CB1 Receptor Research Articles

Identification of different extracts and phytoconstituents of Callistemon viminalis Cheel for their anti-anxiety effects based on pharmacognostic, toxicological, and pharmacological strategies

BackgroundPsychiatric disorders like depression and anxiety are global challenges, exacerbated by the limitations of synthetic medications, including addiction and toxic side effects. MethodsThis study meticulously investigated the pharmacognostic, phytochemical, toxicological, and pharmacological properties of Callistemon viminalis Cheel. Toxicological assessments, including hemocompatibility assays, LD50 studies, FOB analysis, biochemical parameters, and structural integrity of vital organs, were conducted on aqueous, methanolic, chloroform, and petroleum ether extracts of leaves and stems. Phytochemical profiling via qualitative tests and GC-MS screened extracts for molecular docking against key receptors. Categorically screened extracts were evaluated for therapeutic potential against LPS-induced anxiety in mice. ResultsToxicological evaluations on experimental animals demonstrated the safety of various extracts, evidenced by no in vitro and in vivo toxicity. GC-MS identified numerous phytochemicals that passed “Lipinski's Rule of Five.” These compounds were screened for molecular docking, revealing significant binding affinities with CB1, SERT, α2A-AR, and GABAβ2 receptors, suggesting potential therapeutic effects against anxiety. The phytoconstituents with the highest docking scores, particularly in aqueous and methanolic extracts, were further validated for their therapeutic efficacy. Preliminary analysis based on the EPM test and serum cortisol levels confirmed these extracts' superior therapeutic effectiveness. ConclusionIn conclusion, aqueous and methanolic extracts of Callistemon viminalis Cheel’s leaf and stem showed promising potential as therapeutic interventions for anxiety disorders.

The Endocannabinoid Peptide RVD-Hemopressin Is a TRPV1 Channel Blocker.

Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp's role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1's selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp's potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.

Mechanistic insights into the impact of WIN 55, 212-2, a synthetic cannabinoid, on adhesion molecules PECAM-1 and VE-cadherin in HeLa cells: implications on cancer processes

The endocannabinoid (eCB) system comprises endogenous ligands, cannabinoid receptors (CBRs), and their regulatory proteins; its alteration leads to many diseases including cancer. Thus, becomes a therapeutic target for synthetic cannabinoids aimed to control cancer cell proliferation, migration, adhesion, and invasion. However, little is known about adhesion molecules regulation through CBRs activation. The aim of this study was to evaluate the effects of a CB1/CB2 agonist, WIN-55, 212-2 (WIN), on the regulation of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial cadherin (VE-cadherin) in HeLa cells. CBRs expression was evaluated by immunofluorescence staining in HeLa cells and cell viability (thiazolyl blue tetrazolium bromide), cell adhesion (crystal violet), adhesion molecules expression and location (Western blot and immunofluorescence staining assays) were all assessed on cells treated with different WIN concentrations. Receptors CB1, CB2, and G-protein-coupled receptor 55 were expressed in HeLa cells. Additionally, biphasic effects were observed in their metabolic activity and adhesive properties: low WIN concentrations resulted in significant increases whereas, high ones decreased them compared to controls (p < 0.0001), demonstrating that WIN elicits opposite effects depending on the concentration and exposure time. PECAM-1 was detected in HeLa cell’s cytoplasm, membrane, and perinuclear region, whereas VE-cadherin had a nuclear distribution. There were no significant differences in PECAM-1 and VE-cadherin expression and location, suggesting that WIN does not modulate these proteins. These findings support the potential use of WIN due to its anticancer properties without dysregulating adhesion molecules. WIN possible contribution to inhibit cancer progression should be further investigated.

