Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis

Abstract

The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap-junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin-induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide-induced cell death was preceded by G0/G1 cell-cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stressinduced hepatocyte damage. In this context, methyl-␤-cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor, or N-acetyl cysteine, an antioxidant, potently inhibited the anandamide-induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide-induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor.

Intraocular expression and release of high-mobility group box 1 protein in retinal detachment

“…24 Briefly, 2 Â 10 5 R28 cells were cultured in 24-well plates (500 ml medium per well) with or without hydrogen peroxide (1 mM; Merck, Darmstadt, Germany) for 24 h. Then the cells were incubated with MTT (0.5 mg/ml; final concentration) for 3 h. Formazan product was solubilized by the addition of dimethyl sulfoxide for 16 h. Dehydrogenase activity was expressed as absorbance at a test wavelength of 570 nm and at a reference wavelength of 630 nm. Assays were performed in triplicate and repeated three times in independent experiments.…”

“…Activation of ERK-1/2 was analyzed as described previously. 24 In brief, after treatment, whole cells were lysed with SDS sample buffer and an equal volume of protein extracts was loaded onto 12% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane. The membrane was blocked by incubation with 5% non-fat dry milk plus 1% BSA in TBST (0.02% Tween-20 in Trisbuffered saline, pH 7.4) for 1 h at room temperature.…”

Characterization of the Endocannabinoid System in Human Neuronal Cells and Proteomic Analysis of Anandamide-induced Apoptosis

“…Such a pro-apoptotic activity of AEA 4 The abbreviations used are: CB1/2R, type 1/2 cannabinoid receptor; AEA, N-arachidonoylethanolamine (or anandamide); 2-AG, 2-arachidonoylglycerol; ARP5, actin-related protein 2/3 complex subunit 5; BSA, bovine serum albumin; CP55940, 5-(1,10-dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)-cyclohexyl]phenol; DAGL, sn-1-specific diacylglycerol lipase; DAPI, 4Ј,6-diamino-2-phenylindole; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; I-RTX, iodo-resinferatoxin; MAGL, monoacylglycerol lipase; MS, mass spectrometry; MS/MS, tandem MS; NAPE-PLD, N-acylphosphatidylethanolamine phospholipase D; PBS, phosphate-buffered saline; PPIL3b, peptidylprolyl isomerase-like protein 3 isoform PPIL3b appears to be mediated by divergent pathways, each potentiated, reduced, or unaffected by cannabinoid or TRPV1 receptors (6 -9). They can also be dependent on membrane cholesterol content (10,11), generation of reactive oxygen species (12,13), or even release of ethanolamine from AEA catalyzed by FAAH (14). In this context, it should be stressed that data on ECS and AEA-induced apoptosis in human neuronal cells are still very scant.…”

The Endocannabinoid, Anandamide, Induces Cannabinoid Receptor-Independent Cell Death in Renal Proximal Tubule Cells

“…Endocannabinoids (ECs) are endogenous lipids that have been gaining more interest during the last years as the exogenous equivalent is known as Δ 9 -THC (Δ 9 -tetrahydrocannabinol), the active psychotropic natural product of Cannabis [1].…”

“…In peripheral organs like the liver, the endocannabinoid system contributes to cell death mechanisms that involve cyclooxygenase 2 (COX2) [5], as well as hepatic injury and fibrogenesis [6] [7]. The EC anandamide (AEA) can reach an intracellular level of up to 50 µM upon proinflammatory activation of cells [8] [9].…”