Reactive oxygen species regulate signaling pathways induced by M1 muscarinic receptors in PC12M1 cells

Activation of the mi muscarinic receptor subtype in rat pheochromocytoma (PC12) cells stably expressing cloned mi muscarinic acetylcholine receptors was previously shown to induce morphological changes and growth arrest. However, the signaling pathways which lead to these effects were not identified. In an attempt to characterize the intracellular signaling that might be involved in the muscarinic-induced effects, we investigated the role of reactive oxygen species in the regulation of these processes. Stimulation of the muscarinic receptor in these cells increased the intracellular concentrations of reactive oxygen species. Muscarinic activation induced intracellular signaling pathways that involve activation of Ras, extracellular signal-regulated kinase (ERK), and p38. These pathways were partially blocked when reactive oxygen species (ROS) production was prevented by the antioxidant N-acetylcysteine. Other muscarinic-induced signals, such as activation of c-Jun NH2-terminal kinase (JNK) or an increase in the binding activity of the transcription factors nuclear factor-KB and activator protein-1, were inhibited by the antioxidant dicoumarol. N-Acetylcysteine also blocked the growth arrest and changes in cell shape induced by stimulation of the muscarinic receptor in PC12M1 cells. These findings suggest that ROS act as second messengers in muscarinic-induced cellular signaling. Moreover, generation of ROS appears to be an early and critical intermediary event, which occurs immediately after stimulation of the muscarinic receptor and affects in a variety of mechanisms the muscarinic-mediated cellular signaling.