Arachidonyl-2-chloroethylamide Research Articles

SAT025 Characterization Of The Melatonin Type 1 Receptor / Cannabinoid Type 1 Receptor (MT1/CB1) Heteromers

Abstract Disclosure: Y. Cui: None. H. Cherdoud: None. G. Marsicano: None. L. Bellocchio: None. R. Jockers: None. O. Lahuna: None. Membrane receptors belonging to the G proteins coupled receptors (GPCRs), the largest family of proteins in the human genome with more than 800 members. GPCR functions were initially thought to take place at the plasma membrane upon binding of single monomeric receptor unit by their cognate ligand. This scheme was complexified after identification of cross-talks between GPCRs upon heteromerization and identification of functional GPCRs within intracellular organelles as nucleus or endosomes. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we are identifying and characterizing MT1/CB1 heteromers as a new GPCR heteromer. Previously we showed that coexpression of the MT1 and CB1 receptors induced the constitutive formation of MT1/CB1 heteromers using coimmunoprecipitation experiments on transiently transfected HEK293 cells. Using simultaneous PLA (Proximity Ligation Assay) and immunofluorescence experiments, we described the existence of intracellular MT1/CB1 heteromers associated with the mitochondrial network. Since then, we have further characterized the properties of MT1/CB1 heteromers. Coimmunoprecipitation experiments performed on cerebellum from wild-type, MT1 or CB1 knock-out mice enabled us to identify MT1/CB1 heteromers in vivo. We established stable monoclonal HEK293 cell lines expressing both receptors. Transmission Electronic Microscopy (TEM) and PLA experiments showed the presence of MT1/CB1 complexes not only intracellularly but also at the cell surface. Using PLA experiments we identified a penetrating interfering peptide that specifically dissociate the MT1/CB1 complex and thus open the door to experiments targeting specifically the complex. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 complex from the plasma membrane to the late endosomes using a combination of Nanoluciferase complementation experiments and bystander BRET (Bioluminescence Resonance Energy Transfer). Costimulation of HE293 cells expressing both receptors with melatonin and the selective CB1 agonist ACEA (Arachidonyl-2'-chloroethylamide), a derivative of the endocannabinoid AEA (Arachidonylethanolamide or anandamide) resulted in a potentiation of the melatonin-dependent cAMP inhibition. Use of the identified interfering peptide will enable us to identify actions on signaling pathways resulting from a direct cross-talk between MT1 or CB1 subunits. Presentation: Saturday, June 17, 2023

The Cannabinoid Ligand Arachidonyl-2'-Chloroethylamide (ACEA) Ameliorates Depressive and Overactive Bladder Symptoms in a Corticosterone-Induced Female Wistar Rat Model.

There is growing need to increase the knowledge on the cannabinoid ligands in the treatment of overactive bladder. Among potential candidates, arachidonyl-2'-chloroethylamide (ACEA), a selective cannabinoid CB1 receptor agonist is proposed. The aim of this paper was to determine if ACEA, a selective cannabinoid CB1 receptor agonist, could reverse the effects of corticosterone (CORT), characteristic of depressive and bladder overactivity potential. The animals (48 female rats) were divided into four groups: I-control, II-received CORT, III-received ACEA, and IV-received the combination of CORT and ACEA. The conscious cystometry, forced swim test (FST), and locomotor activity measurements were performed 3 days after the last dose of ACEA, followed by ELISA measurements. In group IV, ACEA restored urodynamic parameters that were altered by CORT. CORT prolonged the immobility time in FST and the values were lowered by ACEA. ACEA normalized the expression of c-Fos in all the analyzed central micturition centers (group IV vs. group II). ACEA restored the CORT-induced changes in the biomarkers in urine (BDNF, NGF), bladder detrusor (VAChT, Rho kinase), bladder urothelium (CGRP, ATP, CRF, OCT-3, TRPV1), and hippocampus (TNF-α, IL-1β and Il-6, CRF, IL-10, BDNF, NGF). In conclusion, ACEA was proven to reverse CORT-induced changes in both cystometric and biochemical parameters that are determinants of OAB/depression, which represents an example of an existing link between OAB and depression via cannabinoid receptors.

Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes.

Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)-CB1R agonist, rimonabant-CB1R antagonist, John W. Huffman (JWH133)-CB2R agonist and CB2R antagonist-6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation.

