Agonist 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

Cannabinoid-1 receptor activation modulates lipid mobilization and adipogenesis in the adipose tissue of dairy cows

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the β-adrenergic agonist isoproterenol (1 μM) in the presence of the CB1R agonist arachidonyl-2'-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage.

Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects.

There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1Δ, Dat-Cnr2Δ, and Cx3cr1-Cnr2Δ) of CB1R and CB2R by crossing Cnr1f/f and Cnr2f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the “tetrad” effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2′-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2Δ and Cx3cr1-Cnr2Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2Δ mice and remained intact in Cx3cr1-Cnr2Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

Cannabinoid CB1 receptor agonist ACEA alleviates brain ischemia/reperfusion injury via CB1\u2013Drp1 pathway

Activation of the cannabinoid CB1 receptor induces neuroprotection against brain ischemia/reperfusion injury (IRI); however, the mechanism is still unknown. In this study, we used oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in neuronal cells and middle cerebral artery occlusion (MCAO)-induced brain IRI in rats to mimic ischemic brain injury, and hypothesized that the CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA) would protect ischemic neurons by inhibiting mitochondrial fission via dynamin-related protein 1 (Drp1). We found that OGD/R injury reduced cell viability and mitochondrial function, increased lactate dehydrogenase (LDH) release, and increased cell apoptosis, and mitochondrial fission. Notably, ACEA significantly abolished the OGD/R-induced neuronal injuries described above. Similarly, ACEA significantly reversed MCAO-induced increases in brain infarct volume, neuronal apoptosis and mitochondrial fission, leading to the recovery of neurological functions. The neuroprotective effects of ACEA were obviously blocked by coadministration of the CB1 receptor antagonist AM251 or by the upregulation of Drp1 expression, indicating that ACEA alleviates brain IRI via the CB1–Drp1 pathway. Our findings suggest that the CB1 receptor links aberrant mitochondrial fission to brain IRI, providing a new therapeutic target for brain IRI treatment.

Cannabinoid Receptor Type 1 Agonist ACEA Improves Cognitive Deficit on STZ-Induced Neurotoxicity Through Apoptosis Pathway and NO Modulation.

The cannabinoid system has the ability to modulate cellular and molecular mechanisms, including excitotoxicity, oxidative stress, apoptosis, and inflammation, acting as a neuroprotective agent, by its relationship with signaling pathways associated to the control of cell proliferation, differentiation, and survival. Recent reports have raised new perspectives on the possible role of cannabinoid system in neurodegenerative diseases like Alzheimer disease's (AD). AD is a neurodegenerative disorder characterized by the presence of amyloid plaques, neurofibrillary tangles, neuronal death, and progressive cognitive loss, which could be caused by energy metabolism impairment, changes in insulin signaling, chronic oxidative stress, neuroinflammation, Tau hyperphosphorylation, and Aβ deposition in the brain. Thus, we investigated the presumptive protective effect of the cannabinoid type 1 (CB1)-selective receptor agonist arachidonyl-2'-chloroethylamide (ACEA) against streptozotocin (STZ) exposure stimuli in an in vitro neuronal model (Neuro-2a neuroblastoma cells) and in vivo model (intracerebroventricular STZ injection), experimental models of sporadic AD. Our results demonstrated that ACEA treatment reversed cognitive impairment and increased activity of Akt and ERK triggered by STZ, and increased IR expression and increased the anti-apoptotic proteins levels, Bcl-2. In the in vitro model, ACEA was able to rescue cells from STZ-triggered death and modulated the NO release by STZ. Our study has demonstrated a participation of the cannabinoid system in cellular survival, involving the CB1 receptor, which occurs by positive regulation of the anti-apoptotic proteins, suggesting the participation of this system in neurodegenerative processes. Our data suggest that the cannabinoid system is an interesting therapeutic target for the treatment of neurodegenerative diseases.

