Arecoline Increases the Production of Nitric Oxide and Post-Translational S-Nitrosoproteome in Endothelial Cells

Background: Arecoline is known as a carcinogenic toxicant. The refreshment effect of arecoline is mainly due to the increase in vasodilation and blood flow. This is essential to understand whether arecoline can induce the production of Nitric Oxide (NO•) and regulate the subsequent protein S-nitrosylation in Endothelial Cells (ECs). Objective: The present study is focused on the promotion effect of arecoline in NO• production and the subsequent regulation of S-nitrosoproteome. Methods: The phosphorylation of endothelial nitric oxide synthase serine 1177 residue (peNOS^Ser1177) was investigated by western blot. By using a specific FA-OMe fluorescent probe, the NO• molecules could be observed by fluorescent microscopy or flow cytometry. S-nitrosylated proteins were purified by biotin switch and then subjected to the Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-labeled shotgun proteomic analysis. Results: Our study reveals that a lower concentration of arecoline can increase the phosphorylation of peNOS^Ser1177. Pretreatment of N^G-nitro-L-arginine methyl ester (L-NAME) indicated that arecolineinduced NO• production was mediated by e-NOS. We identified 224 proteins with up-regulated S-nitrosylation and 159 proteins with down-regulated S-nitrosylation. The NO• binding sites of seven representative S-nitrosoproteins were illustrated. The effect of arecoline on the S-nitrosylation of HSP60 chaperonin and calnexin was verified. Conclusion: Our experimental results proved that a lower concentration of arecoline could modulate the production of NO• and the subsequent protein S-nitrosylation. Therefore, it is worthy for further investigation and discussion if these S-nitrosoproteomes are important in maintaining endothelium homeostasis.