Cannabinoid 1 and mu-Opioid Receptor Agonists Synergistically Inhibit Abdominal Pain and Lack Side Effects in Mice.

While effective in treating abdominal pain, opioids have significant side effects. Recent legalization of cannabis will likely promote use of cannabinoids as an adjunct or alternative to opioids, despite a lack of evidence. We aimed to investigate whether cannabinoids inhibit mouse colonic nociception, alone or in combination with opioids at low doses. Experiments were performed on C57BL/6 male and female mice. Visceral nociception was evaluated by measuring visceromotor responses (VMR), afferent nerve mechanosensitivity in flat-sheet colon preparations, and excitability of isolated DRG neurons. Blood oxygen saturation, locomotion, and defecation were measured to evaluate side effects. An agonist of cannabinoid 1 receptor (CB1R), arachidonyl-2'-chloroethylamide (ACEA), dose-dependently decreased VMR. ACEA and HU-210 (another CB1R agonist) also attenuated colonic afferent nerve mechanosensitivity. Additionally, HU-210 concentration-dependently decreased DRG neuron excitability, which was reversed by the CB1R antagonist AM-251. Conversely, cannabinoid 2 receptor (CB2R) agonists did not attenuate VMR, afferent nerve mechanosensitivity, or DRG neuron excitability. Combination of subanalgesic doses of CB1R and µ-opioid receptor agonists decreased VMR; importantly, this analgesic effect was preserved after 6 d of twice daily treatment. This combination also attenuated afferent nerve mechanosensitivity and DRG neuron excitability, which was inhibited by neuronal nitric oxide synthase and guanylate cyclase inhibitors. This combination avoided side effects (decreased oxygen saturation and colonic transit) caused by analgesic dose of morphine. Activation of CB1R, but not CB2R, decreased colonic nociception both alone and in synergy with µ-opioid receptor. Thus, CB1R agonists may enable opioid dose reduction and avoid opioid-related side effects.SIGNIFICANCE STATEMENT One of the most cited needs for patients with abdominal pain are safe and effective treatment options. The effectiveness of opioids in the management of abdominal pain is undermined by severe adverse side effects. Therefore, strategies to replace opioids or reduce the doses of opioids to suppress abdominal pain is needed. This study in mice demonstrates that cannabinoid 1 receptor (CB1R) agonists inhibit visceral sensation. Furthermore, a combination of subanalgesic doses of µ-opioid receptor agonist and CB1R agonist markedly reduce abdominal pain without causing the side effects of high-dose opioids. Thus, CB1R agonists, alone or in combination with low-dose opioids, may be a novel and safe treatment strategy for abdominal pain.

Arachidonyl‐2‐ Chlorethylamide alters the Gene Expression of Renin Angiotensin System Components in Primary Cultures of Astrocytes

ObjectiveTo determine the of effect Arachidonyl‐2′‐chloroethylamide (ACEA) on the gene expression of renin angiotensin system (RAS) components in rat astrocytes.BackgroundWe have previously shown that activation of the Angiotensin type 1 receptor (AT1R) altered the expression of the cannabinoid receptor type 1 (CB1R) in rat astrocytes. Activation of the CB1R can potentially have synergistic or antagonistic effects on AT1R actions. Studies have shown that an overactive RAS contributes to pathological conditions. Thus, we determined the molecular effects of activation of the CB1R by ACEA on RAS components. To investigate this, we determined ACEA and Angiotensin (Ang) II effects alone, then combined, on the mRNA levels of RAS components in astrocytes isolated from the cerebellum of Wistar and Spontaneously hypertensive rats (SHRs). CB1R and RAS components are expressed in the cerebellum.MethodsAstrocytes were prepared from 2‐3 days rat pups. Quiescent cells were treated with 10nM ACEA and or 100nM Ang II for 12‐24 hours. mRNA levels of RAS components were measured by quantitative polymerase chain reaction.ResultsAng II predominantly downregulated RAS components in both Wistar and SHR astrocytes at the time points examined. ACEA downregulated AT1R mRNA in Wistar, increased angiotensinogen mRNA in both Wistar and SHR astrocytes, and increased angiotensin converting enzyme mRNA in SHR astrocytes. The effects of combined treatments were similar to Ang II single treatments.ConclusionCB1R activation altered the mRNA levels of RAS components but did not appear to have considerable impact on Ang II’s effects on these components. Since mRNA levels might not correlate with protein levels, determination of protein levels and/or activity would complement these findings.

