CB1 receptor signaling regulates social anxiety and memory

The endocannabinoid (eCB) system regulates emotion, stress, memory and cognition through the cannabinoid type 1 (CB₁) receptor. To test the role of CB₁ signaling in social anxiety and memory, we utilized a genetic knockout (KO) and a pharmacological approach. Specifically, we assessed the effects of a constitutive KO of CB₁ receptors (CB₁KOs) and systemic administration of a CB₁ antagonist (AM251; 5 mg/kg) on social anxiety in a social investigation paradigm and social memory in a social discrimination test. Results showed that when compared with wild‐type (WT) and vehicle‐treated animals, CB₁KOs and WT animals that received an acute dose of AM251 displayed anxiety‐like behaviors toward a novel male conspecific. When compared with WT animals, KOs showed both active and passive defensive coping behaviors, i.e. elevated avoidance, freezing and risk‐assessment behaviors, all consistent with an anxiety‐like profile. Animals that received acute doses of AM251 also showed an anxiety‐like profile when compared with vehicle‐treated animals, yet did not show an active coping strategy, i.e. changes in risk‐assessment behaviors. In the social discrimination test, CB₁KOs and animals that received the CB₁ antagonist showed enhanced levels of social memory relative to their respective controls. These results clearly implicate CB₁ receptors in the regulation of social anxiety, memory and arousal. The elevated arousal/anxiety resulting from either total CB₁ deletion or an acute CB₁ blockade may promote enhanced social discrimination/memory. These findings may emphasize the role of the eCB system in anxiety and memory to affect social behavior .     

Cannabinoid Type 1 and Type 2 Receptor Antagonists Prevent Attenuation of Serotonin-Induced Reflex Apneas by Dronabinol in Sprague-Dawley Rats

The prevalence of obstructive sleep apnea (OSA) in Americans is 9% and increasing. Increased afferent vagal activation may predispose to OSA by reducing upper airway muscle activation/patency and disrupting respiratory rhythmogenesis. Vagal afferent neurons are inhibited by cannabinoid type 1 (CB₁) or cannabinoid type 2 (CB₂) receptors in animal models of vagally-mediated behaviors. Injections of dronabinol, a non-selective CB₁/CB₂ receptor agonist, into the nodose ganglia reduced serotonin (5-HT)-induced reflex apneas. It is unknown what role CB₁ and/or CB₂ receptors play in reflex apnea. Here, to determine the independent and combined effects of activating CB₁ and/or CB₂ receptors on dronabinol’s attenuating effect, rats were pre-treated with CB₁ (AM251) and/or CB₂ (AM630) receptor antagonists. Adult male Sprague-Dawley rats were anesthetized, instrumented with bilateral electrodes to monitor genioglossus electromyogram (EMGgg) and a piezoelectric strain gauge to monitor respiratory pattern. Following intraperitoneal treatment with AM251 and/or AM630, or with vehicle, serotonin was intravenously infused into a femoral vein to induce reflex apnea. After baseline recordings, the nodose ganglia were exposed and 5-HT-induced reflex apneas were again recorded to confirm that the nerves remained functionally intact. Dronabinol was injected into each nodose ganglion and 5-HT infusion was repeated. Prior to dronabinol injection, there were no significant differences in 5-HT-induced reflex apneas or phasic and tonic EMGgg before or after surgery in the CB₁, CB₂, combined CB₁/CB₂ antagonist, and vehicle groups. In the vehicle group, dronabinol injections reduced 5-HT-induced reflex apnea duration. In contrast, dronabinol injections into nodose ganglia of the CB₁, CB₂, and combined CB₁/CB₂ groups did not attenuate 5-HT-induced reflex apnea duration. However, the CB₁ and CB₂ antagonists had no effect on dronabinol’s ability to increase phasic EMGgg. These findings underscore the therapeutic potential of dronabinol in the treatment of OSA and implicate participation of both cannabinoid receptors in dronabinol’s apnea suppression effect.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.     

Probable involvement of Ca2 +-activated Cl− channels (CaCCs) in the activation of CB1 cannabinoid receptors

AimsRecently, we demonstrated that peripheral antinociception induced by δ opioid receptor is dependent of Ca^2 +-activated Cl^? channels (CaCCs). Because opioid and cannabinoid receptors share some common mechanisms of action, our objective was to identify a possible relationship between CaCCs and the endocannabinoid system.Main methodsTo induce hyperalgesia, rat paws were treated with intraplantar prostaglandin E₂ (PGE₂, 2 μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3 h following injection. Probabilities were calculated using ANOVA/Bonferroni's test, and values that were less than 5% were considered to be statistically significant.Key findingsAdministration of the cannabinoid agonist CB₁ anandamide (12.5, 25 and 50 μg/paw) and the cannabinoid agonist CB₂ PEA (5, 10 and 20 μg/paw) decreased the PGE₂-induced hyperalgesia in a dose-dependent manner. The possibility of the higher doses of anandamide (50 μg) and PEA (20 μg) having a central or systemic effect was excluded because the administration of the drug into the contralateral paw did not elicit antinociception in the right paw. As expected, the antinociceptive effects induced by anandamide and PEA were blocked by the CB₁ and CB₂ receptor antagonists AM251 and AM630, respectively. The peripheral antinociception was induced by anandamide but not PEA and was dose-dependently inhibited by the CaCC blocker niflumic acid (8, 16 and 32 μg).SignificanceThese results provide the first evidence for the involvement of CaCCs in the peripheral antinociception induced by activation of the CB₁ cannabinoid receptor.     

Striatal CB1 and D2 receptors regulate expression of each other,CRIP1A and delta opioid systems

Although biochemical and physiological evidence suggests a strong interaction between striatal CB₁ cannabinoid (CB₁R) and D₂ dopamine (D₂R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB₁R or D₂R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB₁R and D₂R knockdown reduced striatal dopaminergic‐stimulated [³⁵S]GTPγS binding, and D₂R knockdown reduced pallidal WIN55212‐2‐stimulated [³⁵S]GTPγS binding. Decreased D₂R and CB₁R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB₁Rs or D₂Rs, and over‐expression of CRIP1a. Down‐regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D‐Pen2, D‐Pen5]‐enkephalin‐stimulated [³⁵S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB₁R or D₂R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.     

THC Prevents MDMA Neurotoxicity in Mice

The majority of MDMA (ecstasy) recreational users also consume cannabis. Despite the rewarding effects that both drugs have, they induce several opposite pharmacological responses. MDMA causes hyperthermia, oxidative stress and neuronal damage, especially at warm ambient temperature. However, THC, the main psychoactive compound of cannabis, produces hypothermic, anti-inflammatory and antioxidant effects. Therefore, THC may have a neuroprotective effect against MDMA-induced neurotoxicity. Mice receiving a neurotoxic regimen of MDMA (20 mg/kg ×4) were pretreated with THC (3 mg/kg ×4) at room (21°C) and at warm (26°C) temperature, and body temperature, striatal glial activation and DA terminal loss were assessed. To find out the mechanisms by which THC may prevent MDMA hyperthermia and neurotoxicity, the same procedure was carried out in animals pretreated with the CB₁ receptor antagonist AM251 and the CB₂ receptor antagonist AM630, as well as in CB₁, CB₂ and CB₁/CB₂ deficient mice. THC prevented MDMA-induced-hyperthermia and glial activation in animals housed at both room and warm temperature. Surprisingly, MDMA-induced DA terminal loss was only observed in animals housed at warm but not at room temperature, and this neurotoxic effect was reversed by THC administration. However, THC did not prevent MDMA-induced hyperthermia, glial activation, and DA terminal loss in animals treated with the CB₁ receptor antagonist AM251, neither in CB₁ and CB₁/CB₂ knockout mice. On the other hand, THC prevented MDMA-induced hyperthermia and DA terminal loss, but only partially suppressed glial activation in animals treated with the CB₂ cannabinoid antagonist and in CB₂ knockout animals. Our results indicate that THC protects against MDMA neurotoxicity, and suggest that these neuroprotective actions are primarily mediated by the reduction of hyperthermia through the activation of CB₁ receptor, although CB₂ receptors may also contribute to attenuate neuroinflammation in this process.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

Cannabinoid Receptor CB2 Modulates Axon Guidance

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB₂R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB₂R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 ^−/−). The CB₂R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB₂R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 ^−/− mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB₂R’s implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB₁R, but also involves CB₂R.     

Metabolic effects of cannabinoids in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos determined by <Superscript>1</Superscript>H NMR metabolomics

The study of drug-driven biochemical changes is important in order to determine the biomarkers associated with a specific compound activity in an individual biological system. Rodent models have been widely used to study the metabolic changes induced by psychostimulants in a cell, tissue or whole organism. However these models are not suitable for large-scale, high-throughput screening. Here, we used zebrafish embryos to study the metabolic effects of the cannabinoid receptor type 1 (CB₁) agonist (?)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol (?⁹-THC) and antagonist (AM251). The zebrafish embryos were exposed to ?⁹-THC and AM251 at 24 h post fertilization (hpf) for 96 h. Proton nuclear magnetic resonance based metabolomic results show an increase in the level of choline, betaine, taurine, adenosine triphosphate and glucose upon exposure to ?⁹-THC. The levels of excitatory neurotransmitters (glutamate and glutamine) increased at lower doses of ?⁹-THC, whereas toxic dose resulted in reduction of glutamate. In contrast to ?⁹-THC, AM251 caused a dose dependent reduction of betaine, choline, taurine and also reduce the level of glutamate and glutamine. Interestingly, both compounds induce the production of the dopamine precursors, phenylalanine and tyrosine at higher doses. These findings suggest that CB₁ receptor is involved in the regulation of metabolites, which might be involved in the neurotransmission of zebrafish embryos. Furthermore, our results show that zebrafish embryo can be successfully used to provide a detailed overview of general effects of drug on the overall metabolome of an intact organism.     

The hypotensive effect of intrathecally injected (m)VD-hemopressin(α) in urethane-anesthetized rats

Previous studies suggest that cannabinoids system plays an important role in cardiovascular regulation. (m)VD-hemopressin(α) (VD-Hpα), an 11-residue peptide originating from the α₁ chain of hemoglobin, was recently reported as a selective agonist of cannabinoid CB₁ receptor. The present study was undertaken to investigate the intrathecal (i.t.) action of (m)VD-Hpα on blood pressure in urethane-anesthetized rats. Our results demonstrated that injections of (m)VD-Hpα (5–30 nmol, i.t.) produced a dose-dependent decrease in mean arterial pressure (MAP), similar to that of the non-peptidic cannabinoid receptor agonist WIN55212-2 (1.25–10 nmol, i.t.). The hypotensive effect of (m)VD-Hpα was not influenced by the CB₁ receptor antagonist AM251 (20 nmol, i.t.) or the CB₂ receptor antagonist AM630 (20 nmol, i.t.). However, WIN55212-2-induced hypotension was almost completely prevented by i.t. administration of AM251, not by AM630. The spinal hypotension of (m)VD-Hpα and WIN55212-2 was significantly reduced by pretreatment with the α-adrenoceptor antagonist phentolamine (1 mg/kg, i.v.), but not by the β-adrenoceptor antagonist propranolol (2 mg/kg, i.v.) or the muscarinic receptor antagonist atropine (2 mg/kg, i.v.). In addition, l-NAME (50 mg/kg, i.v.), the inhibitor of nitric oxide (NO) synthase, significantly reduced WIN55212-2-induced hypotension, but had no effect on the hypotensive response to (m)VD-Hpα. Collectively, the results show that i.t. administration of (m)VD-Hpα induces a decrease in MAP via a non-CB₁ and non-CB₂ mechanism.     

Predicting the molecular interactions of CRIP1a–cannabinoid 1 receptor with integrated molecular modeling approaches

Cannabinoid receptors are a family of G-protein coupled receptors that are involved in a wide variety of physiological processes and diseases. One of the key regulators that are unique to cannabinoid receptors is the cannabinoid receptor interacting proteins (CRIPs). Among them CRIP1a was found to decrease the constitutive activity of the cannabinoid type-1 receptor (CB₁R). The aim of this study is to gain an understanding of the interaction between CRIP1a and CB₁R through using different computational techniques. The generated model demonstrated several key putative interactions between CRIP1a and CB₁R, including the critical involvement of Lys130 in CRIP1a.     

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

Background

Cannabinoid receptor 1 (CB₁) is expressed in certain types of malignancies. An analysis of CB₁ expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.

Design and Methods

We examined the distribution of CB₁ protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB₁ signaling on cell survival was investigated.

Results

A predominant expression of CB₁ was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB₁-abundance and displayed a dose-dependent decline of viability under CB₁ inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.

Conclusions

The present study identifies CB₁ as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.     

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands

The cannabinoid receptor 1 (CB₁) and CB₂ cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.The CB₁² and CB₂ cannabinoid receptors comprise a two-member subfamily of G-protein-coupled receptors (GPCRs) that are notable as the targets of the tetrahydrocannabinol (THC) derivatives found in marijuana. More recently CB₁ receptors along with other GPCRs have been promoted as therapeutic pharmacological targets in the billion dollar weight loss market for controversial drugs such as rimonabant (SR141716A) and Fen-phen. Thus, an important utility of cannabinoid family receptors to society appears to arise from their role in regulating a broad spectrum of addiction-based behaviors, and the addition of new members to the cannabinoid receptor family may have social and economic implications that reach far beyond the initial scientific discovery. As a consequence, the re-classification of an orphan GPCR as a cannabinoid family member should be done with caution requiring strict criteria of receptor activation by THC derivatives or endogenous cannabinoid compounds and a widespread agreement of the results by the scientific community.Marijuana, one of the most widely abused substances (1), mediates many of its psychotropic effects by targeting CB₁ receptors in the central nervous system, but studies with CB₁ and CB₂ knock-out mice indicate that the complex pharmacological properties on pain, mood, and memory exhibited by exogenous cannabinoids and the endogenous arachidonic acid-based endo-cannabinoids, including anandamide and 2-arachidonoylglycerol (2-AG), are not fully explained by their activation of CB₁ and CB₂ (2–4). The CB₁ and CB₂ receptors are 44% identical and signal through G_i/o-mediated pathways. Activation of either receptor is inhibitory for cAMP production via adenylyl cyclase and stimulatory for mitogen-activated protein kinase (MAPK) (extracellular-regulated protein kinase 1/2 (ERK1/2)) activation (5). However, the failure of these two receptors to account for the full complement of physiological effects observed with cannabinoid ligands has led to the hypothesis that additional cannabinoid-like receptors exist.The orphan GPCR, GPR55, which exhibits only 10–15% homology to the two human cannabinoid receptors (6), is one of a number of plausible cannabinoid family member candidates (7). GPR55 was first identified and mapped to human chromosome 2q37 a decade ago (8). In the human central nervous system, it is predominantly localized to the caudate, putamen, and striatum (8), coupling to Gα₁₃ (9, 10), Gα₁₂, or Gα_q (11).GPR55 has been tested against a number of cannabinoid ligands with mixed results. Observations using a GTPγS functional assay indicate that GPR55 is activated by nanomolar concentrations of the endocannabinoids 2-AG, virodhamine, noladin ether, and palmitoylethanolamine (10) and the atypical cannabinoids Abn-CBD and O-1602 (12) as well as by the drugs CP55,950, HU210, and Δ⁹-THC (11). Exposure of GPR55 to the cannabinoids THC and JWH015 in dorsal root ganglion neurons and in receptor-transfected HEK293 cells correlates with increases of intracellular Ca²⁺ (11). In contrast, GPR55 is insensitive to the CB₁ inverse agonist AM281 and the potent cannabinoid agonist WIN55212-2 but is antagonized by the marijuana constituent CBD (9, 10). However, Oka et al. (13) reported that GPR55 is not a typical cannabinoid receptor, as numerous endogenous and synthetic cannabinoids, including many mentioned above, had no effect on GPR55 activity. They present compelling data suggesting that the endogenous lipid LPI and its 2-arachidonyl analogs are agonists at GPR55 as a result of their abilities to phosphorylate extracellular-regulated kinase and induce calcium signaling (13, 14). Further studies indicate that LPI and the rimonabant-like CB₁ inverse agonist AM251 induce oscillatory Ca²⁺ release through Gα₁₃ and RhoA (9). These reports were all performed in HEK 293 cells, yet each documented a distinct and conflicting chemical space of agonists that recognized GPR55. To resolve these inconsistencies in classification, an alternative approach for identifying GPR55 ligands that is insensitive to the endogenous complement of cellular receptors could circumvent many of the challenges that have arisen in the measurements of G-protein signaling.β-Arrestins are intracellular proteins that bind and desensitize activated GPCRs and in the process form stable receptor/arrestin signaling complexes (15, 16). β-Arrestin redistribution to the activated membrane-bound receptor represents one of the early intracellular events provoked by agonist binding and, consequently, is less prone to a false positive or negative readout as compared with studying a downstream signaling event as a readout of receptor activation. β-arrestin-green fluorescent chimeras can make this process attractive to monitor by forming remarkably sensitive and specific probes of GPCR activation that are independent of downstream G-protein-mediated signaling (17–19). We have determined GPR55 responsiveness to a representative panel of cannabinoid ligands and LPI in the presence (and absence) of a β-arrestin2-green fluorescent protein (βarr2-GFP) biosensor. Our data demonstrate that LPI, the CB₁ inverse agonist/antagonists SR141716A, and AM251 are GPR55 agonists, and the CB₁ agonist CP55940 is a GPR55 antagonist/partial agonist. These data together with our inability to observe activation of GPR55 by Δ⁹-THC and endocannabinoids indicate that GPR55 should be classified as an atypical cannabinoid receptor at best.     

