leukoc. Biol. 2015 Jun; 97(6):1049-70)). “Thus, FAAH and MGL inhibitors increase endocannabinoid accumulation (AEA and 2-AG, respectively) by inhibiting hydrolysis of fatty-acid amides and monoacylglycerols; these enzymes have multiple substrates.” See, Guindon and Hohmann, CNS Neurol. Disord. Drug Targets. 2009 Dec; 8(6): 403–421.
“With respect to the functional role of changes in the ECS signaling during stress, studies have demonstrated that the decline in AEA appears to contribute to the manifestation of the stress response, including activation of the hypothalamic–pituitary–adrenal (HPA) axis and increases in anxiety behavior, while the increased 2-AG signaling contributes to termination and adaptation of the HPA axis, as well as potentially contributing to changes in pain perception, memory and synaptic plasticity. More so, translational studies have shown that the ECS signaling in humans regulates many of the same domains and appears to be a critical component of stress regulation, and impairments in this system may be involved in the vulnerability to stress-related psychiatric conditions, such as depression and posttraumatic stress disorder.” See, Morena et al., Neuropsychopharmacol 41, 80–102 (2016).
Below is a general overview of the synthesis of AEA and A-AG, including the enzymes involved in degradation of AEA and 2-AG, respectively.
The ECS runs through adipose tissue, demonstrating its role in adipogenesis, lipogenesis, and glucose uptake, all of which are stimulated by the CB1 receptor. Cannabinoids are unique in that they are rapidly synthesized as well as broken down soon after being used, which creates fewer long-term side effects. The two main enzymes that break down these endocannabinoids are fatty amide acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL). See Abstract, Petrosino and Di Marzo, Curr. Opin. Investig. Drugs. 2010 Jan;11(1):51-62. “The endogenous cannabinoid system is extremely ubiquitous due to the fact that cannabinoids are both rapidly synthesized and degraded, which creates less long-term side effects.”
AEA degradation in the CNS is primarily by the enzyme fatty acid amino hydrolase (FAAH). “As its name suggests, FAAH degrades multiple fatty acid amides, including palmitoyl and oleoyl ethanolamide. This has important experimental and therapeutic implications as inhibition of FAAH increases levels of these ethanolamides.” A second pathway for anandamide degradation is via oxidation by cyclooxygenase-2 (COX-2), to create prostamides. See, Woodward et al., Br. J. Pharmacol. 2008 Feb; 153(3): 410–419. These compounds have distinct biological actions that are independent of cannabinoid receptors, have their own unique pharmacology and have a significant role as a therapy for intraocular hypertension. The differences in structure between arachidonic acid and anandamide are sufficient to allow the development of COX-2 inhibitors that inhibit anandamide oxidation without affecting prostaglandin formation. See, Hermanson et al., Nat, Neurosci. 2013 Sep; 16(9): 1291–1298. Furthermore, COX-2 is reasonably selective for anandamide over other acyl ethanolamides, so its inhibition offers a more selective way to increase anandamide when compared to inhibition of FAAH. See, Hermanson et al., Trends Pharmacol. Sci. 2014 Jul; 35(7): 358–367. A third potential route of anandamide degradation is via N-acylethanolamine-hydrolyzing acid amidase (NAAA). See, Tsuboi et al., The J. of Biol. Chem. Vol. 280, No. 12, Issue of March 25, pp. 11082–11092, 2005. “Inhibition of FAAH may shunt anandamide metabolism to one of these alternative pathways, altering cell functions that may be independent of cannabinoid receptor engagement.”
2-AG degradation is primarily due to three hydrolytic enzymes, monoacylglycerol lipase (MGL) and alpha/beta domain hydrolases 6 and 12 (ABHD6 and 12). See, Blankman et al., Chem. Biol. 2007 Dec; 14(12): 1347–1356. Additionally, 2-AG can be oxidized by COX-2 and hydrolyzed under some conditions by FAAH. See, Hermanson et al., Trends Pharmacol. Sci. 2014 Jul; 35(7): 358–367.The first three enzymes have different subcellular localizations, which likely define degradation of 2-AG in different cellular compartments. MGL is widespread and in the adult nervous system is primarily localized in synaptic terminals. See, Ludànyi, Neuroscience. 2011 Feb 3; 174: 50–63. It appears to account for the majority of 2-AG hydrolysis in a broad survey of brain 2-AG hydrolytic activity. See, Schlosburg, Nat Neurosci. 2010 Sep; 13(9): 1113–1119. One consequence of MGL inhibition is increased 2-AG signaling at CNS CB1R, however, it also reduces available levels of arachidonic acid, which is required for prostaglandin synthesis. See, Nomura et al., Science. 2011 Nov 11; 334(6057): 809–813. Subsequently, prostaglandin-mediated inflammatory processes are lessened by MGL inhibition.” In contrast to the presynaptic localization of MGL (See, Uchiganshima et al., J. Neurosci. 2007 Apr 4; 27(14): 3663–3676), ABHD6 is primarily localized to dendrites and dendritic spines of excitatory neurons in cortex. See, Marrs et al., Nat. Neurosci. 2010 Aug; 13(8): 951–957. Inhibition of ABHD6 also increases 2-AG signaling through CB1 receptors in the CNS. See, Naydenov et al., Neuron. 2014 Jul 16; 83(2): 361–371. ABHD6 also has significant functions outside of the CNS e.g., (79, 80), which will complicate the application of ABHD6-based therapies to CNS disorders. See, Thomas et al., Cell Rep. 2013 October 31; 5(2). ABHD12 is the other major hydrolytic enzyme suggested to be involved in the hydrolysis of 2-AG in brain. While its role in 2-AG metabolism in vivo is not firmly established, it plays a significant role in the degradation of long chain lyso-phosphatidylserines. See, Blankman et al., Proc. Natl. Acad. Sci. U S A. 2013 Jan 22; 110(4): 1500–1505.
Reviewing the experimental studies to understand how ECS functions through the regulation of neurotransmitter function, apoptosis, mitochondrial function, and ion-gated channels, researchers have concluded the following (See, Sallaberry and Astern, JYI, June 2018, Vol. 34, Issue 6, p 48-55):
The ECS is one of the, if not the most, important systems in our body. Its role in the homeostatic function of our body is undeniable, and its sphere of influence is incredible. Additionally, it also plays a major role in apoptotic diseases, mitochondrial function, and brain function.
Its contribution is more than maintaining homeostasis; it also has a profound ability in regulation. Working in a retrograde fashion and with a generally inhibitory nature, ECS can act as a “kill switch.” However, it has been shown to play an inhibitory or stimulatory role based on the size of the influx of cannabinoids, resulting in a bimodal regulation. Furthermore, due to the nature of the rate of degradation of cannabinoids, it does not have as many long-term side effects as most of the current drugs on the market.
The ECS may not only provide answers for diseases with no known cures, but it could change the way we approach medicine. This system would allow us to change our focus from invasive pharmacological interventions (i.e. SSRIs for depression, benzodiazepines for anxiety, chemotherapies for cancer) to uncovering the mystery of why the body is failing to maintain homeostasis. Understanding the roles of ECS in these diseases confers a new direction for medicine which may eradicate the use of some of the less tolerable therapeutics.
The Federal Food, Drug, and Cosmetic Act require that we inform you that the efficacy of any cannabinoid, including, but not limited to, CBD or CBN, products has not been confirmed by FDA-approved research as a treatment for any medical condition. The information in this document is not intended to diagnose, treat, cure or prevent any disease.
Originally published by FP Botanicals on November 7, 2020.