Dual action of the cannabinoid receptor 1 ligand arachidonyl-2\u2032-chloroethylamide on calcitonin gene-related peptide release

BackgroundBased on the current understanding of the role of neuropeptide signalling in migraine, we explored the therapeutic potential of a specific cannabinoid agonist. The aim of the present study was to examine the effect of the synthetic endocannabinoid (eCB) analogue, arachidonyl-2′-chloroethylamide (ACEA), on calcitonin gene-related peptide (CGRP) release in the dura and trigeminal ganglion (TG), as cannabinoids are known to activate Gi/o-coupled cannabinoid receptors type 1 (CB1), resulting in neuronal inhibition.MethodsThe experiments were performed using the hemi-skull model and dissected TGs from male Sprague-Dawley rats. CGRP release was induced by either 60 mM K+ (for depolarization-induced stimulation) or 100 nM capsaicin (for transient receptor potential vanilloid 1 (TRPV1) -induced stimulation) and measured using an enzyme-linked immunosorbent assay. The analysis of CGRP release data was combined with immunohistochemistry in order to study the cellular localization of CB1, cannabinoid receptor type 2 (CB2), CGRP and receptor activity modifying protein 1 (RAMP1), a subunit of the functional CGRP receptor, in the TG.ResultsCB1 was predominantly expressed in neuronal somas in which colocalization with CGRP was observed. Furthermore, CB1 exhibited colocalization with RAMP1 in neuronal Aδ-fibres but was not clearly expressed in the CGRP-immunoreactive C-fibres. CB2 was mainly expressed in satellite glial cells and did not show substantial colocalization with either CGRP or RAMP1. Without stimulation, 140 nM ACEA per se caused a significant increase in CGRP release in the dura but not TG, compared to vehicle. Furthermore, 140 nM ACEA did not significantly modify neither K+- nor capsaicin-induced CGRP release. However, when the TRPV1 blocker AMG9810 (1 mM) was coapplied with ACEA, K+-induced CGRP release was significantly attenuated in the TG and dura.ConclusionsResults from the present study indicate that ACEA per se does not exhibit antimigraine potential due to its dual agonistic properties, resulting in activation of both CB1 and TRPV1, and thereby inhibition and stimulation of CGRP release, respectively.

A239 COMBINED CANNABINOID RECEPTOR 1 AND MU-OPIOID RECEPTOR AGONISTS SYNERGISTICALLY INHIBIT VISCERAL PAIN IN VIVO WITHOUT ADVERSE SIDE EFFECTS OR TOLERANCE

Abstract Background Previously we have shown that both cannabinoid 1 receptor (CB1R) and mu-opioid receptor (MOR) agonists inhibit mechanosensitivity of colonic nociceptive nerves. However, it is unknown whether agonists of cannabinoid and opioid receptors have a synergistic interaction, such that very low doses of these agonists can be employed to reduce visceral pain and side effects. Aims To determine the effects of combined cannabinoid and opioid receptor agonists on visceral pain and its side effects. Methods Telemetric transmitters were surgically implanted into the abdominal cavity of C57/BL6 mice, with electrodes sutured to the external oblique muscle to measure the visceromotor response (VMR) to colorectal distention using a 4F arterial embolectomy catheter (volume range 20–80 µL). Mice were injected intraperitoneally with vehicle, arachidonyl-2’-chloroethylamide (ACEA), a selective CB1R agonist (0.3, 1, 3 mg/kg), HU-308, a selective CB2R agonist (0.3, 1, 3mg/kg), morphine, a MOR agonist (0.3, 3 mg/kg), or a combination, 30-minutes prior to distention. To assess side effects, pulse oximetry, heart rate, fecal pelleting, and locomotion (via an open-field maze) were measured. Data was analyzed with one or two-way ANOVA with post-hoc Bonferroni multiple comparisons test. Results ACEA dose-dependently reduced VMR (P<0.01 vs. vehicle, N=5); at the 80 µL distention, reductions of 35% (P<0.01) and 62% (P<0.01) were observed for 1 mg/kg and 3 mg/kg respectively. In contrast, HU-308 did not reduce VMR at any dose (P=0.17, N=8). Morphine (0.3 mg/kg) did not reduce VMR (P>0.99), while a higher dose (3 mg/kg) attenuated VMR (P<0.01 vs. vehicle, N=5); reductions of 32% (P<0.01) and 61% (P<0.01) at 60 and 80 µL distentions respectively. Interestingly, a combination of sub-analgesic doses of ACEA (0.3 mg/kg) and morphine (0.3 mg/kg) significantly reduced VMR (P<0.01 vs vehicle, N=5); compared to vehicle, VMR was reduced by 21% at 40 µL (P<0.05), 27% at 60 µL (P<0.01) and 60% at 80 µL (P<0.01). Chronic administration (2x/day for 6 days) of this combination did not alter the magnitude of its inhibitory effect (P=0.67, N=5), suggesting tolerance did not develop. While analgesic doses of morphine (3mg/kg) decreased oxygen saturation (P<0.01), heart rate (P<0.01) and fecal pelleting (i.e., GI motility; P<0.05), the sub-analgesic combination of ACEA and morphine had no effect (P>0.99 for all, N=5). Locomotion was unchanged by the agonists alone or combined (P=0.17). Conclusions A combination of sub-analgesic doses of CB1R and MOR agonists significantly inhibits visceral pain in vivo, without development of sides effects or tolerance with chronic use. Thus, combining low doses of CB1R and MOR agonists may be an effective visceral pain management strategy. Funding Agencies NSERC

Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet.

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2’-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

The Impact of CB1 Receptor on Nuclear Receptors in Skeletal Muscle Cells.

Cannabinoids are abundant signaling compounds; their influence predominantly arises via engagement with the principal two G-protein-coupled cannabinoid receptors, CB1 and CB2. One suggested theory is that cannabinoids regulate a variety of physiological processes within the cells of skeletal muscle. Earlier publications have indicated that expression of CB1 receptor mRNA and protein has been recognized within myotubes and tissues of skeletal muscle from both murines and humans, thus representing a potentially significant pathway which plays a role in the control of skeletal muscular activities. The part played by CB1 receptor activation or inhibition with respect to these functions and relevant to targets in the periphery, especially skeletal muscle, is not fully delineated. Thus, the aim of the current research was to explore the influence of CB1 receptor stimulation and inhibition on downstream signaling of the nuclear receptor, NR4A, which regulates the immediate impacts of arachidonyl-2′-chloroethylamide (ACEA) and/or rimonabant in the cells of skeletal muscle. Murine L6 skeletal muscle cells were used in order to clarify additional possible molecular signaling pathways which contribute to alterations in the CB1 receptor. Skeletal muscle cells have often been used; it is well-documented that they express cannabinoid receptors. Quantitative real-time probe-based polymerase chain reaction (qRT-PCR) assays are deployed in order to assess the gene expression characteristics of CB1 receptor signaling. In the current work, it is demonstrated that skeletal muscle cells exhibit functional expression of CB1 receptors. This can be deduced from the qRT-PCR assays; triggering CB1 receptors amplifies both NR4A1 and NR4A3 mRNA gene expression. The impact of ACEA is inhibited by the selective CB1 receptor antagonist, rimonabant. The present research demonstrated that 10 nM of ACEA notably amplified mRNA gene expression of NR4A1 and NR4A3; this effect was suppressed by the addition of 100 nM rimonabant. Furthermore, the CB1 receptor antagonist led to the downregulation of mRNA gene expression of NR4A1, NR4A2 and NR4A3. In conclusion, in skeletal muscle, CB1 receptors were recognized to be important moderators of NR4A1 and NR4A3 mRNA gene expression; these actions may have possible clinical benefits. Thus, in skeletal muscle cells, a possible physiological expression of CB1 receptors was identified. It is as yet unknown whether these CB1 receptors contribute to pathways underlying skeletal muscle biological function and disease processes. Further research is required to fully delineate their role(s).

Angiotensin II and Arachidonyl‐2′‐chloroethylamide effects on Nuclear factor kappa B and Prostaglandin E2