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB₁ receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D₂ receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D₂ receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC₅₀: 3.25 × 10^?8 M). The CB₁ receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB₁ receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB₁ receptors and D₂ autoreceptors in regulating rat striatal dopamine release.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling

Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB₁)‐deficient mice (CB₁^?/?) and their wild‐type littermates (CB₁^+/+) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB₁^?/?. Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin‐releasing hormone (CRH) signaling. Pharmacological blockade of CB₁ receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1^?/?) or type 2 (CRHR2^?/?), and in respective wild‐type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear‐alleviating effects of CB₁. As CRHR1^?/? are known to be severely impaired in stress‐induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB₁‐mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB₁ in principal neurons of the forebrain (CaMK‐CB₁^?/?), or in cortical glutamatergic neurons (Glu‐CB₁^?/?), showed a similar phenotype as CB₁^?/?, thus indicating that endocannabinoid‐controlled glutamatergic transmission plays an essential role in acute fear adaptation.     

Effect of a synthetic cannabinoid agonist on the proliferation and invasion of gastric cancer cells

Although cannabinoids are associated with antineoplastic activity in a number of cancer cell types, the effect in gastric cancer cells has not been clarified. In the present study, we investigated the effects of a cannabinoid agonist on gastric cancer cell proliferation and invasion. The cannabinoid agonist WIN 55,212‐2 inhibited the proliferation of human gastric cancer cells in a dose‐dependent manner and that this effect was mediated partially by the CB₁ receptor. We also found that WIN 55,212‐2 induced apoptosis and down‐regulation of the phospho‐AKT expression in human gastric cancer cells. Furthermore, WIN 55,212‐2 treatment inhibited the invasion of gastric cancer cells, and down‐regulated the expression of MMP‐2 and VEGF‐A through the cannabinoid receptors. Our results open the possibilities in using cannabinoids as a new gastric cancer therapy. J. Cell. Biochem. 110: 321–332, 2010. © 2010 Wiley‐Liss, Inc.     

Activation and induction of cytosolic phospholipase A2 by IL‐1β in human tracheal smooth muscle cells: Role of MAPKs/p300 and NF‐κB

Cytosolic phospholipase A₂ (cPLA₂) plays a pivotal role in mediating agonist‐induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL‐1β. However, the mechanisms underlying IL‐1β‐induced cPLA₂ expression and PGE₂ synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL‐1β‐induced cPLA₂ protein and mRNA expression, PGE₂ production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL‐1β‐induced cPLA₂ expression was also inhibited by pretreatment with a NF‐κB inhibitor, helenalin or transfection with siRNA of NIK, IKKα, or IKKβ. IL‐β‐induced NF‐κB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA₂ expression induced by IL‐1β. Moreover, p300 was associated with the cPLA₂ promoter, which was dynamically linked to histone H4 acetylation stimulated by IL‐1β. These results suggest that in HTSMCs, activation of MAPKs, NF‐κB, and p300 are essential for IL‐1β‐induced cPLA₂ expression and PGE₂ secretion. J. Cell. Biochem. 109: 1045–1056, 2010. © 2010 Wiley‐Liss, Inc.