am-356 and glyceryl-2-arachidonate

In the early stages, prostate cancer is androgen‑ dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.

Topics: Adenocarcinoma; Apoptosis; Arachidonic Acids; Cell Cycle; Drug Screening Assays, Antitumor; Endocannabinoids; Glycerides; Humans; Male; MAP Kinase Signaling System; Neoplasm Proteins; Piperidines; Polyunsaturated Alkamides; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Tumor Cells, Cultured

The functional presence of type-2 cannabinoid receptors (CB2Rs) in layer II/III pyramidal neurons of the rat medial prefrontal cortex (mPFC) was recently demonstrated. In the present study, we show that the application of the endocannabinoids (eCBs) 2-arachidonoylglycerol (2-AG) and methanandamide [a stable analog of the eCB anandamide (AEA)] can activate CB2Rs of mPFC layer II/III pyramidal neurons, which subsequently induces a Cl(-) current. In addition, we show that action potential (AP) firing evoked by 20-Hz current injections results in an eCB-mediated opening of Cl(-) channels via CB2R activation. This AP-evoked synthesis of eCBs is dependent on the Ca(2+) influx through N-type voltage-gated calcium channels. Our results indicate that 2-AG is the main eCB involved in this process. Finally, we demonstrate that under physiologically relevant intracellular Cl(-) conditions, 20-Hz AP firing leads to a CB2R-dependent reduction in neuronal excitability. Altogether, our data indicate that eCBs released upon action potential firing can modulate, through CB2R activation, neuronal activity in the mPFC. We discuss how this may be a mechanism to prevent excessive neuronal firing.

Topics: Action Potentials; Animals; Arachidonic Acids; Calcium Channels, N-Type; Chlorides; Endocannabinoids; Glycerides; Mice; Mice, Inbred C57BL; Prefrontal Cortex; Pyramidal Cells; Receptor, Cannabinoid, CB2

In this study, we have ascertained the presence and functionality in mouse embryonic stem cells (ESCs) of members of the endocannabinoid system that have been proposed as possible modulators of the survival and differentiation of various type of stem cells. We show that mouse ESCs, in addition to classical CB(1) and CB(2) cannabinoid receptors, express the transient receptor potential vanilloid receptor, at mRNA, protein, and binding levels. Remarkably, we demonstrate that ESCs have the mRNA, protein, and enzyme activity to synthesize and degrade the prominent endocannabinoids anandamide (through N-acyl-phosphatidylethanolamine-specific phospholipase D and fatty acid amide hydrolase) and 2-arachidonoylglycerol (through diacylglycerol lipase and monoacylglycerol lipase). In addition, both endocannabinoids were detected in ESCs that were also shown to constitutively release a fatty acid amide hydrolase-activating compound. Finally, we document that the stimulation of ESCs by methanandamide, a nonhydrolysable analog of anandamide, does not lead to overt alteration of the expression of Oct3/4, Nanog, and Cdx2, genes that are involved in early cell fate in the preimplantation embryo and stemness, or of the expression patterns of Brachyury and Hnf4, genes that are used as late markers of lineage differentiation capability of ESC-derived embryoid bodies. Similarly ineffective on the expression of the tested stemness genes was 2-arachidonoylglycerol. Taken together, these results confirm and extend the notion that ESCs express several functional members of the endocannabinoid system, but they leave open the question about their role in stem cells as modulators of stemness and differentiation potential.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Biomarkers; Blotting, Western; Cannabinoid Receptor Modulators; Cell Lineage; Culture Media, Conditioned; Embryonic Stem Cells; Endocannabinoids; Enzyme Activators; Fibroblasts; Gene Expression Regulation; Glycerides; Mice; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cannabinoid compounds affect synaptic activity and plasticity in numerous brain areas by activating CB1 receptors (CB1). In hippocampus, varying results have been obtained on the extent and site of cannabinoid actions on excitatory transmission, ranging from no effect to complete obliteration of synaptic responses. Here we used the rat hippocampal slice preparation to study and compare the effect of various synthetic and endogenous CB1 ligands on excitatory synaptic transmission. The full CB1 agonist WIN55212-2 (WIN2) greatly decreased excitatory synaptic transmission by 62%. The effect of WIN-2 was concentration dependent (EC50 of 200 nM) and completely prevented by CB1 antagonists. The nondegradable partial CB1 agonist R1-methanandamide (mAEA) decreased transmission by 25% and the endocannabinoids 2-arachidonylglycerol (2-AG) and anandamide (AEA) had no significant effect. The action of AEA was improved by inhibiting its degradation but not its transport. The effect of 2-AG was enhanced upon inhibition of COX-2 but remained unchanged with blockade of monoacylglycerol lipase (MAGL). The observed effects were prevented by CB1 antagonists regardless of the ligand used, and paired-pulse paradigms pointed to presynaptic mechanisms of cannabinoid action. Our results show that cannabinoid effects on neuronal activity differ widely according to the CB1 ligand used. We observed large differences between full (synthetic) and partial (endogenous) CB1 agonists in altering synaptic transmission, notably because of the involvement of active degradation mechanisms.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; In Vitro Techniques; Ligands; Male; Monoacylglycerol Lipases; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission

Status epilepticus is a serious neurological disorder associated with a significant morbidity and mortality. Antiepileptic drugs such as diazepam, phenobarbital and phenytoin are the mainstay of status epilepticus treatment. However, over 20% of status epilepticus cases are refractory to the initial treatment with two or more antiepileptic drugs. Endocannabinoids have been implicated as playing an important role in regulating seizure activity and seizure termination. This study evaluated the effects of the major endocannabinoids methanandamide and 2-arachidonylglycerol (2-AG) on status epilepticus in the low-Mg(2+) hippocampal neuronal culture model. Status epilepticus in this model was resistant to treatment with phenobarbital and phenytoin. Methanandamide and 2-AG inhibited status epilepticus in a dose-dependent manner with an EC(50) of 145+/-4.15 nM and 1.68+/-0.19 microM, respectively. In addition, the anti-status epilepticus effects of methanandamide and 2-AG were mediated by activation of the cannabinoid CB(1) receptor since they were blocked by the cannabinoid CB(1) receptor antagonist AM251. These results provide the first evidence that the endocannabinoids, methanandamide and 2-AG, are effective inhibitors of refractory status epilepticus in the hippocampal neuronal culture model and indicate that regulating the endocannabinoid system may provide a novel therapeutic approach for treating refractory status epilepticus.

Topics: Animals; Anticonvulsants; Arachidonic Acids; Cannabinoid Receptor Modulators; Cells, Cultured; Endocannabinoids; Glycerides; Hippocampus; Magnesium; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Status Epilepticus

The endocannabinoids, N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Whilst these lipid mediators are known to modulate vascular tone, the extent to which they are inactivated via local metabolism in the vasculature remains unclear.. In rat isolated small mesenteric arteries, the regulatory role of FAAH, MGL and cyclooxygenase (COX) in relaxant responses to anandamide and 2-AG was evaluated by using inhibitors of these enzymes. Relaxations to non-hydrolysable analogues of endocannabinoids and arachidonic acid were also examined.. Relaxation to anandamide but not 2-AG was potentiated by the selective FAAH inhibitor, URB597 (1 microM). In contrast, MAFP (10 microM; an inhibitor of FAAH and MGL) enhanced responses to both anandamide and 2-AG. Inhibition of COX-1 by indomethacin (10 microM) potentiated relaxations to 2-AG, whereas inhibition of COX-2 by nimesulide (10 microM) potentiated anandamide-induced relaxation. With the exception of MAFP, effects of FAAH and COX inhibitors were dependent on the endothelium. Relaxation to methanandamide and noladin ether, the non-hydrolysable analogues of anandamide and 2-AG respectively, were insensitive to the enzyme inhibitors.. This study shows that local activity of FAAH, MGL and COX, which is present largely in the endothelium, limits the vasodilator action of endocannabinoids in rat small mesenteric arteries. Despite the differential roles played by these enzymes on relaxation to anandamide versus 2-AG, our results suggest that inhibitors of these enzymes enhance the vascular impact of endocannabinoids.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Enzyme Inhibitors; Glycerides; Hydrolases; In Vitro Techniques; Lectins; Lectins, C-Type; Male; Membrane Proteins; Mesenteric Artery, Superior; Organophosphonates; Polyunsaturated Alkamides; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Receptors, Cell Surface; Vasodilation; Vasodilator Agents

Hepatic myofibroblasts are central for the development of liver fibrosis associated with chronic liver diseases, and blocking their accumulation may prevent fibrogenesis. Cannabinoids are the active components of marijuana and act via 2 G-protein-coupled receptors, CB1 and CB2. Here, we investigated whether liver fibrogenic cells are a target of cannabinoids.. CB2 receptors were characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultures of hepatic stellate cells and hepatic myofibroblasts by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and GTPgammaS assays. Functional studies were performed in cultured hepatic myofibroblasts and activated hepatic stellate cells. Carbon tetrachloride-induced liver fibrosis was studied in mice invalidated for CB2 receptors.. In liver biopsy specimens from patients with active cirrhosis of various etiologies, CB2 receptors were expressed in nonparenchymal cells located within and at the edge of fibrous septa in smooth muscle alpha-actin-positive cells. In contrast, CB2 receptors were not detected in normal human liver. CB2 receptors were also detected in cultured hepatic myofibroblasts and in activated hepatic stellate cells. Their activation triggered potent antifibrogenic effects, namely, growth inhibition and apoptosis. Growth inhibition involved cyclooxygenase-2, and apoptosis resulted from oxidative stress. Finally, mice invalidated for CB2 receptors developed enhanced liver fibrosis following chronic carbon tetrachloride treatment as compared with wild-type mice.. These data constitute the first demonstration that CB2 receptors are highly up-regulated in the cirrhotic liver, predominantly in hepatic fibrogenic cells. Moreover, this study also highlights the antifibrogenic role of CB2 receptors during chronic liver injury.

Topics: Adult; Aged; Animals; Apoptosis; Arachidonic Acids; Cell Division; Cells, Cultured; Dronabinol; Endocannabinoids; Female; Fibroblasts; Glycerides; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Knockout; Middle Aged; Myocytes, Smooth Muscle; Rats; Receptor, Cannabinoid, CB2

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Calcium; Cannabinoids; Cell Line; Cyclohexanols; Cytoplasm; DNA, Complementary; Dronabinol; Endocannabinoids; Endoplasmic Reticulum; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Fura-2; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Hippocampus; Humans; Immunosuppressive Agents; Morpholines; Naphthalenes; Neurons; Pertussis Toxin; Piperidines; Protein Binding; Protein Conformation; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Time Factors; Type C Phospholipases

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by

Topics: Anilides; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cell Division; Cells, Cultured; Cinnamates; Endocannabinoids; Esters; Glioma; Glycerides; Humans; Ketones; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Solubility; Tumor Cells, Cultured

Cannabinoids, including the endogenous cannabinoid or endocannabinoid, anandamide, modulate several gastrointestinal functions. To date, the gastrointestinal effects of the second putative endocannabinoid 2-arachidonoylglycerol (2-AG) have not been studied. In the present study using a shrew (Cryptotis parva) emetic model, 2-AG (0.25-10 mg/kg, i.p.) potently and dose-dependently increased vomiting frequency (ED(50) = 1.13 mg/kg) and the number of animals vomiting (ED(50) = 0.48 mg/kg). In contrast, neither anandamide (2.5-20 mg/kg) nor methanandamide (5-10 mg/kg) induced a dose-dependent emetogenic response, but both could partially block the induced emetic effects. Delta(9)-Tetrahydrocannabinol and its synthetic analogs reduced 2-AG-induced vomiting with the rank order potency: CP 55,940 > WIN 55,212-2 > Delta(9)-tetrahydrocannabinol. The nonpsychoactive cannabinoid, cannabidiol, was inactive. Nonemetic doses of SR 141716A (1-5 mg/kg) also blocked 2-AG-induced vomiting. The 2-AG metabolite arachidonic acid also caused vomiting. Indomethacin, a cyclooxygenase inhibitor, blocked the emetogenic effects of both arachidonic acid and 2-AG. CP 55,940 also blocked the emetic effects of arachidonic acid. 2-AG (0.25-10 mg/kg) reduced spontaneous locomotor activity (ED(50) = 11 mg/kg) and rearing frequency (ED(50) = 4.3 mg/kg) in the shrew, whereas such doses of both anandamide and methanandamide had no effect on locomotor parameters. The present study indicates that: 1) 2-AG is an efficacious endogenous emetogenic cannabinoid involved in vomiting circuits, 2) the emetic action of 2-AG and the antiemetic effects of tested cannabinoids are mediated via CB(1) receptors, and 3) the emetic effects of 2-AG occur in lower doses relative to its locomotor suppressant actions.

Topics: Animals; Antiemetics; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Emetics; Endocannabinoids; Female; Glycerides; Humans; Male; Motor Activity; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Shrews

There are conflicting reports in the literature as to whether palmitoylethanolamide affects the function of mast cell-related cell lines in vitro, in contrast to the well-documented effects of this compound upon mast cell function in vivo. In the present study, we have reinvestigated the effects of palmitoylethanolamide upon antigen-induced release of [3H]serotonin and beta-hexosaminidase from rat basophilic leukemia RBL-2H3 cells and compared these effects with those of 2-arachidonoylglycerol, anandamide and R1-methanandamide. RBL-2H3 cells were sensitized with a monoclonal anti-DNP IgE, after which they were stimulated with antigen (DNP-HSA). Palmitoylethanolamide produced a small, but significant reduction in antigen-stimulated [3H]serotonin release at high concentrations, whereas anandamide was without effect. In contrast, 2-arachidonoylglycerol and methanandamide increased the antigen-stimulated release of both [3H]serotonin and beta-hexosaminidase. It is concluded that in RBL-2H3 cells, these cannabimimetic fatty acid derivatives do not have potent stabilizing effects upon antigen-induced degranulation.

Topics: Adjuvants, Immunologic; Amides; Animals; Arachidonic Acids; beta-N-Acetylhexosaminidases; Endocannabinoids; Enzyme Induction; Ethanolamines; Glycerides; Immunoglobulin E; Inflammation Mediators; Leukemia; Ligands; Mast Cells; Palmitic Acids; Polyunsaturated Alkamides; Rats; Serotonin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonylglycerol increased cell viability in cerebral cortical neuron cultures subjected to 8 h of hypoxia and glucose deprivation. This effect was observed at nanomolar concentrations, was reproduced by a non-hydrolyzable analog of anandamide, and was unaltered by CB1 or CB2 cannabinoid receptor antagonists. Like synthetic cannabinoids, endocannabinoids can protect neurons from hypoxic injury, and may represent endogenous neuroprotective molecules in cerebral ischemia.

Topics: Animals; Arachidonic Acids; Brain Ischemia; Cannabinoid Receptor Modulators; Cannabinoids; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Neurons; Neuroprotective Agents; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoids; Electrophysiology; Endocannabinoids; Glycerides; Hippocampus; Ion Transport; Leukotriene C4; Long-Term Potentiation; Neurons; Potassium; Rats; Receptors, Cannabinoid; Receptors, Drug