Overexpression Of Fatty Acid Amide Hydrolase Research Articles

The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

Upregulation of Anandamide Hydrolysis in the Basolateral Complex of Amygdala Reduces Fear Memory Expression and Indices of Stress and Anxiety.

Increased anandamide (AEA) signaling through inhibition of its catabolic enzyme fatty acid amide hydrolase (FAAH) in the basolateral complex of amygdala (BLA) is thought to buffer against the effects of stress and reduces behavioral signs of anxiety and fear. However, examining the role of AEA signaling in stress, anxiety, and fear through pharmacological depletion has been challenging due to the redundant complexity of its biosynthesis and the lack of a pharmacological synthesis inhibitor. We developed a herpes simplex viral vector to rapidly yet transiently overexpress FAAH specifically within the BLA to assess the impact of suppressing AEA signaling on stress, fear, and anxiety in male rats. Surprisingly, FAAH overexpression in BLA dampened stress-induced corticosterone release, reduced anxiety-like behaviors, and decreased conditioned fear expression. Interestingly, depleting AEA signaling in the BLA did not prevent fear conditioning itself or fear reinstatement. These effects were specific to the overexpression of FAAH because they were reversed by intra-BLA administration of an FAAH inhibitor. Moreover, the fear-suppressive effects of FAAH overexpression were also mitigated by intra-BLA administration of a low dose of a GABAA receptor antagonist, but not an NMDA/AMPA/kainate receptor antagonist, suggesting that they were mediated by an increase in GABAergic neurotransmission. Our data suggest that a permissive AEA tone within the BLA might gate GABA release and that loss of this tone through elevated AEA hydrolysis increases inhibition in the BLA, which in turn reduces stress, anxiety, and fear. These data provide new insights on the mechanisms by which amygdalar endocannabinoid signaling regulates emotional behavior.SIGNIFICANCE STATEMENT Amygdala endocannabinoid signaling is involved in the regulation of stress, anxiety, and fear. Our data indicate that viral-mediated augmentation of anandamide hydrolysis within the basolateral amygdala reduces behavioral indices of stress, anxiety, and conditioned fear expression. These same effects have been previously documented with inhibition of anandamide hydrolysis in the same brain region. Our results indicate that the ability of anandamide signaling to regulate emotional behavior is nonlinear and may involve actions at distinct neuronal populations, which could be influenced by the basal level of anandamide. Modulation of anandamide signaling is a current clinical therapeutic target for stress-related psychiatric illnesses, so these data underscore the importance of fully understanding the mechanisms by which anandamide signaling regulates amygdala-dependent changes in emotionality.

Impaired anandamide/palmitoylethanolamide signaling in hippocampal glutamatergic neurons alters synaptic plasticity, learning, and emotional responses

Endocannabinoid signaling via anandamide (AEA) is implicated in a variety of neuronal functions and considered a promising therapeutic target for numerous emotion-related disorders. The major AEA degrading enzyme is fatty acid amide hydrolase (FAAH). Genetic deletion and pharmacological inhibition of FAAH reduce anxiety and improve emotional responses and memory in rodents and humans. Complementarily, the mechanisms and impact of decreased AEA signaling remain to be delineated in detail. In the present study, using the Cre/loxP system combined with an adeno-associated virus (AAV)-mediated delivery system, FAAH was selectively overexpressed in hippocampal CA1-CA3 glutamatergic neurons of adult mice. This approach led to specific FAAH overexpression at the postsynaptic site of CA1-CA3 neurons, to increased FAAH enzymatic activity, and, in consequence, to decreased hippocampal levels of AEA and palmitoylethanolamide (PEA), but the levels of the second major endocannabinoid 2-arachidonoyl glycerol (2-AG) and of oleoylethanolamide (OEA) were unchanged. Electrophysiological recordings revealed an enhancement of both excitatory and inhibitory synaptic activity and of long-term potentiation (LTP). In contrast, excitatory and inhibitory long-term depression (LTD) and short-term synaptic plasticity, apparent as depolarization-induced suppression of excitation (DSE) and inhibition (DSI), remained unaltered. These changes in hippocampal synaptic activity were associated with an increase in anxiety-like behavior, and a deficit in object recognition memory and in extinction of aversive memory. This study indicates that AEA is not involved in hippocampal short-term plasticity, or eLTD and iLTD, but modulates glutamatergic transmission most likely via presynaptic sites, and that disturbances in this process impair learning and emotional responses.