The multicomponent Passerini reaction as a means of accessing diversity in structure, activity and properties: Soft and hard vanilloid/cannabinoid modulators

A growing body of evidence points to the existence of a crosstalk between the endovanilloid (EV)- and the endocannabinoid (EC) systems, leading to the concept of a single system based on a shared set of endogenous ligands and regulation mechanisms. The EV/EC system encompasses the ion channel TRPV1, the G protein coupled receptors CB1 and CB2, their endogenous ligands and the enzymes for biosynthesis and inactivation. Disorders in which the EV/EC interaction is involved are inflammation, pain, neurodegenerative diseases and disorders of bones and skin.In the present paper, with the aim of targeting the EV/EC system, the Passerini reaction is used in a diversity-oriented approach to generate a series of α-acyloxycarboxamides bearing different substructures that resemble endogenous ligands. Compounds have been screened for activity on TRPV1, CB1 and CB2 and metabolic stability in skin cells, liver subcellular fractions and plasma. This protocol allowed to generate agents characterized by a diverse activity on TRPV1, CB1 and CB2, as well as heterogeneous metabolic stability that could allow different routes of administration, from soft drugs for topical treatment of skin diseases to hard drugs for systemic use in inflammation and pain. Compared to natural mediators, these compounds have a better drug-likeness. Among them, 41 stands out as an agonist endowed with a well-balanced activity on both TRPV1 and CB2, high selectivity over TRPM8, TRPA1 and CB1, metabolic stability and synthetic accessibility.

Abstract P173: Pivotal Role for the Kinin B1R in CB1 Receptor Mediated Neuroinflammation and Pressor Response

The cannabinoid receptors CB1R and CB2R are involved in blood pressure (BP) regulation and CB1R is implicated in the incidence of hypertension. CB1R is highly expressed in the brain but both receptors are also expressed in the heart. Prior research has demonstrated that CB1R mediates significant cardiovascular responses, including elevated BP, heightened plasma norepinephrine levels, and increased sympathetic nerve activity. Furthermore, the activation of the kinin B1 receptor (B1R) mediates similar effects when studied independently. However, the role of B1R in CB1R-mediated BP regulation and the associated signaling mechanisms remain unknown. In this study, we tested the hypothesis that B1R plays a pivotal role in CB1R-mediated pressor response, oxidative stress and neuroinflammation. To test this, we used both in vivo mouse models and in vitro primary hypothalamic neurons. Male C57BL/6NJ wild-type (WT) and B1R knockout mice (B1RKO) underwent surgical carotid artery and jugular vein catheterization to measure BP in conscious state. Following stabilization of BP for at least 90 minutes, the CB1R agonist WIN55,212-2 (300 ug/kg; i.v., WIN) or its vehicle was administered, and recordings continued for 45 minutes. WIN increased BP in WT mice (25±7 mmHg vs baseline), but this effect was blunted in B1RKO mice (n=3, p&lt;0.05). Moreover, administration of WIN led to an augmentation of CB1R expression in the paraventricular nucleus of WT mice, but not in B1RKO mice, thereby indicating the involvement of B1R in CB1R signaling (p&lt;0.05, n=3 mice/group). WT, but not B1RKO, mice treated with WIN displayed a significant increase in oxidative stress levels (p&lt;0.05, n=3 mice/group), as indicated by dihydroethidium staining. To further confirm the role of B1R blockade on CB1R signaling, we cultured primary hypothalamic neurons, as neurons express the highest levels of CB1R in the brain. Neurons were stimulated with WIN (1 uM) both with and without a B1R selective antagonist (SSR240612, 10uM). The results indicated that WIN incubation for 24 hours increased CB1R expression, inflammation, and oxidative stress compared to vehicle treated, and these responses were abrogated by prior B1R blockade (p&lt;0.05, n=5/group), consistent with the in vivo observations. These data provide novel evidence that B1R is implicated in CB1R-mediated proinflammatory responses and serves as a potential therapeutic target to reduce CB1R induced effects.

Cannabis sativa L. Extract Alleviates Neuropathic Pain and Modulates CB1 and CB2 Receptor Expression in Rat.

