Received for publication October 20, 2004.
Revised December 17, 2004.
Accepted for publication December 20, 2004.

Involvement of protein kinase A in cannabinoid receptor-mediated protection from oxidative neuronal injury

Abstract

CB1 cannabinoid receptors (CB1R) 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 following cerebral ischemia and neurodegenerative disorders, in mouse cortical neuron cultures. The CB1R agonist Win 55212-2 reduced neuronal death, measured by lactate dehydrogenase release, in cultures treated with 50 µM FeCl₂, and its protective effect was attenuated by the CB1R antagonist, SR 141716A. The endocannabinoid, anandamide, reproduced the effect of Win 55212-2, as did the antioxidant Trolox. Neuronal injury was more severe following in vitro or in vivo administration of FeCl₂ to CB1R-knockout compared to 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 H-89 (=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 appears to be required for the neuroprotective effect of CB1R activation against oxidative neuronal injury.

Key words: cannabinoid, endocannabinoid, ischemia, neuroprotection, oxidative injury, protein kinase A

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