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.

Schlüsselwörter

Anandamide - Anti-Fibrotic Therapy - Cell Death - Endocannabinoids - Hepatic Stellate Cells - Hepatocytes