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.

背景：槟榔碱被称为致癌性毒物。槟榔碱的提神作用主要是由于血管舒张和血流量的增加。这对于了解槟榔碱是否可以诱导一氧化氮（NO•）的产生并调节随后在内皮细胞（EC）中的蛋白质S-亚硝基化至关重要。

目的：本研究的重点是槟榔碱在NO·生产中的促进作用及随后的S-亚硝基蛋白质组调控。

方法：采用western blot方法检测内皮一氧化氮合酶丝氨酸1177残基（peNOS ^Ser1177）的磷酸化。通过使用特定的FA-OMe荧光探针，可以通过荧光显微镜或流式细胞术观察NO•分子。通过生物素转换纯化S-亚硝基化的蛋白质，然后进行等压标记，用于相对和绝对定量（iTRAQ）标记的shot弹枪蛋白质组学分析。

结果：我们的研究表明，较低浓度的槟榔碱可以增加peNOS ^Ser1177的磷酸化。N个预处理^ģ -硝基-L-精氨酸甲酯（L-NAME）指出，arecolineinduced NO•产量通过e-NOS介导的。我们确定了224个蛋白的S-亚硝基化上调和159个蛋白的S-亚硝化下调。说明了七个代表性S-亚硝基蛋白的NO•结合位点。验证了槟榔碱对HSP60伴侣蛋白和钙粘蛋白S-亚硝基化的影响。

结论：我们的实验结果证明，较低浓度的槟榔碱可以调节NO•的产生以及随后的蛋白S-亚硝基化。因此，这些S-亚硝基蛋白质组在维持内皮稳态方面是否重要，值得进一步研究和讨论。