Interactions between CB ₁ receptors and TRPV1 channels mediated by 12-HPETE are cytotoxic to mesencephalic dopaminergic neurons

Background and purposes: We recently proposed the existence of neurotoxic interactions between the cannabinoid type 1 (CB ₁) receptor and transient receptor potential vanilloid 1 (TRPV1) channels in rat mesencephalic cultures. This study seeks evidence for the mediator(s) and mechanisms underlying the neurotoxic interactions between CB ₁ receptors and TRPV1 in vitro and in vivo. Experimental approach: The mediator(s) and mechanism(s) for the interactions between CB ₁ receptors and TRPV1 were evaluated by cell viability assays, immunocytochemistry, Fura-2 calcium imaging, mitochondrial morphology assay, ELISA and Western blot assay in vitro in neuron-enriched mesencephalic cultures. Injections into the substantia nigra and subsequent cell counts were also used to confirm these interactions in vivo. Key results: The neurotoxic interactions were mediated by 12(S)- hydroperoxyeicosatetraenoic acid (12(S)-HPETE), an endogenous TRPV1 agonist. CB ₁ receptor agonists (HU210 and WIN55,212-2) increased the level of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a downstream metabolite of 12(S)-HPETE, which stimulates TRPV1-mediated death of mesencephalic neurons, both in vitro and in vivo. The neurotoxicity was mediated by increased intracellular Ca ²⁺ concentration ([Ca ²⁺] _i) through TRPV1, consequently leading to mitochondrial damage and was attenuated by baicalein, a 12-lipoxygenase inhibitor. Conclusion and implications: Activation of CB ₁ receptors in rat mesencephalic neurons was associated with biosynthesis of 12(S)-HPETE, which in turn stimulated TRPV1 activity, leading to increased [Ca ²⁺] _i, mitochondrial damage and neuronal death.