Inhibition of mitochondrial permeability transition pore opening contributes to cannabinoid type 1 receptor agonist ACEA-induced neuroprotection

Cannabinoid type 1 (CB1) receptor agonist arachidonyl-2-chloroethylamide (ACEA) induces neuroprotection against brain ischemia, and the mechanism, however, is still elusive. In this study, we used bilateral common carotid artery occlusion (BCCAO) in mice and oxygen-glucose deprivation (OGD) in primary cultured neurons to mimic brain ischemic injury, and hypothesized that cannabinoid CB1 receptor agonist ACEA protects ischemic neurons via inhibiting the opening of mitochondrial permeability transition pore (MPTP). In vivo, we found that BCCAO treatment reduced the neurological functions, increased the number of apoptotic neuronal cells and deteriorated the mitochondrial morphology in the ischemic brain tissue. And in vitro, we observed that OGD injury reduced cell viability, mitochondrial function and anti-oxidant SOD2 expression, increased lactate dehydrogenase (LDH), mitochondrial cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) releases, elevated the cell apoptosis and mitochondrial superoxide level. And the CB1 receptor agonist ACEA significantly abolished the BCCAO and OGD-induced neuronal injury above. However, the MPTP opener atractyloside (Atr) markedly reversed the ACEA-induced neuroprotective effects, inhibited the mitochondrial Cyto C and AIF releases and relieved the mitochondrial swelling, but the MPTP inhibitor cyclosporin A (CsA) did not cause significant effects on the ACEA-induced neuroprotection above. These findings indicated that inhibition of MPTP opening may be involved in the cannabinoid CB1 receptor agonist ACEA-induced neuroprotection.

Cannabinoid Receptor Type 1 Agonist ACEA Protects Neurons from Death and Attenuates Endoplasmic Reticulum Stress-Related Apoptotic Pathway Signaling.

Neurodegeneration is the result of progressive destruction of neurons in the central nervous system, with unknown causes and pathological mechanisms not yet fully elucidated. Several factors contribute to neurodegenerative processes, including neuroinflammation, accumulation of neurotoxic factors, and misfolded proteins in the lumen of the endoplasmic reticulum (ER). Endocannabinoid signaling has been pointed out as an important modulatory system in several neurodegeneration-related processes, inhibiting the inflammatory response and increasing neuronal survival. Thus, we investigated the presumptive protective effect of the selective cannabinoid type 1 (CB1) receptor agonist arachidonyl-2'-chloroethylamide (ACEA) against inflammatory (lipopolysaccharide, LPS) and ER stress (tunicamycin) stimuli in an in vitro neuronal model (Neuro-2a neuroblastoma cells). Cell viability analysis revealed that ACEA was able to protect against cell death induced by LPS and tunicamycin. This neuroprotective effect occurs via the CB1 receptor in the inflammation process and via the transient receptor potential of vanilloid type-1 (TRPV1) channel in ER stress. Furthermore, the immunoblotting analyses indicated that the neuroprotective effect of ACEA seems to involve the modulation of eukaryotic initiation factor 2 (eIF2α), transcription factor C/EBP homologous protein (CHOP), and caspase 12, as well as the survival/death p44/42 MAPK, ERK1/2-related signaling pathways. Together, these data suggest that the endocannabinoid system is a potential therapeutic target in neurodegenerative processes, especially in ER-related neurodegenerative diseases.

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.

Orphan nuclear receptor ERRγ is a key regulator of human fibrinogen gene expression.

Fibrinogen, 1 of 13 coagulation factors responsible for normal blood clotting, is synthesized by hepatocytes. Detailed roles of the orphan nuclear receptors regulating fibrinogen gene expression have not yet been fully elucidated. Here, we identified estrogen-related receptor gamma (ERRγ) as a novel transcriptional regulator of human fibrinogen gene expression. Overexpression of ERRγ specially increased fibrinogen expression in human hepatoma cell line. Cannabinoid receptor types 1(CB1R) agonist arachidonyl-2'-chloroethylamide (ACEA) up-regulated transcription of fibrinogen via induction of ERRγ, whereas knockdown of ERRγ attenuated fibrinogen expression. Deletion analyses of the fibrinogen γ (FGG) gene promoter and ChIP assays revealed binding sites of ERRγ on human fibrinogen γ gene promoter. Moreover, overexpression of ERRγ was sufficient to increase fibrinogen gene expression, whereas treatment with GSK5182, a selective inverse agonist of ERRγ led to its attenuation in cell culture. Finally, fibrinogen and ERRγ gene expression were elevated in liver tissue of obese patients suggesting a conservation of this mechanism. Overall, this study elucidates a molecular mechanism linking CB1R signaling, ERRγ expression and fibrinogen gene transcription. GSK5182 may have therapeutic potential to treat hyperfibrinogenemia.

The Role of BK Channels in Antiseizure Action of the CB1 Receptor Agonist ACEA in Maximal Electroshock and Pentylenetetrazole Models of Seizure in Mice.

