Muscarinic M1 receptor represents the predominant mAChR in the CNS. Previously, we showed that antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cellular lines and specifically interacts with cell surface membrane of human neuroblastoma SK-N-NE cells activating PLC transduction pathway. Further, we demonstrated that small SOD1 amount is contained in large core dense vesicle and is secreted in response to depolarization. In this study, performed in SK-N-BE cells, we investigated whether SOD1 induced activation of PLC transduction mechanism involves muscarinic M1 receptor.

Human neuroblastoma SK-N-BE cells were grown in DMEM; SOD1, M1 muscarinic receptor, P-AKT and P-ERK1/2 were detected by western blotting technique from cell lysates with specific polyclonal rabbit antibodies

Transfection of siRNAs was carried out by MicroPorator (MP-100) Digital Bio Technology, a pipette-type electroporation.

Intracellular calcium was measured by single cell computer-assisted videoimaging. SK-N-BE cells, grown on glass coverslips, were loaded with 10 mM Fura-2 acetoxymethyl ester (Fura-2AM).

The data obtained demonstrate that SOD1 interacts with muscarinic M1 receptor in human neuroblastoma SK-N-BE cells causing an activation of a trasductional mechanism that involves PLC and PKC. This effects is dose and time dependent on downstream signal molecules ERK 1/2 and AKT. In addition, our results indicate that in presence of M1 antagonist, pyrenzepine or after silencing of M1 receptor by siRNA experiment, SOD1 activation of ERK1/2 and AKT is remarkably reduced.

Our results clearly indicate that SOD1 carries out an inedited role as a putative extracellular messenger activating M1 muscarinic receptor signaling.