(17)O NMR spectroscopy-assisted in vitro bioactivity studies of the intermediates formed via Na(2)S and RSNO cross-linking reactions

The cross-linking reaction between sulfide and S-nitrosothiol moieties has been intensively investigated and thionitrite/thionitrous acid (SNO(−)/HSNO) as well as nitrosopersulfide (SSNO(−)) were reported to be the intermediates that could serve as reservoirs for nitric oxide (NO). However, debate still exists regarding the stability and biological activity of SNO(−)/HSNO and SSNO(−). In order to investigate the chemical properties and biological activity of SNO(−) and SSNO(−), we set out to re-characterize the reaction intermediates using UV-Vis and (15)N NMR spectroscopy techniques, as well as a new (17)O NMR approach. The effects of SNO(−) and SSNO(−) on cellular NO and cGMP levels were assessed via cell culture experiments, and also the effects of SNO(−) and SSNO(−) on cell proliferation, migration, and capillary-like structure formation were evaluated with human umbilical vein endothelial cells (HUVEC). Through this work, the characteristic peaks and half-lives of SNO(−) and SSNO(−) were elucidated under various preparation conditions. The biological assays demonstrated that SSNO(−) increased the cellular NO and cGMP levels and also facilitated cell proliferation, migration and stimulated angiogenesis, while in contrast SNO(−) did not exhibit these effects.