Cannabis sativa L. (CSL) extract has pain-relieving potential due to its cannabinoid content, so the effects of two CSL extracts on alleviating neuropathic pain were investigated in vivo. Methods and groups: Male Wistar rats (n = 130) were divided into groups and received vincristine (0.1 mg/kg) and gabapentin (60 mg/kg) to induce and relieve neuropathic pain or CSL extracts (D and B). The mRNA and protein expression of the cannabinoid receptors type 1 and 2 (CB1R, CB2R) were evaluated in the cerebral cortex, hippocampus, and lymphocytes. Behavioural tests (Tail-Flick and von Frey) were performed on all animals. VK-induced neuropathic pain was accompanied by decreased CB1R protein level and CB2R mRNA expression in the cortex. Gabapentin relieved pain and increased CB1R protein levels in the hippocampus compared to the vincristine group. Hippocampus CB1R protein expression increased with the administration of extract D (10 mg/kg, 40 mg/kg) and extract B (7.5 mg/kg, 10 mg/kg) compared to VK group. In the cerebral cortex CSL decreased CB1R protein expression (10 mg/kg, 20 mg/kg, 40 mg/kg of extract B) and mRNA level (5 mg/kg, 7.5 mg/kg of extract B; 20 mg/kg of extract D) compared to the VK-group.CB2R protein expression increased in the hippocampus after treatment with extract B (7.5 mg/kg) compared to the VK-group. In the cerebral cortex extract B (10 mg/kg, 20 mg/kg) increased CB2R protein expression compared to VK-group. Alterations in cannabinoid receptor expression do not fully account for the observed behavioural changes in rats. Therefore, additional signalling pathways may contribute to the initiation and transmission of neuropathic pain. The Cannabis extracts tested demonstrated antinociceptive effects comparable to gabapentin, highlighting the antinociceptive properties of Cannabis extracts for human use.

Homodimerization of CB2 cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we show that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB2 receptor (CB2R) homodimerization by simultaneously binding both protomers of the CB2R-CB2R homodimer. Computational and pharmacological experiments showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This results in unique pharmacological properties, including increased potency in Gi-mediated signaling and enhanced recruitment of β-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB2R activity, potentially leading to improved therapeutic outcomes.

Olfactory bulb astrocytes link social transmission of stress to cognitive adaptation in male mice

Emotions and behavior can be affected by social chemosignals from conspecifics. For instance, olfactory signals from stressed individuals induce stress-like physiological and synaptic changes in naïve partners. Direct stress also alters cognition, but the impact of socially transmitted stress on memory processes is currently unknown. Here we show that exposure to chemosignals produced by stressed individuals is sufficient to impair memory retrieval in unstressed male mice. This requires astrocyte control of information in the olfactory bulb mediated by mitochondria-associated CB1 receptors (mtCB1). Targeted genetic manipulations, in vivo Ca2+ imaging and behavioral analyses reveal that mtCB1-dependent control of mitochondrial Ca2+ dynamics is necessary to process olfactory information from stressed partners and to define their cognitive consequences. Thus, olfactory bulb astrocytes provide a link between social odors and their behavioral meaning.

Basal forebrain-lateral habenula inputs and control of impulsive behavior.