PO-294 Role of fatty acid amide hydrolase (FAAH) in breast development and cancer

IntroductionThere are three major stages of breast development – embryonic, pubertal, and reproductive. During this complex developmental cycling, the epithelial compartment undergoes several rounds of proliferation, remodelling and cell death to eventually form an organised and functional mammary tree. The very nature of the pathways controlling these processes makes them susceptible to promote tumorigenic processes and ultimately lead to breast carcinoma.Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the endocannabinoid anandamide and related amidated signalling lipids. Here, we aimed at analysing the expression FAAH in breast physio-pathology.Material and methodsThe expression of FAAH was analysed by immunohistochemistry in a tissue microarray (TMA) containing ≈600 human breast cancer samples and in public microarrays data bases. Cell lines derived from different breast cancer subtypes have been analysed for FAAH expression and the effect of a Knock-out (CRISPR) and overexpression of FAAH examined. FACS and immunofluorescence were also performed to further assess the identity of cell populations. FAAH effects on breast CSC activity was examinated using mammosphere formation and aldehyde dehydrogenase (ALDH) activity assays.Results and discussionsWe have found that FAAH shows remarkable variations in expression within the human breast epithelial hierarchy. Particularly, FAAH expression is restricted to a certain subset of mature cells in normal breast tissue (i.e. luminal cells), with negligible levels being detected in the progenitor/stem cell subset, In line with this idea, pharmacological inhibition of FAAH with URB597 in the mammary stem cell line HC11 delayed differentiation to mature epithelial cells. In addition, we observed significant differences in duct formation between FAAH KO and WT mice during pubertal development.Our results also correlate low FAAH expression with more undifferentiated phenotypes, high histologic grade, absence of oestrogen receptor and triple-negative phenotype in breast cancer. Consistent with this, we found low FAAH mRNA levels associated with metastasis and poor prognosis gene signatures. Finally, pharmacoinhibition and knocking-out of FAAH in breast cancer lines, enriched the cultures in cancer stem cells.ConclusionTogether, our data suggest that FAAH plays a role in breast cell differentiation, both in normal development and in oncologic contexts.

The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system

Cannabidiol (CBD), the main non-psychotomimetic component of the plant Cannabis sativa, exerts therapeutically promising effects on human mental health such as inhibition of psychosis, anxiety and depression. However, the mechanistic bases of CBD action are unclear. Here we investigate the potential involvement of hippocampal neurogenesis in the anxiolytic effect of CBD in mice subjected to 14d chronic unpredictable stress (CUS). Repeated administration of CBD (30mg/kg i.p., 2h after each daily stressor) increased hippocampal progenitor proliferation and neurogenesis in wild-type mice. Ganciclovir administration to GFAP-thymidine kinase (GFAP-TK) transgenic mice, which express thymidine kinase in adult neural progenitor cells, abrogated CBD-induced hippocampal neurogenesis. CBD administration prevented the anxiogenic effect of CUS in wild type but not in GFAP-TK mice as evidenced in the novelty suppressed feeding test and the elevated plus maze. This anxiolytic effect of CBD involved the participation of the CB1 cannabinoid receptor, as CBD administration increased hippocampal anandamide levels and administration of the CB1-selective antagonist AM251 prevented CBD actions. Studies conducted with hippocampal progenitor cells in culture showed that CBD promotes progenitor proliferation and cell cycle progression and mimics the proliferative effect of CB1 and CB2 cannabinoid receptor activation. Moreover, antagonists of these two receptors or endocannabinoid depletion by fatty acid amide hydrolase overexpression prevented CBD-induced cell proliferation. These findings support that the anxiolytic effect of chronic CBD administration in stressed mice depends on its proneurogenic action in the adult hippocampus by facilitating endocannabinoid-mediated signalling.

Fatty acid amide hydrolase but not monoacyl glycerol lipase controls cell death induced by the endocannabinoid 2-arachidonoyl glycerol in hepatic cell populations

