Discovery and Neuropharmacology of Endogenous Cannabinoids
The discovery of endogenous cannabinoid substances in the brain has a rich history that began with the synthesis and radiolabeling of synthetic and potent cannabinoids that allowed the identification and characterization of cannabinoid receptors in the rat brain. The pharmacological potency of cannabinoids correlated well with their affinity for the cannabinoid binding site (Figure 8.13). The cannabinoid receptor distribution in rat brain sections showed the densest binding in the basal ganglia and cerebellum, with intermediate levels in the hippocampus, amygdala, and cortex. Low levels were found in brainstem areas. This corresponds well with the low levels of lethality of cannabinoids in terms of respiratory depression (Figure 8.14).
The identification of a brain binding site and early work that showed that cannabinoids inhibited the enzyme adenylate cyclase led to the hypothesis that the cannabinoid receptor was G-protein linked. Serendipitous cloning of the cannabinoid receptor that had homology with other G-protein receptors confirmed this hypothesis. The mRNA distribution of the receptor clone paralleled the distribution of the cannabinoid receptor. This cannabinoid receptor was named CB1 and belongs to the G-protein-coupled receptor subfamily.
WITHDRAWAL FROM CHRONIC USE OF SPICE
WITHDRAWAL FROM CHRONIC USE OF SPICE
A single case report describes chronic synthetic cannabinoid use in a 20-year-old man who smoked 3 g of Spice Gold daily for eight months. He found Spice relaxing, sedative, and to have cannabis-like psychoactive effects. However, he developed tolerance and the following signs of withdrawal during his hospital admission: “inner unrest, drug craving, nocturnal nightmares, profuse sweating, nausea, tremor, headache,” hypertension, and tachycardia.
From: Zimmermann US, Winkelmann PR, Pilhatsch M, Nees JA, Spanagel R, Schulz K. Withdrawal phenomena and dependence syndrome after the consumption of spice gold. Deutsches Ärzteblatt International, 2009, (106), 464–467.
CANNABIS AS A DOPING DRUG
CANNABIS AS A DOPING DRUG
Should cannabis be considered a “doping” drug in sports and continue to be banned? There are clear arguments that cannabis can have some beneficial effects in reducing anxiety, improving muscle relaxation, and reducing fearful memories. However, cannabis can impair performance and cause memory loss, executive function deficits, and motor impairment, in addition to the possibility of impairing lung function. Thus, cannabis can both improve performance and pose a health risk, two of the criteria for inclusion in the Prohibited List according to the Code of the World Doping Agency. Presumably, cannabis ingestion also violates the spirit of the sport, the third criterion. Thus, compelling arguments exist for considering cannabis a doping drug (for further reading, see Bergamaschi and Crippa, 2013).
The receptor-mediated action of cannabinoids was based on the synthesis and radiolabeling of potent cannabinoids and the establishment of binding in brain membranes. The initial site of Δ9-THC binding was hypothesized to be the CB1 receptor, which is widely distributed throughout the brain but is particularly concentrated in the extrapyramidal motor system. The CB1 receptor was the first to be cloned and localized to brain structures. Subsequently, the CB2 receptor was cloned and initially localized peripherally (outside the brain) and more recently within the brain. CB1 receptors mediate many of the psychoactive effects of cannabinoids. CB2 receptors in the periphery are mainly expressed in the immune system. However, CB2 receptors have recently been identified in the brain and may have functional effects in modulating the mesocorticolimbic dopamine system.
Both CB1 and CB2 receptors are coupled to the G-proteins Gi and Go, and this interaction inhibits adenylate cyclase. This inhibition leads to a subsequent reduction of cAMP and an increase in mitogen-activated protein kinase. Cannabinoids also enhance the activation of A-type potassium channels, enhance outward potassium current, inhibit voltage-activated N-type calcium channels, and inhibit presynaptic P/Q calcium channels. The activation of CB1 receptors inhibits the presynaptic release of other neurotransmitters via several molecular pathways, including the inhibition of adenylyl cyclase.
FIGURE 8.13 ED50 values (μmol/kg) plotted against the KI (nM) value for 29 different cannabinoids. The potency of these cannabinoid analogs administered intravenously in the mouse was assessed using four pharmacological measures: spontaneous activity, antinociception, hypothermia, and catalepsy. The solid line represents the linear regression of the relationship between the ED50 and the KI for each measure across 29 compounds. The dashed line represents the linear regression that would be obtained if the mean ED50 value of the four measures was plotted instead of that shown for each individual behavior, so it is the same in each panel. These data show the relationships between the ED50 for different cannabinoids as a function of their binding to the CB1 receptor. ED50 refers to the dose that produces a given effect in 50% of the population. Binding affinity in this study was determined using a radiolabeled ligand (known as a hot ligand). The drug in question in the figure involves binding site competition between a hot ligand and a cold ligand (29 different untagged cannabinoid ligands). Ki (nM) refers to the dose that displaces the binding of a tritiated high-potency synthetic cannabinoid CB1 receptor agonist by 50%. Notice that similar functions are generated for each of the different behaviors, suggesting a common site of action at the CB1 receptor. [Taken with permission from Compton DR, Rice KC, De Costa BR, Razdan RK, Melvin LS, Johnson MR, Martin BR, Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities, Journal of Pharmacology and Experimental Therapeutics, 1993, (265),218–226.]
