title: Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways.
abstract: Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.
authors.affiliation: Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA.
authors.forename: V A
authors.initials: VA
authors.lastname: Movsesyan
authors.forename: B A
authors.initials: BA
authors.lastname: Stoica
authors.forename: A G
authors.initials: AG
authors.lastname: Yakovlev
authors.forename: S M
authors.initials: SM
authors.lastname: Knoblach
authors.forename: P M
authors.initials: PM
authors.lastname: Lea
authors.forename: I
authors.initials: I
authors.lastname: Cernak
authors.forename: R
authors.initials: R
authors.lastname: Vink
authors.forename: A I
authors.initials: AI
authors.lastname: Faden
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D063387:Cannabinoid Receptor Antagonists; D061945:Caspase Inhibitors; D020169:Caspases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D063388:Endocannabinoids; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D021381:Protein Transport; D051381:Rats; D043882:Receptors, Cannabinoid / Q000378:metabolism; D011955:Receptors, Drug / Q000037:antagonists & inhibitors / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D002118:Calcium; D002154:Calpain; D063387:Cannabinoid Receptor Antagonists; D061945:Caspase Inhibitors; D020169:Caspases; D045304:Cytochromes c; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D016194:Receptors, N-Methyl-D-Aspartate; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide