anandamide and Necrosis

We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.

Topics: Animals; Arachidonic Acids; Autophagosomes; Benzamides; CA3 Region, Hippocampal; Carbamates; Dentate Gyrus; Endocannabinoids; Endoplasmic Reticulum; Excitatory Amino Acid Agonists; Golgi Apparatus; Kainic Acid; Lysosomes; Male; Microscopy, Electron; Necrosis; Nerve Degeneration; Polyunsaturated Alkamides; Pyramidal Cells; Rats; Rats, Wistar; Status Epilepticus

The functional existence of the emerging endocannabinoid system (ECS), one of the new neuroendocrine players in cutaneous biology, is recently described in the human skin. In this study, using human eccrine sweat gland-derived immortalized NCL-SG3 model cells and a wide array of cellular and molecular assays, we investigated the effects of prototypic endocannabinoids (anandamide, 2-arachidonoylglycerol) on cellular functions. We show here that both endocannabinoids dose-dependently suppressed proliferation, induced apoptosis, altered expressions of various cytoskeleton proteins (e.g., cytokeratins), and upregulated lipid synthesis. Interestingly, as revealed by specific agonists and antagonists as well as by RNA interference, neither the metabotropic cannabinoid receptors (CB) nor the "ionotropic" CB transient receptor potential ion channels, expressed by these cells, mediated the cellular actions of the endocannabinoids. However, the endocannabinoids selectively activated the mitogen-activated protein kinase signaling pathway. Finally, other elements of the ECS (i.e., enzymes involved in the synthesis and degradation of endocannabinoids) were also identified on NCL-SG3 cells. These results collectively suggest that cannabinoids exert a profound regulatory role in the biology of the appendage. Therefore, from a therapeutic point of view, upregulation of endocannabinoid levels might help to manage certain sweat gland-derived disorders (e.g., tumors) characterized by unwanted growth.

Topics: Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Cell Line; Cell Proliferation; Cell Survival; Cytoskeleton; Dose-Response Relationship, Drug; Endocannabinoids; Epithelial Cells; Gene Expression Regulation; Glycerides; Humans; Lipids; Models, Biological; Necrosis; Polyunsaturated Alkamides; Receptors, Cannabinoid; RNA Interference; Sweat Glands; Tetrazolium Salts; Thiazoles

Anandamide (AEA), the most extensively studied endocannabinoid, and its putative cannabinoid receptors, CB1 and CB2, exert a variety of physiological and pharmacological effects in chronic liver diseases, such as hyperdynamic circulation. Anandamide selectively blocks proliferation and induces cell death in hepatic stellate cells (HSC), the key cell type of liver fibrogenesis. However, its precise molecular mechanism in rat HSC has not been fully elucidated.. CB1 and CB2 mRNA transcriptions were evaluated by reverse transcription polymerase chain reaction; CB1, CB2, phosphoinositide 3-kinases (PI3K) and protein kinase B (PKB) protein expressions were investigated by western blot and/or immunofluorescence. Cell death was detected by Annexin V-PE/7AAD flow cytometry, lipid raft content by confocal microscopic analysis, cell viability by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nuclear morphological changes by Hoechst 33258 fluorochrome, and inflammatory cytokines interleukin (IL)-2 and IL-6, and tumor necrosis factor-alpha (TNF-alpha) by enzyme-linked immunosorbent assay.. CB1 and CB2 receptors were detectable in HSC. AEA caused HSC growth inhibition in a concentration-dependent manner. Furthermore, a high concentration of AEA (20 micromol/L) triggered potent cell death-induced necrosis but not apoptosis. None of these effects were blocked by CB1 or CB2 receptor antagonist, but by methyl-beta-cyclodextrin (MCD; 10 mmol/L), a cholesterol depletory agent. AEA significantly inhibited PI3K/PKB activity, and increased IL-2, IL-6 and TNF-alpha release.. These results demonstrated that AEA induced HSC necrosis through lipid rafts: a possible role of PI3K/PKB signaling pathway downregulation and inflammatory factors production. Cholesterol depletion abolished the effects of AEA on HSC necrosis.

Topics: Animals; Arachidonic Acids; Blotting, Western; Cell Line; Cell Nucleus Shape; Cell Proliferation; Cholesterol; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Hepatic Stellate Cells; Inflammation Mediators; Interleukin-2; Interleukin-6; Membrane Microdomains; Microscopy, Confocal; Necrosis; Phosphatidylinositol 3-Kinases; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.. Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot.. The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05).. AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.