The discovery of the cannabinoid receptors immediately raised the possibility that endogenous ligands for this receptor existed, resulting in the discovery of an arachidonic acid derivative called arachidonoylethanolamide (later termed anandamide) that bound to the CB1 receptor (Figure 8.15). Numerous subsequent studies found that anandamide binds competitively to the CB1 receptor, inhibits adenylate cyclase, inhibits voltage-sensitive calcium channels, and produces various behavioral and pharmacological effects that are similar to the effects of Δ9-THC, including antinociception, hypomotility, catalepsy, and hypothermia. However, relative to other cannabinoids, anandamide produces only weak and transient behavioral effects, possibly because of its rapid breakdown.
FIGURE 8.14 Autoradiographic film images that show cannabinoid receptor localization (A) in the rat. The sagittal section of the rat brain (B) shows the locations of neurons that express the mRNA at this level. High levels of receptor protein are visible in basal ganglia structures, including the globus pallidus (GP), entopeduncular nucleus (Ep), and substantia nigra pars reticulata (SNR). High binding is also seen in the cerebellum. Moderate binding is found in the hippocampus (Hipp), cortex, and caudate putamen (CPu). Low binding is found in the brain stem and thalamus. Notice that the GP, Ep, and SNR do not contain CB1 mRNA-expressing cells (B). This is because the receptors in these areas are on axons (large arrows in panel A) and terminals, and the mRNA-expressing cells of origin reside in the caudate and putamen. [Taken with permission from Freund TF, Katona I, Piomelli D. Role of endogenous cannabinoids in synaptic signaling. Physiological Reviews, 2003, (83), 1017–1066.]
After the identification of anandamide, a second endogenous cannabinoid (endocannabinoid) was found: 2-arachidonylglycerol (2-AG). 2-AG is an anandamide analog with a glycerol backbone. It has high affinity for the CB1 receptor and is found in the brain in amounts 1000 times higher than anandamide. Both of these endocannabinoids are lipid-like compounds that are synthesized and released from neurons, bind to cannabinoid receptors, activate transduction mechanisms, and have a reuptake system, although questions remain regarding the alleged anandamide transporter (Figure 8.16). However, endocannabinoids do not fulfill all the requirements of a “classic” neurotransmitter. They are not synthesized in the cytosol of neurons; they are not stored in synaptic vesicles to be secreted by exocytosis following excitation of nerve terminals by action potentials. Instead, endocannabinoids are synthesized in postsynaptic elements of neurons when required in response to depolarization by receptor-stimulated synthesis from membrane lipid precursors and released from cells immediately after their production (Figure 8.17). Once released, endocannabinoids act on cannabinoid receptors and may be taken back into cells via an energy-independent transport system. Once inside the cells, both anandamide and 2-AG can be broken down. This non-synaptic release mechanism and the rapid breakdown of both endocannabinoids suggest that these compounds may locally regulate the effects of primary neurotransmitters (Figure 8.18).
Advances in the understanding of the neuropharmacology of endocannabinoids have been aided by the identification of drugs that can block the formation, hypothesized reuptake, and inactivation of both anandamide and 2-AG. Anandamide reuptake is theorized to be blocked by the hypothesized anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM 404). The effects of exogenous anandamide are potentiated by AM 404, which has been shown to elevate the levels of circulating anandamide and produce behavioral effects, but it is not entirely clear that these effects are mediated by reuptake blockade. Inhibitors of anandamide amidohydrolase can also increase the levels of anandamide by blocking its breakdown. Another enzyme implicated in the function of anandamide is fatty acid amide hydrolase (FAAH), a membrane-associated hydrolase that breaks down anandamide. Genetically engineered mice that lack FAAH are unable to degrade anandamide. These mice also show an enhanced response to exogenous anandamide administration, including hypoactivity, analgesia, catalepsy, and hypothermia. In the brain, FAAH is localized in the cell body and dendritic areas of neurons that are postsynaptic to CB1-expressing axons, suggesting that FAAH may participate in cannabinoid signaling mechanisms by inactivating locally released endocannabinoids (for further reading, see Freund et al., 2003).
FIGURE 8.15Chemical structures of 2-arachidonoylglycerol, structural analogs, and synthetic cannabinoids.
FIGURE 8.16Signal transduction mechanisms stimulated by CB1 receptors in a presynaptic nerve terminal. [Taken with permission from Ameri A. The effects of cannabinoids on the brain. Progress in Neurobiology, 199, (58), 315–348.]
Key enzymes that regulate the generation and degradation of the other major endogenous cannabinoid, 2-AG, have been elucidated. The enzyme responsible for the synthesis of 2-AG is diacylglycerol lipase α (see Figure 8.17). The deletion of diacylglycerol lipase α in mice completely eliminated cannabinoid-based neuroplasticity and negative feedback function, suggesting that 2-AG is likely the predominant neurotransmitter for currently identified roles of the endogenous cannabinoid system. The enzyme primarily responsible for the breakdown of 2-AG is monoacylglycerol lipase (MAGL), and the blockade or elimination of this enzyme using a constitutional genetic knockout approach in mice produces an accumulation of excessive 2-AG in the brain and cannabinoid receptor-mediated effects on analgesia, hypothermia, and locomotor activity. Long-term excessive 2-AG accumulation eventually leads to cannabinoid tolerance, receptor downregulation, and even physical withdrawal typically seen with THC. Using a combination of FAAH (the anandamide-metabolizing enzyme) and MAGL inhibition to elevate anandamide and 2-AG simultaneously produces mild THC-like behavioral effects in mice and fully substitutes for THC in the drug discrimination paradigm, suggesting that the two endogenous cannabinoids act on separate but interacting neuronal populations to produce cannabinoid-like effects.