Deficits in impulse control are observed in several neurocognitive disorders, including attention deficit hyperactivity (ADHD), substance use disorders (SUDs), and those following traumatic brain injury (TBI). Understanding brain circuits and mechanisms contributing to impulsive behavior may aid in identifying therapeutic interventions. We previously reported that intact lateral habenula (LHb) function is necessary to limit impulsivity defined by impaired response inhibition in rats. Here, we examine the involvement of a synaptic input to the LHb on response inhibition using cellular, circuit, and behavioral approaches. Retrograde fluorogold tracing identified basal forebrain (BF) inputs to LHb, primarily arising from ventral pallidum and nucleus accumbens shell (VP/NAcs). Glutamic acid decarboxylase and cannabinoid CB1 receptor (CB1R) mRNAs colocalized with fluorogold, suggesting a cannabinoid modulated GABAergic pathway. Optogenetic activation of these axons strongly inhibited LHb neuron action potentials and GABA release was tonically suppressed by an endogenous cannabinoid in vitro. Behavioral experiments showed that response inhibition during signaled reward omission was impaired when VP/NAcs inputs to LHb were optogenetically stimulated, whereas inhibition of this pathway did not alter LHb control of impulsivity. Systemic injection with the psychotropic phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC), also increased impulsivity in male, and not female rats, and this was blocked by LHb CB1R antagonism. However, as optogenetic VP/NAcs pathway inhibition did not alter impulse control, we conclude that the pro-impulsive effects of Δ9-THC likely do not occur via inhibition of this afferent. These results identify an inhibitory LHb afferent that is controlled by CB1Rs that can regulate impulsive behavior.

Activation of CB2 Receptors by (-)-Cannabichromene but Not (+)-Cannabichromene.

Introduction: Cannabichromene (CBC) is a minor constituent of cannabis that is a selective cannabinoid CB2 receptor agonist and activator of TRPA1. To date, it has not been shown whether (-)-CBC, (+)-CBC, or both can mediate these effects. In this study, we investigate the activity of the CBC enantiomers at CB1, CB2, and Transient receptor potential ankyrin 1 (TRPA1) receptors in vitro. Materials and Methods: CBC enantiomers were purified from synthetic CBC by chiral chromatography, and their optical activity was confirmed by spectroscopy. Human CB1 and CB2 receptor activity was measured using a fluorescent assay of membrane potential in stably transfected AtT20 cells. TRPA1 activation was measured using a fluorescent assay of intracellular calcium in stably transfected HEK293 cells. Results: The (-)-CBC activated CB2 with an EC50 of 1.5 µM, to a maximum of 60% of (-)CP55940. (+)-CBC did not activate CB2 at concentrations up to 30 µM. Only 30 µM (-)-CBC produced detectable activation of CB1, (+)-CBC was inactive. Both (-)-CBC and (+)-CBC activated TRPA1; at 30 µM (-)-CBC produced an activation 50% of that of the reference agonist cinnamaldehyde (300 µM), 30 µM (+)-CBC activated TRPA1 to 38% of the cinnamaldehyde maximum. Discussion: It is unclear whether (-)-CBC is the sole or even the predominant enantiomer of CBC enzymatically synthesized in cannabis. This study shows that (-)-CBC is the active isomer at CB2 receptors, while both isomers activate TRPA1. The results suggest that medicinal preparations of CBC that target cannabinoid receptors would be most effective when (-)-CBC is the dominant isomer.

Endocannabinoids regulate enteric neuron-glia networks and visceral hypersensitivity following inflammation through a glial-dependent mechanism.

Acute gastrointestinal (GI) inflammation induces neuroplasticity that produces long-lasting changes in gut motor function and pain. The endocannabinoid system is an attractive target to correct pain and dysmotility, but how inflammation changes endocannabinoid control over cellular communication in enteric neurocircuits is not understood. Enteric glia modulate gut neurons that control motility and pain and express monoacylglycerol lipase (MAGL) which controls endocannabinoid availability. We used a combination of in situ calcium imaging, chemogenetics, and selective drugs to study how endocannabinoid mechanisms affect glial responses and subsequent enteric neuron activity in health and following colitis in Wnt1Cre;GCaMP5g-tdT;GFAP::hM3Dq mice. Trpv1Cre;GCaMP5gtdT mice were used to study nociceptor sensitivity and Sox10CreERT2;Mgllf/f mice were used to test the role of glial MAGL in visceral pain. The data show that endocannabinoid signaling regulates neuro-glial signaling in gut neurocircuits in a sexually dimorphic manner. Inhibiting MAGL in healthy samples decreased glial responsiveness but this effect was lost in females following colitis and converted to an excitatory effect in males. Manipulating CB1 and CB2 receptors revealed further sex differences amongst neuro-glia signaling that were impacted following inflammation. Inflammation increased gut nociceptor sensitivity in both sexes but only females exhibited visceral hypersensitivity in vivo. Blocking MAGL normalized nociceptor responses in vitro and deleting glial Mgll in vivo rescued visceral hypersensitivity in females. These results show that sex and inflammation impact endocannabinoid mechanisms that regulate intercellular enteric glia-neuron communication. Further, targeting glial MAGL could provide therapeutic benefits for visceral nociception in a sex-dependent manner.