Objective : To determine the effect of Angiotensin (Ang) II and Arachidonyl-2′-chloroethylamide (ACEA) on nuclear factor kappa B (NFkB) activation and prostaglandin E2 (PGE2) production in rat astrocytes. Background :Neuroinflammation is a hallmark of numerous diseases including neurogenic hypertension. NFkB and PGE2 have been shown to mediate proinflammatory processes. We investigated the effect of Ang II and ACEA, a selective agonist of the CB1R, on NFkB phosphorylation and PGE2 production in astrocytes. Further, we investigated whether differences in NFkB phosphorylation and PGE2 production were observed in Wistar astrocytes compared to astrocytes isolated from the spontaneously hypertensive rat (SHR). Methods Brainstem and cerebellum astrocytes were prepared from 2-3day old rat pups. Quiescent cells were treated with 100nM Ang II or 10nM ACEA for varying time intervals. Phosphorylated (p)NFkB protein levels were measured by western blotting and PGE2 levels by ELISA. Results Ang II and ACEA had contrasting effects on NFkB phosphorylation and PGE2 production. ACEA showed significant NFkB activation predominantly in Wistar astrocytes whereas Ang II showed significant NFkB activation mainly in SHR astrocytes at similar time points. Ang II increased PGE2 production with higher levels produced in SHR compared to Wistar astrocytes. ACEA both increased and decreased PGE2 production depending on the brain region. Conclusion These results provide insight on early molecular changes in astrocytes from Wistar as compared to prehypertensive SHRs. We demonstrated that both Ang II and ACEA induced NFkB activation in astrocytes. However, Ang II potently induced PGE2 production compared to ACEA. It is plausible that ACEA could potentially counter these Ang II effects on PGE2 production. Further studies are required to understand these early molecular changes in SHRs, and the molecular mechanisms by which both Ang II and ACEA elicit their effects.

Cannabinoid receptor type 1 antagonist inhibits progression of obesity-associated nonalcoholic steatohepatitis in a mouse model by remodulating immune system disturbances.

ScopeThis study investigated whether AM251, a cannabinoid receptor type 1 (CB1) antagonist, ameliorates hepatic levels of metabolic abnormalities and inflammatory responses in a murine nonalcoholic steatohepatitis (NASH) model via reversal of disturbances in the immune system.Methods and ResultsFifteen‐week‐old male obese db/db mice were randomly assigned to the following two groups: no treatment and treatment with AM251 at 5 mg/kg for 15 days. C57BL/6J‐Lean mice were utilized as the control group. Plasma parameters, liver histopathology, and hepatic status were measured. For the in vitro study, macrophage‐derived RAW264.7 cells were cultured with AM251 or CB1 small interfering RNA (siRNA) before challenge with arachidonyl‐2′‐chloroethylamide (ACEA) or a high concentration of fatty acids (HFFAs). The db/db mice exhibited an increase in CB1 levels, lipid droplet accumulation, mitogen‐activated protein kinase‐related inflammatory responses, and macrophage and neutrophil infiltration in the liver tissues. Flow cytometry analysis revealed an elevation in macrophages and T helper cells, plus a decrease in natural killer T cells and regulatory T cells in the liver tissues of the db/db mice; treatment with 5 mg/kg AM251 reversed these changes. Moreover, in vitro experiments revealed that administration of 3.3 μM AM251 or CB1 siRNA prevented 1 mM HFFA‐ and 1 μΜ ACEA‐induced inflammatory cytokine protein expression in the RAW264.7 cells.ConclusionThese findings suggested that a blockade caused by CB1 reduced obesity‐associated NASH progression via correction of immune system dysregulations and elevated inflammatory responses in the liver tissues.

Role of 2-Arachidonoyl-Glycerol and CB1 Receptors in Orexin-A-Mediated Prevention of Oxygen-Glucose Deprivation-Induced Neuronal Injury.

Orexin-A (OX-A) protects the brain against oxidative stress-mediated ischemic injury. Since the endocannabinoid 2-arachidonoylglycerol (2-AG) and cannabinoid type-1 (CB1) receptors were previously shown to mediate some of the effects of OX-A exerted through the orexin-1 receptor (OX-1R), we investigated the involvement of 2-AG in OX-A-induced neuroprotection following oxygen and glucose deprivation (OGD) in mouse cortical neurons. OGD-induced reactive oxygen species (ROS) accumulation and neuronal death were prevented by both OX-A and arachidonyl-2′-chloroethylamide (ACEA), a synthetic CB1 receptor agonist, in a manner sensitive to OX-1R and CB1 receptor antagonists, SB334867 and AM251. OX-A stimulated 2-AG biosynthesis in cortical neurons. In neurons isolated from monoacylglycerol lipase (MAGL, a 2-AG hydrolyzing enzyme) null mice, 10-fold higher 2-AG concentrations were found and OGD failed to induce ROS production and cell death, whereas AM251 restored these noxious effects. OX-A-induced neuroprotection was mediated by the phosphoinositide-3-kinase/Akt (PI3K/Akt) survival pathway since both OX-A and ACEA induced phosphorylation of Akt and prevented OGD-induced cytochrome c release from the mitochondria, in a manner counteracted by SB334867 or AM251. Administration of OX-A reduced infarct volume and elevated brain 2-AG levels in a mouse model of transient ischemia. These results suggest that 2-AG and CB1 receptor mediate OX-A prevention of ischemia-induced neuronal apoptosis.