The anticonvulsant effect of cannabinoid compound has been shown in various models of seizure. On the other hand, there are controversial findings about the role of large conductance calcium-activated potassium (BK) channels in the pathogenesis of epilepsy. Also, there is no data regarding the effect of co-administration of cannabinoid type 1 (CB1) receptor agonists and BK channels antagonists in the acute models of seizure in mice. In this study, the effect of arachidonyl-2'-chloroethylamide (ACEA), a CB1 receptor agonist, and a BK channel antagonist, paxilline, either alone or in combination was investigated. Both pentylenetetrazole (PTZ) and maximal electroshock (MES) acute models of seizure were used to evaluate the protective effects of drugs. Mice were randomly selected in different groups: (i) control group; (ii) groups that received different doses of either paxilline or ACEA; and (iii) groups that received combinations of ACEA and paxillin at different doses. In MES model, prevention of hindlimb tonic extension (HLTE) was considered as protective effect. In PTZ model, the required dose of PTZ (mg/kg) to induce tonic-clonic seizure with loss of righting reflex was considered as seizure threshold. In PTZ model, while administration of ACEA per se (5 and 10 mg/kg) caused protective effect against seizure; however, co-administration of ACEA and ineffective doses of paxilline attenuated the antiseizure effects of paxilline. In MES model, while pretreatment by ACEA showed protective effects against seizure; however, co-administration of paxilline and ACEA caused an antagonistic interaction for their antiseizure properties. Our results showed a protective effect of ACEA in both PTZ and MES acute models of seizure. This effect was attenuated by co-administration with paxilline, suggesting the involvement of BK channels in antiseizure activity of ACEA.

CB1 Receptors in the Nucleus of the Solitary Tract Decrease the Duration of the LCR in Neonatal Rat Pups

The laryngeal chemoreflex (LCR) is stimulated by the presence of fluid in the larynx, eliciting signal transduction in the superior laryngeal nerve to the nucleus of the solitary tract (NTS) in the brainstem. LCR activation stimulates survival reflexes to clear the airway, including apnea, bradycardia, coughing, and swallowing. Because LCR activation is pronounced in neonatal animals, the LCR has been implicated in the cessation of breathing characteristic of Sudden Infant Death Syndrome (SIDS). Transient receptor potential vanilloid 1 (TRPV1) receptors are ligand-gated ion channels in the NTS that prolong the LCR. TRPV1 receptors increase presynaptic calcium, cannabinoid1 receptors, co-expressed with TRPV1 in the NTS, reduce presynaptic calcium levels. We hypothesized that if TRPV1 is capable of elongating the LCR, then CB1 activation is capable of shortening the LCR. Therefore, the primary objective of this experiment was to characterize the role that CB1 plays to decrease the LCR. To determine if CB1 inhibition of TRPV1 was capable of shortening the LCR, the CB1 agonist arachidonyl-2′-chloroethylamide (ACEA) was microinjected into the NTS of p8 to p18 Sprague-Dawley rat pups. The LCR was recorded prior to and after injections. CB1 activation significantly (p<0.05) shortened the LCR. From these data, we can conclude that CB1 activation in the NTS, and the resulting TRPV1 inactivation in the NTS, plays a significant role in the central respiratory control of the LCR in neonatal rat pups. We are currently investigating the interaction between activation of TRPV1 and CB1 receptors.

Cholinergic and endocannabinoid neuromodulatory effects overlap on neurons of the pedunculopontine nucleus of mice.

The pedunculopontine nucleus (PPN) is a part of the reticular activating system and one of the main sources of the cholinergic fibers in the midbrain, while it is also subject to cholinergic modulation. This nucleus is known to be a structure that controls sleep-wake cycles, arousal, and locomotion. Neurons of the PPN are targets of several neuromodulatory mechanisms, which elicit heterogeneous pharmacological responses including hyperpolarization and depolarization, whereas lack of response can also be observed. In agreement with previous findings, we found that PPN neurons respond to the muscarinic agonist carbachol in a heterogeneous manner: they were depolarized and showed increased firing rate, decreased firing frequency, and were hyperpolarized, or showed no response. The heterogeneity of the muscarinic activation was similar to our previous observations with type 1 cannabinoid (CB1) receptor agonists; therefore, we investigated whether muscarinic and endocannabinoid modulatory mechanisms elicit the same action on a certain neuron. To achieve this, whole-cell patch clamp experiments were conducted on midbrain slices containing the PPN. Carbachol was applied first and, after recording the changes in the membrane potential and the firing frequency and achieving washout, the CB1 receptor agonist arachidonyl-2'-chloroethylamide (ACEA) was applied. A marked but not full overlap was observed: all neurons depolarized by carbachol were depolarized by the CB1 receptor agonist ACEA, and all neurons lacking response to carbachol lacked response to ACEA as well. However, neurons hyperpolarized by carbachol were depolarized, hyperpolarized, or not affected by the ACEA. These results indicate that endocannabinoid and muscarinic modulatory effects involve similar mechanisms of action.

Activation of cannabinoid receptor 1 inhibits increased bladder activity induced by nerve growth factor

Nerve growth factor (NGF) is an important mediator of inflammatory pain, in part by sensitizing afferent nerve fibers, and expression of NGF is increased during bladder inflammation. We investigated whether intravesical instillation of the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2′-chloroethylamide (ACEA) affects NGF-induced increased bladder activity in female C57BL/6J wild-type (WT) mice. We also examined the effects of intravesical NGF in female fatty acid amide hydrolase knock-out (FAAH KO) mice. We found that CB1 and tyrosine kinase A (trkA, the high-affinity NGF receptor) were present in L6 dorsal root ganglion (DRG) afferent neurons and in bladders of both genotypes. Intravesical NGF increased bladder activity that was inhibited by intravesical ACEA in WT mice. The inhibitory effects of ACEA were reversed by the selective CB1 antagonist AM 251. Intravesical NGF failed to affect bladder activity in FAAH KO mice, and treatment with AM251, restored the stimulatory effects of NGF on the bladder in FAAH KO mice. These results indicate that activation of CB1 inhibits increased bladder activity induced by NGF.

The interaction between ghrelin and cannabinoid systems in penicillin-induced epileptiform activity in rats

The majority of experimental and clinical studies show that ghrelin and cannabinoids are potent inhibitors of epileptic activity in various models of epilepsy. A number of studies have attempted to understand the connection between ghrelin and cannabinoid signalling in the regulation of food intake. Since no data show a functional interaction between ghrelin and cannabinoids in epilepsy, we examined the relationship between these systems via penicillin-induced epileptiform activity in rats. Doses of the CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA) (2.5 and 7.5 µg), the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide (AM-251) (0.25 and 0.5 µg) and ghrelin (0.5 and 1 µg) were administered intracerebroventricularly (i.c.v.) 30 minutes after the intracortical (i.c.) application of penicillin. In the interaction groups, the animals received either an effective dose of ACEA (7.5 µg, i.c.v.) or a non-effective dose of ACEA (2.5 µg, i.c.v.) or effective doses of AM-251 (0.25, 0.5 µg, i.c.v.) 10 minutes after ghrelin application. A 1 µg dose of ghrelin suppressed penicillin-induced epileptiform activity. The administration of a 0.25 µg dose of AM-251 increased the frequency of penicillin-induced epileptiform activity by producing status epilepticus-like activity. A 7.5 µg dose of ACEA decreased the frequency of epileptiform activity, whereas a non-effective dose of ACEA (2.5 µg) did not change it. Effective doses of AM-251 (0.25, 0.5 µg) reversed the ghrelin's anticonvulsant activity. The application of non-effective doses of ACEA (2.5 µg) together with ghrelin (0.5 µg) within 10 minutes caused anticonvulsant activity, which was reversed by the administration of AM-251 (0.25 µg). The electrophysiological evidence from this study suggests a possible interaction between ghrelin and cannabinoid CB1 receptors in the experimental model of epilepsy.

Activation of CB1 inhibits NGF-induced sensitization of TRPV1 in adult mouse afferent neurons

Transient receptor potential vanilloid 1 (TRPV1)-containing afferent neurons convey nociceptive signals and play an essential role in pain sensation. Exposure to nerve growth factor (NGF) rapidly increases TRPV1 activity (sensitization). In the present study, we investigated whether treatment with the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2′-chloroethylamide (ACEA) affects NGF-induced sensitization of TRPV1 in adult mouse dorsal root ganglion (DRG) afferent neurons. We found that CB1, NGF receptor tyrosine kinase A (trkA), and TRPV1 are present in cultured adult mouse small- to medium-sized afferent neurons and treatment with NGF (100ng/ml) for 30min significantly increased the number of neurons that responded to capsaicin (as indicated by increased intracellular Ca2 + concentration). Pretreatment with the CB1 agonist ACEA (10nM) inhibited the NGF-induced response, and this effect of ACEA was reversed by a selective CB1 antagonist. Further, pretreatment with ACEA inhibited NGF-induced phosphorylation of AKT. Blocking PI3 kinase activity also attenuated the NGF-induced increase in the number of neurons that responded to capsaicin. Our results indicate that the analgesic effect of CB1 activation may in part be due to inhibition of NGF-induced sensitization of TRPV1 and also that the effect of CB1 activation is at least partly mediated by attenuation of NGF-induced increased PI3 signaling.

Inhibition and enhancement of contextual fear memory destabilization.

The reactivation of a memory can result in its destabilization, necessitating a process of memory reconsolidation to maintain its persistence. Here we show that the destabilization of a contextual fear memory is potentiated by the cannabinoid CB1 receptor agonist Arachidonyl-2-chloroethylamide (ACEA). Co-infusion of ACEA and the IkappaB kinase (IKK) inhibitor sulfasalazine (Sulf) into the dorsal hippocampus impaired contextual fear memory reconsolidation. This observation was achieved under behavioral conditions that, by themselves, did not result in a reconsolidation impairment by Sulf alone. Moreover, we show that the destabilization of a contextual fear memory is dependent upon neuronal activity in the dorsal hippocampus, but not memory expression per se. The effect on contextual fear memory destabilization of intra-hippocampal ACEA was replicated by systemic injections, allowing an amnestic effect of MK-801. These results indicate that memory expression and destabilization, while being independent from one another, are both dependent upon memory reactivation. Moreover, memory destabilization can be enhanced pharmacologically, which may be of therapeutic potential.

Endocannabinoids regulate adipokine production and the immune balance of omental adipose tissue in human obesity

(1) To investigate whether modulation of the cannabinoid type 1 receptor (CB1R) directly regulates the production of adiponectin (ApN) and other adipokines in omental adipose tissue (OAT) of obese subjects, (2) to establish in which cellular fraction of OAT the effects of CB1R blockade take place and (3) to unravel the underlying mechanisms. OAT was obtained from 30 obese subjects (body mass index: 40.6±1.3 kg m(-2)) undergoing abdominal surgery. Primary cultures of explants or of freshly isolated adipocytes or stromal-vascular cells (SVCs) were used. In OAT explants, the CB1R blocker Rimonabant upregulated ApN gene expression. mRNA abundance of omentin that exhibits insulin-sensitizing properties was upregulated as well. Conversely, mRNA levels of two pro-inflammatory cytokines, macrophage inflammatory protein (MIP)-1β and interleukin (IL)-7 were downregulated. We next examined where these effects took place within OAT. CB1R expression was similar in both cellular fractions. In isolated mature adipocytes, blockade of CB1R reproduced the increase of ApN mRNA and the decrease of IL-7 mRNA, while inducing a rise of ApN secretion into the medium. In isolated SVC, gene expression of omentin, which is restricted to this fraction, was augmented, while that of MIP-1β was diminished. Finally, we deciphered the mechanisms leading to ApN regulation by the endocannabinoid system (ES). We first established that ApN regulation was actually mediated by the CB1R: ApN gene expression was upregulated by Rimonabant and downregulated by the CB1R agonist arachidonyl-2-chloroethylamide (ACEA). Upregulation of ApN by Rimonabant was unaltered by inhibiting cAMP production. However, downregulation of ApN by ACEA was fully reversed by an inhibitor of p38 mitogen-activated protein kinase (p38MAPK) and ACEA increased p38MAPK phosphorylation. Blockade of CB1R attenuates the inflammatory state in both cellular fractions of OAT either by increasing ApN and omentin production or by decreasing mRNAs of MIP-1β and IL-7. ApN regulation by the ES partly involves p38MAPK.

