Nobiletin differentially regulates muscarinic and nicotinic receptor-mediated ERK signaling

Nobiletin, a citrus polymethoxyflavone with neuroprotective properties, activates extracellular signal-regulated kinase 1/2 (ERK1/2); however, its effects on cholinergic receptor-mediated ERK signaling remain unclear. This study aimed to investigate the effects of nobiletin on the muscarinic and nicotinic receptor-dependent ERK signaling in neuronal nitric oxide synthase-expressing PC12 cells. The results showed that nobiletin inhibited acetylcholine- and bethanechol-induced ERK1/2 phosphorylation, an effect attenuated by increasing agonist concentrations, consistent with a competitive-like pattern. In contrast, nobiletin inhibited nicotine-induced ERK1/2 phosphorylation, which was not overcome by increasing nicotine concentrations, consistent with a noncompetitive-like pattern. Nobiletin also selectively suppressed nicotine-induced intracellular Ca²⁺ level increase without affecting ATP-, KCl-, or Ca²⁺ ionophore-induced Ca²⁺ responses, suggesting selective inhibition of nicotinic receptor-mediated Ca²⁺ entry. Although it modestly increased basal ERK1/2 phosphorylation, nobiletin suppressed both muscarinic and nicotinic receptor-mediated ERK1/2 activation. These findings suggest that nobiletin differentially regulates muscarinic and nicotinic receptor-mediated ERK signaling, reveal novel pharmacological actions of nobiletin, and provide insight into the regulation of neuronal cholinergic signaling by citrus polymethoxyflavones.