Arachidonyl-2′-chloroethylamide (ACEA), a synthetic agonist of cannabinoid receptor, increases CB1R gene expression and reduces dyskinesias in a rat model of Parkinson's disease

l-Dopa is the most effective drug used for Parkinson's disease (PD), but after long-term treatment, the vast majority of PD patients develop abnormal involuntary movements (AIMs) termed l-Dopa-induced dyskinesia (LID). Cannabinoid receptors in the basal ganglia can modulate motor functions, but their role in the treatment of LID is controversial. Therefore, the aim of this study is to evaluate the motor behavior and mRNA expression of the cannabinoid receptor-1 (CB1R), encoded by the Cnr1 gene, in the striatum and globus pallidus of a 6-hydroxydopamine rat model of PD. The evaluated rats had 6-hydroxydopamine-induced injury, LID, and LID treated with arachidonyl-2′-chloroethylamide (ACEA), a cannabinoid receptor agonist. Contralateral turns and AIMs were recorded to assess motor behavior. Gene expression was quantified by reverse transcription coupled with quantitative polymerase chain reaction using TaqMan probes. Behavioral evaluations demonstrated that dyskinetic rats treated with ACEA had a significant reduction in AIMs compared to the dyskinetic group. The expression of CB1R mRNA was significantly decreased in the 6-hydroxydopamine-injured and dyskinetic rats, compared to intact rats. The striata of dyskinetic rats treated with ACEA exhibited highly significant increases in CB1R mRNA expression. Contrary to results in the striatum, a lower CB1R expression was observed in globus pallidus from dyskinetic ACEA-treated group. In summary, significant differences in mRNA expression of CB1R were found between the evaluated groups of rats, suggesting the occurrence of compensatory mechanisms that may result in the ACEA-mediated reduction of dyskinesias in a rat model of PD.

An inverse agonist of estrogen-related receptor γ regulates 2-arachidonoylglycerol synthesis by modulating diacylglycerol lipase expression in alcohol-intoxicated mice.

Chronic alcohol feeding increases the levels of 2-arachidonoylglycerol (2-AG) in the liver, which activates hepatic cannabinoid receptor type 1 (CB1R), leading to oxidative liver injury. 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL). However, the mechanisms regulating hepatic DAGL gene expression and 2-AG production are largely unknown. In this study, we show that CB1R-induced estrogen-related receptor γ (ERRγ) controls hepatic DAGL gene expression and 2-AG levels. Arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1R agonist, significantly upregulated ERRγ, DAGLα, and DAGLβ, and increased 2-AG levels in the liver (10mg/kg) and hepatocytes (10μM) of wild-type (WT) mice. ERRγ overexpression upregulated DAGLα and DAGLβ expressions and increased 2-AG levels, whereas ERRγ knockdown abolished ACEA-induced DAGLα, DAGLβ, and 2-AG in vitro and in vivo. Promoter assays showed that ERRγ positively regulated DAGLα and DAGLβ transcription by binding to the ERR response element in the DAGLα and DAGLβ promoters. Chronic alcohol feeding (27.5% of total calories) induced hepatic steatosis and upregulated ERRγ, leading to increased DAGLα, DAGLβ, or 2-AG in WT mice, whereas these alcohol-induced effects did not occur in hepatocyte-specific CB1R knockout mice or in those treated with the ERRγ inverse agonist GSK5182 (40mg/kg in mice and 10μM in vitro). Taken together, these results indicate that suppression of alcohol-induced DAGLα and DAGLβ gene expressions and 2-AG levels by an ERRγ-specific inverse agonist may be a novel and attractive therapeutic approach for the treatment of alcoholic liver disease.