Topics: Amidohydrolases; Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Cholesterol; Endocannabinoids; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Necrosis; p38 Mitogen-Activated Protein Kinases; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction

The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro.. We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation.. We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium.. Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.

Topics: Animals; Apoptosis; Arachidonic Acids; Blastocyst; Cannabinoid Receptor Modulators; Cell Proliferation; Embryonic Development; Endocannabinoids; Female; In Situ Nick-End Labeling; Necrosis; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Sheep; Signal Transduction; Uterus

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH(-/-) mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 microm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH(-/-) mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.

Topics: Adenoviridae; Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Bile Ducts; Blotting, Western; Calcium Channel Blockers; Cell Death; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acid Transport Proteins; Glutathione; Hepatocytes; Lipid Metabolism; Liver; Male; Mice; Mice, Inbred C57BL; Necrosis; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although AEA levels are elevated in liver fibrosis, its role in fibrogenesis remains unclear. This study investigated effects of AEA in primary hepatic stellate cells (HSCs). Anandamide blocked HSC proliferation at concentrations of 1 to 10 micromol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25-100 micromol/L), AEA rapidly and dose-dependently induced cell death in primary culture-activated and in vivo-activated HSCs, with over 70% cell death after 4 hours at 25 micromol/L. In contrast to treatment with Fas ligand or gliotoxin, AEA-mediated death was caspase independent and showed typical features of necrosis such as rapid adenosine triphosphate depletion and propidium iodide uptake. Anandamide-induced reactive oxygen species (ROS) formation, and an increase in intracellular Ca(2+). Pretreatment with the antioxidant glutathione or Ca(2+)-chelation attenuated AEA-induced cell death. Although the putative endocannabinoid receptors CB1, CB2, and VR1 were expressed in HSCs, specific receptor blockade failed to block cell death. Depletion of membrane cholesterol by methyl-beta-cyclodextrin inhibited AEA binding, blocked ROS formation and intracellular Ca(2+)-increase, and prevented cell death. In primary hepatocytes, AEA showed significantly lower binding and failed to induce cell death even after prolonged treatment. In conclusion, AEA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca(2+) and ROS. The anti-proliferative effects and the selective killing of HSCs, but not hepatocytes, indicate that AEA may be used as a potential anti-fibrogenic tool.

Topics: Animals; Apoptosis; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Cell Death; Cells, Cultured; Cholesterol; Endocannabinoids; Hepatocytes; Humans; JNK Mitogen-Activated Protein Kinases; Liver Cirrhosis; Male; Necrosis; NF-kappa B; Polyunsaturated Alkamides; Rats; Reactive Oxygen Species; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Drug; Tumor Necrosis Factor-alpha

It has been demonstrated that the endogenous cannabinoid receptor ligand, anandamide, and other N-acylethanolamines (NAEs), accumulate during neuronal injury in vitro, a process that may be linked to the neuroprotective effects of NAEs. The crucial step for generation of NAEs is the synthesis of the corresponding precursors, N-acylethanolamine phospholipids (NAPEs). However, it is unknown whether this key event for NAE formation is regulated differently in the context of insults causing necrotic or apoptotic neuronal death. To address this question, we monitored a range of cortical NAPE species in three infant rat models of in vivo neurodegeneration: (i) necrosis caused by intrastriatal injection of NMDA (25 nmol); (ii) apoptosis induced by systemic administration of the NMDA-receptor antagonist (+)MK-801 (3 x 0.5 mg/kg, i.p.); and (iii) apoptosis following focal necrosis triggered by concussive head trauma. A marked increase of all NAPE species was observed in both hemispheres 4 and 24 h after NMDA-induced injury, with a relatively larger increase in N-stearoyl-containing NAPE species. Thus, the percentage of the anandamide precursor fell from 1.1 to 0.5 mol %. In contrast, administration of (+)MK-801 did not alter cortical NAPE levels. Concussion head trauma resulted in a similar but less pronounced upregulation of NAPE levels at both 4 and 24 h as compared to NMDA injections. Increased levels of NAPE 24 h post-trauma possibly reflect that necrosis is still ongoing at this time point. Consequently, our data suggest that excitotoxic necrotic mechanisms of neurodegeneration, as opposed to apoptotic neurodegeneration, have a profound effect on in vivo NAE precursor homeostasis.

Topics: Animals; Apoptosis; Arachidonic Acids; Brain Injuries; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Endocannabinoids; Ethanolamines; Male; N-Methylaspartate; Necrosis; Neurodegenerative Diseases; Neurons; Phospholipids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Species Specificity; Wounds, Nonpenetrating