The endogenous cannabinoids anandamide (N-arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) are upregulated during liver fibrogenesis and selectively induce cell death in hepatic stellate cells (HSCs), the major fibrogenic cells in the liver, but not in hepatocytes. In contrast to HSCs, hepatocytes highly express the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) that protects them from AEA-induced injury. However, the role of the major 2-AG-degrading enzyme monoacylglycerol lipase (MGL) in 2-AG-induced hepatic cell death has not been investigated. In contrast to FAAH, MGL protein expression did not significantly differ in primary mouse hepatocytes and HSCs. Hepatocytes pretreated with selective MGL inhibitors were not sensitized towards 2-AG-mediated death, indicating a minor role for MGL in the cellular resistance against 2-AG. Moreover, while adenoviral MGL overexpression failed to render HSCs resistant towards 2-AG, FAAH overexpression prevented 2-AG-induced death in HSCs. Accordingly, 2-AG caused cell death in hepatocytes pretreated with the FAAH inhibitor URB597, FAAH−/− hepatocytes, or hepatocytes depleted of the antioxidant glutathione (GSH). Moreover, 2-AG increased reactive oxygen species production in hepatocytes after FAAH inhibition, indicating that hepatocytes are more resistant to 2-AG treatment due to high GSH levels and FAAH expression. However, 2-AG was not significantly elevated in FAAH−/− mouse livers in contrast to AEA. Thus, FAAH exerts important protective actions against 2-AG-induced cellular damage, even though it is not the major 2-AG degradation enzyme in vivo. In conclusion, FAAH-mediated resistance of hepatocytes against endocannabinoid-induced cell death may provide a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retianl pigment epithelial cells

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.

Fatty Acid Amide Hydrolase but not Monoacyl Glycerol Lipase Is a Crucial Determinant of Endocannabinoid-induced Cell Death in Hepatic Cell Populations

Background: We have shown that the endcannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) selectively induce cell death in activated hepatic stellate cells (HSCs), but not in hepatocytes. In contrast to HSCs, hepatocytes highly express the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) that protects them from AEA-induced injury. However, the hepatic expression pattern and function of the 2-AG-degrading enzyme monoacyl glycerol lipase (MGL) has not been investigated. Methods: MGL expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. MGL or FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. MGL or FAAH overexpression was achieved by adenoviral infection. Results: In contrast to FAAH, MGL mRNA, protein and activity did not significantly differ in hepatocytes and HSCs. Hepatocytes which had been pretreated with the MGL inhibitor URB754 were not sensitized towards 2-AG-mediated death, indicating a minor role for MGL in the resistance against 2-AG. Moreover, MGL overexpression did not render HSCs resistant towards 2-AG. Conversely, FAAH overexpression prevented 2-AG-induced death in HSCs. Accordingly, 2-AG induced necrotic cell death in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH and 2-AG induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597. Conclusion: The main 2-AG degrading enzyme MGL is not differentially expressed in hepatocytes or HSCs and does not appear to play a crucial role for the resistance of hepatocytes against 2-AG-induced cell death. In contrast, the high expression of the primary AEA-metabolizing enzyme FAAH in hepatocytes and its absence in HSCs accounts for the differential susceptibility of these hepatic cell populations towards cell death mediated by endocannabinoids.

Overexpression of a fatty acid amide hydrolase compromises innate immunity in Arabidopsis

N-acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and pathological conditions in mammals. N-acylethanolamine signaling is terminated by the action of diverse hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly susceptible to non-host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared with the wild-type. Gene expression studies revealed that transcripts of a number of plant defense genes, as well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild-type plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in turn compromises innate immunity to bacterial pathogens.

Expression and function of fatty acid amide hydrolase in prostate cancer

The hydrolysis of endocannabinoids has profound effects on the function of the endocannabinoid signaling system in the regulation of prostate carcinoma cells. Prostate carcinoma cells exhibit a wide range of hydrolysis activity for 2-arachidonoylglycerol (2-AG), the major endocannabinoid. However, enzyme(s) responsible for 2-AG hydrolysis and their functions in prostate cancer have not been characterized. In this study, we demonstrated that fatty acid amide hydrolase (FAAH) was differentially expressed in normal and prostate carcinoma cells. In PC-3 cells, overexpression of FAAH resulted in increased FAAH protein, 2-AG hydrolysis, cell invasion and cell migration. Conversely, small-interfering RNA (siRNA) knockdown of FAAH in LNCaP cells decreased FAAH protein, 2-AG hydrolysis and cell invasion. Furthermore, CAY10401, a FAAH inhibitor, decreased cell invasion and it enhanced the reduction of invasion in FAAH siRNA-transfected LNCaP cells. Immunohistochemistry staining of commercial tissue microarrays (TMAs) demonstrated FAAH staining in 109 of 157 cores of prostate adenocarcinomas but weak staining in 1 of 8 cores of normal prostate tissues. These results suggest that FAAH regulates 2-AG hydrolysis and invasion of prostate carcinoma cells and is potentially involved in prostate tumorigenesis.

Role in Anxiety Behavior of the Endocannabinoid System in the Prefrontal Cortex

In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due to TRPV1 stimulation or the lack of CB1 activation, respectively.