Arachidonoyl‐2‐chloroethylamide alleviates inflammatory pain in a sex‐dependent manner using the formalin test

Previous studies have shown that certain cannabinoids have analgesic effects. Specifically, activation of cannabinoid receptor (CB) 1 has been demonstrated to produce analgesia in multiple animal models. Arachidonyl‐2‐chloroethylamide (ACEA) is a synthetic agonist for CB1 receptors. The activation of CB1 with ACEA could enable chronic inflammatory pain patients to manage this discomfort. ACEA has not been extensively studied in animal models; therefore, not much is known about the dose effectiveness, development of tolerance, or sex‐specific effects.To study the analgesic effects of ACEA, we treated male and female C57BL6J mice prior to the formalin pain test. Administration of formalin (2.5% intraplantar) produces a biphasic pain response, modeling both acute and inflammatory pain. We believe that administration of ACEA at a dose of 0.5 mg per kg intraperitoneal will reduce inflammatory pain in this model. Also, we suggest that daily pretreatment with ACEA prior to the formalin test will cause desensitization to this compound; therefore, the initial analgesic effect will not be seen. To examine sex‐specific effects, we also tested and compared male and female mice. Moreover, vaginal lavage was performed daily on female mice to track the estrous cycle.Preliminary data suggests that females experience higher pain sensitivity compared with males when ACEA 0.5 mg per kg was used. Furthermore, our preliminary data shows that chronic administration in females results in estrous cycle changes. We concluded that there is a sex‐dependent difference in analgesic response to ACEA, and that it may be hormonally‐mediated.Support or Funding InformationFunded by NIDA DA044999‐01A1 (JG) and Texas Tech University Health Sciences Center School of Medicine #121035 (JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determining the insulin secretion potential for certain specific G-protein coupled receptors in MIN6 pancreatic beta cells

Background/aimThe polypeptide hormone insulin is essential for the maintenance of whole-body fuel homeostasis, and defects in insulin secretion and/or action are associated with the development of type 1 and type 2 diabetes. The aim of this study was to assess the role of some G-protein coupled receptors (GPCRs), GPR54, GPR56, and GPR75, and cannabinoid receptors CB1R and CB2R, in the regulation of pancreatic β-cell function.Materials and methods Insulin secretion from mouse insulinoma β-cell line (MIN6) monolayers was assessed via insulin radioimmunoassay (RIA). Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the expression of some specific GPCRs and the other receptors by MIN6 pancreatic β-cells. Results The agonists were not found to be toxic for the MIN6 pancreatic β-cells within the range of the doses used in this study, whereas insulin secretion altered depending on the ligands and receptors. In addition, arachidonyl-2’-chloroethylamide (ACEA), carbachol, chemokine (C-C motif) ligand-5 (CCL5), and exendin as well as phorbol myristate acetate (PMA) ligands showed significant increases in the insulin secretion of MIN6 pancreatic β-cells.Conclusion Understanding the normal β-cell function and identifying the defects in β-cell function that lead to the development of diabetes will generate new therapeutic targets.

Levetiracetam combined with ACEA, highly selective cannabinoid CB1 receptor agonist changes neurogenesis in mouse brain

The aim of the study was to evaluate the impact of second generation antiepileptic drug levetiracetam (LEV) with arachidonyl-2′-chloroethylamide (ACEA) on proliferating neural precursor cells in mouse brain. Additionally, we established the relationship between treatment with ACEA in combination with LEV and hippocampal neurogenesis in mouse brain. All experiments were performed on male CB57/BL mice injected i.p. with LEV (10 mg/kg), ACEA (10 mg/kg) and PMSF (30 mg/kg) for 10 days. Experiments were provided in two stages: stage 1- an acute response of proliferating neural precursor cells to ACEA and LEV administration (Ki-67 staining), stage 2 – a long term response to ACEA and LEV administration (BrDU, NeuN, GFAP staining). Results indicate that ACEA + PMSF and ACEA + PMSF + LEV significantly increased the total number of Ki-67 positive cells comparing to the control group. PMSF and LEV administered alone and in combination had no significant impact on cell proliferation compared to the control group. Results from neurogenesis study indicated that ACEA + PMSF administered alone and in combination with LEV increased the total number of BrDU cells compared to the control group, although LEV on its own decreased the number of BrDU cells. Moreover, the combination of ACEA + PMSF + LEV significantly increased the total number of newborn neurons compared to the control group. In turn, LEV significantly decreased the process of neurogenesis. Astrocytes were considerably reduced in all treated groups as compare to the control mice. These data provide substantial evidence that LEV administered chronically decreases the proliferation and differentiation of newly born cells while combination of LEV + ACEA significantly increases the level of newborn neurons in the dentate subgranular zone.

CB1 Agonism Alters Addiction-Related Behaviors in Mice Lacking Mu or Delta Opioid Receptors.

Opioids are powerful analgesics but the clinical utility of these compounds is reduced by aversive outcomes, including the development of affective and substance use disorders. Opioid systems do not function in isolation so understanding how these interact with other neuropharmacological systems could lead to novel therapeutics that minimize withdrawal, tolerance, and emotional dysregulation. The cannabinoid system is an obvious candidate as anatomical, pharmacological, and behavioral studies point to opioid-cannabinoid interactions in the mediation of these processes. The aim of our study is to uncover the role of specific cannabinoid and opioid receptors in addiction-related behaviors, specifically nociception, withdrawal, anxiety, and depression. To do so, we tested the effects of a selective CB1 agonist, arachidonyl-2-chloroethylamide (ACEA), on mouse behavior in tail immersion, naloxone-precipitated withdrawal, light-dark, and splash tests. We examined cannabinoid-opioid interactions in these tests by comparing responses of wildtype (WT) mice to mutant lines lacking either Mu or Delta opioid receptors. ACEA, both acute or repeated injections, had no effect on nociceptive thresholds in WT or Mu knockout (KO) mice suggesting that analgesic properties of CB1 agonists may be restricted to chronic pain conditions. The opioid antagonist, naloxone, induced similar levels of withdrawal in all three genotypes following ACEA treatment, confirming an opioidergic contribution to cannabinoid withdrawal. Anxiety-like responses in the light-dark test were similar across WT and KO lines; neither acute nor repeated ACEA injections modified this behavior. Similarly, administration of the Delta opioid receptor antagonist, naltrindole, alone or in combination with ACEA, did not alter responses of WT mice in the light-dark test. Thus, there may be a dissociation in the effect of pharmacological blockade vs. genetic deletion of Delta opioid receptors on anxiety-like behavior in mice. Finally, our study revealed a biphasic effect of ACEA on depressive-like behavior in the splash test, with a prodepressive state induced by acute exposure, followed by a shift to an anti-depressive state with repeated injections. The initial pro-depressive effect of ACEA was absent in Mu KO mice. In sum, our findings confirm interactions between opioid and cannabinoid systems in withdrawal and reveal reduced depressive-like symptoms with repeated CB1 receptor activation.

A novel role of cannabinoids in synaptogenesis

Synapses play a major role in signalling transduction in the nervous system. They display extensive activity-driven plasticity during development, learning and memory. Here we have explored a new role of endogenous cannabinoids and their CHI receptors in synapse formation, remodelling, and maintenance. Endogenous cannabinoids and their CHI receptors have been known to regulate neurotransmitter release at the level of the synapse and have also been implicated in several developmental events. Recent/y, if was reported that endogeneous cannabinoids decrease functional synapses in pyramidal neurons. We show here that endogenous cannabinoids and their CHI receptors regulate the dendritic and axonal filopodia formation (synapse precursors) and synaptogenesis obtained from embryonic mice cortical cultures. Stimulating the cortical cultures with the synthetic CHI receptor agonist, arachidonyl-2'-chloroethylamide (ACEA), produces a significant decrease in filopodia number at DIV8, and subsequently a lower synaptic contact density at DIV10 compared with the control group. On the other hand, inhibiting the action of endogenous cannabinoids and their CHI receptors by the inverse agonist AM251 or by the pure antagonist 02050 increases filopodia density at DIV8, and elevates synaptic density formation at DIV10. Furthermore, we found that this increase was reversed when cultures were pre-treated with H89, KT5720 (both inhibitors of Prote in Kinase A (PKA)) or DCCjb antibody, (an antibody which blocks the function of Deleted in Colorectal Cancer Receptor). Interestingly, a decrease of DCC receptors present at the surface of the neurons was observed when treated with ACEA. Conversely, an externalisation of DCC was observed when CHI receptors were antagonised by AM251 or 02050 and this effect was prevented when neurons were pretreated using H89, KT5720. This confirms the previous observations showing that the activation of adenylate cyclase and P KA pathway produces a netrinl-DCC dependent increase in synaptogenesis.

Effects of the cannabinoid CB1 agonist ACEA on salicylate ototoxicity, hyperacusis and tinnitus in guinea pigs

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2′-chloroethylamide (ACEA), a highly-selective CB1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6–10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis.