Homologues and isomers of noladin ether,a putative novel endocannabinoid: interaction with rat cannabinoid CB(1) receptors

Two regioisomers and 13 analogues of the putative endocannabinoid noladin ether (2-arachidonyl glyceryl ether, 2-AGE, 1) were synthesized and tested for their interaction with CB(1) receptors in rat brain membranes. The results showed that a C-20 tetra-unsaturated moiety is necessary for high affinity, and that a series of alkyl glyceryl ethers of potential occurrence in brain tissues have less affinity than 2-AGE for CB(1) receptors.     

CB2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB₂ cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB₂ receptors in the brain need to be clarified. The aim of our work was to study the μ-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB₂ receptor antagonist SR144528 in brainstem of mice deficient in either CB₁ or CB₂ receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB₂ cannabinoid antagonist SR144528, suggesting a CB₂ receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [³⁵S]GTPγS binding assay to analyze the capability of μ-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB₁ wild-type and CB₁ knockout mice after a single injection of SR144528 at 0.1 mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB₁ wild-type and CB₁ knockout mice. In vitro addition of 1 μM SR144528 caused a decrease in the maximal stimulation of DAMGO in [³⁵S]GTPγS binding assays in CB₂ wild-type brainstem membranes whereas no significant changes were observed in CB₂ receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB₂ cannabinoid receptors.     

Noladin ether, a putative novel endocannabinoid: inactivation mechanisms and a sensitive method for its quantification in rat tissues

The occurrence of the novel proposed endocannabinoid, noladin ether (2-arachidonyl glyceryl ether, 2-AGE) in various rat organs and brain regions, and its inactivation by intact C6 glioma cells, were studied. 2-AGE was measured by isotope dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry, with a detection limit of 100 fmol. A compound with the same mass and chromatographic/chemical properties as 2-AGE was found in whole brain, with the highest amounts in the thalamus and hippocampus. Synthetic [(3)H]2-AGE was inactivated by intact rat C6 glioma cells by a time- and temperature-dependent process consisting of cellular uptake and partial incorporation into phospholipids. Further data suggested that 2-AGE is taken up by cells via the anandamide/2-arachidonoyl glycerol (2-AG) membrane transporter(s), and biosynthesized in a different way as compared to 2-AG.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

The CB2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the mitochondrial intrinsic pathway

Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway.     

Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors.

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.     

MBC94, a conjugable ligand for cannabinoid CB 2 receptor imaging

Cannabinoid CB 2 receptor is a particularly attractive target for noninvasive imaging of neuroinflammation and monitoring of therapeutic efficacy. Its expression is low to undetectable in healthy brain and induced in resident microglial cells (the macrophage of the brain) after cerebral ischemia, injury, and in neuroinflammatory disease. Additionally, immune cells migrating across the blood-brain barrier typically express CB 2 receptors, which adds to the expression pool of this target and provides a reliable indicator of inflammation in the brain. Here, we synthesized a novel conjugable CB 2 receptor ligand, mbc94, which has a terminal amino group that allows for facile conjugation to imaging moieties. A near-infrared (NIR) dye labeled mbc94, NIRmbc94, was developed for CB 2 targeted imaging. Preliminary evidence, including in vitro fluorescence imaging and a competition study, showed that NIRmbc94 specifically labeled CB 2-expressing cells.     

Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2

A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.     

Expression of CB2 cannabinoid receptor in Pichia pastoris

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.     

Sulfamoyl benzamides as novel CB2 cannabinoid receptor ligands

Sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Starting from a screening hit 8 that had modest affinity for the cannabinoid CB₂ receptor, a parallel synthesis approach and initial SAR are described, leading to compound 27 with 120-fold functional selectivity for the CB₂ receptor. This compound produced robust antiallodynic activity in rodent models of postoperative pain and neuropathic pain without traditional cannabinergic side effects.     

Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes

To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.     

Echinacea alkylamides modulate TNF-alpha gene expression via cannabinoid receptor CB2 and multiple signal transduction pathways

Echinacea plant preparations are widely used in the prevention and treatment of common cold. However, so far no molecular mechanism of action has been proposed. We analyzed the standardized tincture Echinaforce and found that it induced de novo synthesis of tumor necrosis factor alpha (TNF-alpha) mRNA in primary human monocytes/macrophages, but not TNF-alpha protein. Moreover, LPS-stimulated TNF-alpha protein was potently inhibited in the early phase but prolonged in the late phase. A study of the main constituents of the extract showed that the alkylamides dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (1/2), trienoic (3) and dienoic acid (4) derivatives are responsible for this effect. The upregulation of TNF-alpha mRNA was found to be mediated by CB2 receptors, increased cAMP, p38/MAPK and JNK signaling, as well as NF-kappaB and ATF-2/CREB-1 activation. This study is the first to report a possible molecular mechanism of action of Echinacea, highlighting the role of alkylamides as potent immunomodulators and potential ligands for CB2 receptors.     

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233)

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.