CB1 Agonist ACEA Protects Neurons and Reduces the Cognitive Impairment of AβPP/PS1 Mice

The present study shows that chronic administration of the cannabinoid receptor type 1 (CB1) receptor agonist arachidonyl-2-chloroethylamide (ACEA) at pre-symptomatic or at early symptomatic stages, at a non-amnesic dose, reduces the cognitive impairment observed in double AβPP(swe)/PS1(1dE9) transgenic mice from 6 months of age onwards. ACEA has no effect on amyloid-β (Aβ) production, aggregation, or clearance. However, ACEA reduces the cytotoxic effect of Aβ42 oligomers in primary cultures of cortical neurons, and reverses Aβ-induced dephosphorylation of glycogen synthase kinase-3β (GSK3β) in vitro and in vivo. Reduced activity of GSK3β in ACEA-treated mice is further supported by the reduced amount of phospho-tau (Thr181) in neuritic processes around Aβ plaques. In addition, ACEA-treated mice show decreased astroglial response in the vicinity of Aβ plaques and decreased expression of the pro-inflammatory cytokine interferon-γ in astrocytes when compared with age-matched vehicle-treated transgenic mice. Our present results show a beneficial effect of ACEA at both the neuronal, mediated at least in part by GSK3β inhibition, and glial levels, resulting in a reduction of reactive astrocytes and lower expression of interferon-γ. As a consequence, targeting the CB1 receptor could offer a versatile approach for the treatment of Alzheimer's disease.

Contrasting protective effects of cannabinoids against oxidative stress and amyloid-β evoked neurotoxicity in vitro

Cannabinoids have been widely reported to have neuroprotective properties in vitro and in vivo. In this study we compared the effects of CB1 and CB2 receptor-selective ligands, the endocannabinoid anandamide and the phytocannabinoid cannabidiol, against oxidative stress and the toxic hallmark Alzheimer's protein, β-amyloid (Aβ) in neuronal cell lines. PC12 or SH-SY5Y cells were selectively exposed to either hydrogen peroxide, tert-butyl hydroperoxide or Aβ, alone or in the presence of the CB1 specific agonist arachidonyl-2′-chloroethylamide (ACEA), CB2 specific agonist JWH-015, anandamide or cannabidiol. Cannabidiol improved cell viability in response to tert-butyl hydroperoxide in PC12 and SH-SY5Y cells, while hydrogen peroxide-mediated toxicity was unaffected by cannabidiol pretreatment. Aβ exposure evoked a loss of cell viability in PC12 cells. Of the cannabinoids tested, only anandamide was able to inhibit Aβ-evoked neurotoxicity. ACEA had no effect on Aβ-evoked neurotoxicity, suggesting a CB1 receptor-independent effect of anandamide. JWH-015 pretreatment was also without protective influence on PC12 cells from either pro-oxidant or Aβ exposure. None of the cannabinoids directly inhibited or disrupted preformed Aβ fibrils and aggregates. In conclusion, the endocannabinoid anandamide protects neuronal cells from Aβ exposure via a pathway unrelated to CB1 or CB2 receptor activation. The protective effect of cannabidiol against oxidative stress does not confer protection against Aβ exposure, suggesting divergent pathways for neuroprotection of these two cannabinoids.

L-Type Calcium Channel Mediates Anticonvulsant Effect of Cannabinoids in Acute and Chronic Murine Models of Seizure

The anticonvulsant activities of cannabinoid compounds have been shown in various models of seizure and epilepsy. At least, part of antiseizure effects of cannabinoid compounds is mediated through calcium (Ca(2+)) channels. The L-type Ca(2+) channels have been shown to be important in various epilepsy models. However, there is no data regarding the role of L-type Ca(2+) channels in protective action of cannabinoids on acute and chronic models of seizure. In this study, the effects of cannabinoid compounds and L-type Ca(2+) channels blockers, either alone or in combination were investigated using acute model of pentylenetetrazole (PTZ)-induced seizure in mice and chronic model electrical kindling of amygdala in rats. Pretreatment of mice with both cannabinoid CB1 receptor agonist arachidonyl-2'-chloroethylamide (ACEA) and endocannabinoid degradating enzyme inhibitor cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597) produced a protective effect against PTZ-induced seizure. Administration of various doses of the two L-type Ca(2+) channel blockers verapamil and diltiazem did not alter PTZ-induced seizure threshold. However, co-administration of verapamil and either ACEA or URB597 attenuated the protective effect of cannabinoid compounds against PTZ-induced seizure. Also, pretreatment of mice with diltiazem blocked the anticonvulsant activity of both ACEA and URB597. Moreover, (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2), the non-selective cannabinoid CB1 and CB2 receptor agonist showed anticonvulsant effect in amygdala-kindled rats. However, co-administration of WIN55,212-2 and verapamil attenuated the protective properties of WIN55,212-2. Our results showed that the anticonvulsant activity of cannabinoid compounds is mediated, at least in part, by L-type Ca(2+) channels in these two models of convulsion and epilepsy.