Preclinical Assessment of a Cannabinoid CB2 Receptor Antagonist in a Murine Model of Cerebral Malaria

Malaria is a most important parasitic disease due to its highest impact worldwide. It results in around 200 million clinical cases and 0,5-1 million deaths per year, mainly due to cerebral malaria (CM), a life-threatening neurological syndrome that predominantly affects predominantly children under five years old. CM follows neurological alterations leading to the death if left untreated, and, even when it is treated, it is fatal in 15-20% of cases. Moreover, among the survivors, more than 10% of the children develop neurological sequelae. Consequently, there is an urgent need to find therapies to attenuate these neurological signs. Recent evidence has proposed the endocannabinoid system, which plays an important neuromodulatory function in the central nervous system (CNS), also including immunomodulation preferentially exerted by CB2 receptor. Previous studies have shown that the genetic ablation of this receptor improved mice survival against CM, suggesting a potential for the pharmacological treatment of CM with selective antagonists of this receptor. Considering this background, we investigated CM therapy by a classic CB2 antagonist SR144528 in a murine model of the disease. First, we carried out binding studies with SR144528 to confirm its pharmacodynamic profile (binding affinity [Ki] value = 2.34 ± 0.61 nM; and efficacy [IC50] = 96.17 ± 1.41 nM, at the CB2 receptor). Second, P. berghei ANKA infected C57BL/6 mice were treated daily with SR144528 and assessed for parasitemia growth and neurological alterations. 30% of the treated mice showed partial recovery of CM symptoms with 20% increased survival, but finally succumbing to hyperparasitemia and severe anemia. These preliminary preclinical results suggest that, although part of the CM course might be modulated by the pharmacological blockade of the CB2 receptor, other elements trigger the lethal outcome. Thus, while our hypothesis could not be completely validated in this CM model, we detail here all obtained results for further research.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of d-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter d-serine, by its precursor l-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards l-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

Effects of ∆-9 tetrahydrocannabinol on the small intestine altered by high fructose diet: A Histopathological study

The consumption of fructose is increasing day by day. Understanding the impact of increasing fructose consumption on the small intestine is crucial since the small intestine processes fructose into glucose. ∆9-Tetrahydrocannabinol (THC), a key cannabinoid, interacts with CB1 and CB2 receptors in the gastrointestinal tract, potentially mitigating inflammation. Therefore, this study aimed to investigate the effects of the high-fructose diet (HFD) on the jejunum of rats and the role of THC consumption in reversing these effects. Experiments were conducted on Sprague–Dawley rats, with the experimental groups as follows: control (C), HFD, THC, and HFD + THC. The HFD group received a 10% fructose solution in drinking water for 12 weeks. THC groups were administered 1.5 mg/kg/day of THC intraperitoneally for the last four weeks. Following sacrification, the jejunum was evaluated for mucus secretion capacity. IL-6, JNK, CB2 and PCNA expressions were assessed through immunohistochemical analysis and the ultrastructural alterations via transmission electron microscopy. The results showed that fructose consumption did not cause weight gain but triggered inflammation in the jejunum, disrupted the cell proliferation balance, and increased mucus secretion in rats. Conversely, THC treatment displayed suppressed inflammation and improved cell proliferation balance caused by HFD. Ultrastructural examinations showed that the zonula occludens structures deteriorated in the HFD group, along with desmosome shrinkage. Mitochondria were found to be increased due to THC application following HFD. In conclusion, the findings of this research reveal the therapeutic potential of THC in reversing HFD-related alterations and provide valuable insights for clinical application.Graphical abstract

Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid’s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.

Acute rimonabant treatment prevents anhedonia and memory loss in rats submitted to mild restraint stress

Stress-related disorders are becoming increasingly common and are often associated with cognitive impairments. Within this context, the endocannabinoid (ECB) system, particularly the type 1 cannabinoid (CB1) receptor, seems to play a decisive role in restoring body homeostasis. There is consistent evidence in the literature that disrupted CB1-mediated neurotransmission can ultimately contribute to stress-related diseases. Therefore, the present study aimed to evaluate the participation of CB1 receptors in the integrity of stress-induced peripheral and behavioral responses. For this purpose, male adult Wistar rats underwent physical restraint (1 h/day, for 7 days), followed by a single administration of rimonabant (CB1 receptor antagonist, 3 mg/Kg, intraperitonial) at the end of stress protocol. Animals were then subjected to evaluation of neuroendocrine responses, behavioral tests and quantification of Iba-1 (microglial) immunoreactivity in the parvocellular subdivisions of the paraventricular nucleus of the hypothalamus (PVN). No effects of restraint stress or rimonabant administration were detected on body mass variation. However, stress significantly increased adrenal relative mass and corticosterone secretion, and reduced thymus relative size. The stress effects on adrenal size and corticosterone plasma levels were absent in rimonabant-treated rats, but the thymus size was further reduced in the restraint-rimonabant group. Restraint stress also induced anhedonia, a depression-like behavior, and reduced object recognition index, indicating memory recovery impairment. Treatment with the CB1 antagonist significantly reversed stress-induced anhedonia and memory deficit. In the PVN, restraint stress reduced the number of Iba-1 positive cells in the medial parvocellular region of vehicle- but not rimonabant-treated animals. Taken together, these results indicate that the acute inhibition of the CB1-mediated endogenous pathway restores stress-induced depression-like behaviors and memory loss, suggesting a role for endocannabinoids in the neuro-immune-endocrine interplay at both peripheral and hypothalamic levels.

Comparison of Agonist Activity between CB1 and CB2 Receptors with Orthosteric Site Mutations

Human endocannabinoid signaling is primarily mediated by the cannabinoid receptors, CB1 and CB2, which are G protein-coupled receptors (GPCRs). These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug targets due to their potential in treating pain and inflammation. However, because of their homology and shared signaling mechanisms, investigating the individual physiological roles of these receptors and designing subtype-selective ligands has been challenging. Using active-state CB1 and CB2 structures as guides, homologous residues within the orthosteric pocket of each receptor were mutated to alanine to test whether they equally impair CB1 and CB2 activity in response to two high-affinity, nonselective agonists (CP55,940 and AM12033). Interestingly, mutating the Y5.39 position impairs CB1 but not CB2 function. Conversely, mutating residue C6.47 improves CB1 but impairs CB2 signaling. The F7.35A mutation leads to a decrease in CP55,940 potency at CB1 and impairs internalization; however, AM12033 gains potency and promotes CB1 internalization. In CB2, mutation of F7.35A decreases the potency of CP55,940 and neither agonist induces internalization. These observations provide some insight into functional sensitivity of CB1 and CB2 to different agonists when conserved residues are mutated in the orthosteric pocket.

Regulation of Cannabinoid and Opioid Receptor Levels by Endogenous and Pharmacological Chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), δ opioid receptor (DOR), and CB1R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

From Classical to Alternative Pathways of 2-Arachidonoylglycerol Synthesis: AlterAGs at the Crossroad of Endocannabinoid and Lysophospholipid Signaling.

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.