Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of δ-opioid receptor

We have previously reported that N-desmethylclozapine (NDMC), a major clozapine metabolite, acts as a δ-opioid receptor agonist. Here, we show that in different cellular systems NDMC regulates protein kinase B/Akt (Akt) signaling through the activation of δ-opioid receptors. In Chinese hamster ovary cells transfected with the human δ-opioid receptor (CHO/DOR), NDMC induced a time- and concentration-dependent phosphorylation of Akt at Thr308 and glycogen synthase kinase-3β (GSK-3β) at Ser9 and these effects were fully blocked by the δ-opioid receptor antagonist naltrindole. NDMC-induced Akt and GSK-3β phosphorylations were completely prevented by pertussis toxin, the Src tyrosine kinase inhibitor PP2 and the selective insulin-like growth factor-I (IGF-I) receptor tyrosine kinase inhibitor tyrphostin AG 1024. NDMC stimulated IGF-I receptor β subunit tyrosine phosphorylation and this effect was prevented by either naltrindole or PP2. Blockade of phosphatidylinositol 3-kinase (PI3K) α, but not PI3Kγ, suppressed NDMC-induced Akt and GSK-3β phosphorylation, whereas inhibition of Akt curtailed the stimulation of GSK-3β phosphorylation. In rat nucleus accumbens, NDMC induced Akt and GSK-3β phosphorylation either in vitro or in vivo and these effects were prevented by naltrindole. NDMC also regulated Akt and GSK-3β phosphorylation through δ-opioid receptors in NG108-15 cells. In these cells NDMC counteracted oxidative stress-induced apoptosis and the effect was lost following PI3K inhibition. These data demonstrate that in different cell systems NDMC can stimulate Akt signaling by activating Gi/Go-coupled δ-opioid receptors, which, at least in CHO/DOR cells, regulate PI3Kα through Src-dependent transactivation of the IGF-I receptor, and indicate that through this mechanism NDMC can exert neuroprotective effects.