Anandamide induces necrosis in primary hepatic stellate cells

The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although A-EA 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 I to 10 mu mol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25-100 mu mol/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 mu mol/L. In contrast to treatment with Fas ligand or gliotoxin, A-EA-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 Ca2+. Pretreatment with the antioxidant glutathione or Ca2+-chelation attenuated A-EA-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 Ca2+-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, A-EA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca2+ 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.