Fatty Acid Amide Hydrolase Determines Anandamide-induced Cell Death in the Liver

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.

The Endocannabinoid Anandamide Selectively Induces Cell Death in Hepatoma Cell Lines

Background: Endocannabinoids mediate antineoplastic effects in several cancers including breast and prostate cancer. We have shown that the endocannabinoid anandamide (AEA) does not induce cell death in primary hepatocytes due to high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH). Aim: To determine whether the endocannabinoid AEA induces growth arrest and cell death in hepatoma cell lines. Methods: Rat and human hepatoma cell lines (FaO, McA-RH7777, HepG2, Hep3B, PCL/PRF/5) and primary rat hepatocytes were treated with varying concentrations of AEA. Cell death was determined by LDH release, western blot for caspase 3 and PARP cleavage and propidium iodide (PI) uptake. Proliferation was analyzed by 3H-thymidine uptake assay. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. FAAH expression was analyzed by western blot and real time PCR. FAAH overexpression was achieved by adenoviral infection. FAAH activity was measured colorimetrically and inhibited with the specific inhibitor URB597. Glutathione was depleted by BSO. Results: AEA induced dose-dependent cell death in all human and rat hepatoma cell lines, but not in primary rat hepatocytes. The rat hepatoma cell line FaO expressed almost no FAAH and was highly sensitive to AEA-induced cell death (60% at 50μM after 4h), whereas the rat hepatoma McA-RH cell line expressed higher levels and activity of FAAH and required longer AEA treatment to efficiently induce cell death (50% at 50μM after 24h). Overexpression of FAAH rendered FaO cells resistant to AEA whereas inhibition of FAAH activity strongly increased AEA sensitivity in McA-RH cells. Accordingly, FAAH overexpression inhibited, and FAAH inhibition increased AEA-induced cell death in human hepatoma cells. AEA markedly induced ROS formation in hepatoma cells. Blocking AEA-induced ROS formation by GSH pretreatment reduced AEA-induced cell death whereas GSH-depletion further increased AEA sensitivity of all hepatoma cells. Sublethal concentrations of AEA inhibited FCS-induced 3H-thymidine uptake (p<0.05). Conclusion: Anandamide induces ROS-dependent cell death in all investigated hepatoma cell lines, but not in primary hepatocytes. This higher sensitivity of hepatoma cells is due to the lower expression of FAAH. In addition, sublethal doses of AEA block proliferation in hepatoma cells. Modulation of the endocannabinoid system may represent a new option for the treatment of primary hepatocellular cancer.

Endocannabinoid Degradation and Oxidative Defense Mechanisms Determine Anandamide-induced Cell Death in Liver Cell Populations

Background: We have shown that the endogenous cannabinoid anandamide (AEA) induces reactive oxygen species (ROS)-dependent cell death in primary hepatic stellate cells (HSCs), but not in primary hepatocytes. Aim: To determine cellular factors that control AEA-induced cell death in hepatic cell populations. Methods: Fatty acid amide hydrolase (FAAH) expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. Glutathione (GSH) levels were measured by the DTNB method. mRNA expression of the endocannabinoid receptors CB1, CB2 and VR1 was determined by real time PCR. ROS production was monitored by DCFDA fluorescence. Results: FAAH mRNA, FAAH protein and FAAH activity were extremely high in primary rat hepatocytes, but virtually absent in primary rat HSCs. Hepatocytes from FAAH -/- mice, or wildtype hepatocytes which had been pretreated with the FAAH inhibitor URB597 were susceptible to AEA-mediated death (45% and 40% at 50 μM after 24h, respectively). Conversely, FAAH overexpression prevented AEA-induced death in HSCs. Anandamide induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH by BSO. GSH levels were 10-fold higher in hepatocytes than in HSCs. GSH depletion rendered hepatocytes susceptible to AEA-mediated death while GSH pretreatment prevented cell death in HSCs. The combination of FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24h at 50 μM AEA and lowered the threshold for AEA-induced cell death to 500 nM. AEA-induced hepatocyte death after BSO and URB597 pretreatment was purely necrotic and occurred independently of the endocannabinoid receptors CB1, CB2 and VR1 which were virtually absent (CB1, CB2) or expressed at low levels (VR1) in hepatocytes. Conclusion: FAAH and GSH determine endocannabinoid-induced necrosis in hepatocytes and HSCs, independently of expression of cannabinoid receptors. High expression of FAAH and high intracellular GSH levels efficiently prevent AEA-induced cell death in hepatocytes. FAAH-mediated endocannabinoid degradation in hepatocytes leads to a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.