title: Involvement of protein kinase A in cannabinoid receptor-mediated protection from oxidative neuronal injury.
abstract: CB1 cannabinoid receptors (CB1Rs) are involved in protecting the brain from ischemia and related disorders. However, the underlying protective mechanisms are incompletely understood. We investigated the effect of CB1R activation on oxidative injury, which has been implicated in neuronal death after cerebral ischemia and neurodegenerative disorders, in mouse cortical neuron cultures. The CB1R agonist Win 55212-2 [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] reduced neuronal death, measured by lactate dehydrogenase release, in cultures treated with 50 microM FeCl2, and its protective effect was attenuated by the CB1R antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-cichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. The endocannabinoid anandamide reproduced the effect of Win 55212-2, as did the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Neuronal injury was more severe after in vitro or in vivo administration of FeCl2 to CB1R-knockout compared with wild-type mice. Win 55212-2 reduced the formation of reactive oxidative species in cortical neuron cultures treated with FeCl2, consistent with an antioxidant action. Pertussis toxin reduced CB1R-mediated protection, which points to a protective mechanism that involves signaling through G(i/o) proteins. Since CB1R-activated G protein signaling inhibits protein kinase A but activates phosphatidylinositol 3-kinase (PI3K), we tested the involvement of these pathways in CB1R-mediated neuroprotection. Dibutyryl-cyclic adenosine monophosphate (dbcAMP) blocked protection by Win 55212-2, whereas the PI3K inhibitor wortmannin did not, and the effect of dbcAMP was inhibited by the protein kinase A inhibitor H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide] (> or =10 nM). CB1R-induced, SR141716A-, pertussis toxin-, and dbcAMP-sensitive protection was also observed for two other oxidative insults, exposure to H2O2 or buthionine sulfoximine. Therefore, receptor-stimulated inhibition of protein kinase A seems to be required for the neuroprotective effect of CB1R activation against oxidative neuronal injury.
authors.affiliation: Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA. dgreenberg@buckinstitute.org
authors.forename: Sun Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Seok Joon
authors.initials: SJ
authors.lastname: Won
authors.forename: Xiao Ou
authors.initials: XO
authors.lastname: Mao
authors.forename: Kunlin
authors.initials: K
authors.lastname: Jin
authors.forename: David A
authors.initials: DA
authors.lastname: Greenberg
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D000975:Antioxidants; D048588:Benzoxazines; D003994:Bucladesine / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002186:Cannabinoids / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005290:Ferric Compounds / Q000633:toxicity; D007150:Immunohistochemistry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D018696:Neuroprotective Agents; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D043882:Receptors, Cannabinoid / Q000187:drug effects* / Q000235:genetics; D000077285:Rimonabant
chemical_list: D000963:Antimetabolites; D000975:Antioxidants; D048588:Benzoxazines; D002186:Cannabinoids; D002712:Chlorides; D005290:Ferric Compounds; D009025:Morpholines; D009281:Naphthalenes; D018696:Neuroprotective Agents; D010880:Piperidines; D011720:Pyrazoles; D043882:Receptors, Cannabinoid; D019328:Buthionine Sulfoximine; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D003994:Bucladesine; D007770:L-Lactate Dehydrogenase; D017868:Cyclic AMP-Dependent Protein Kinases; D000077285:Rimonabant; C024555:ferric chloride