Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione

Abstract

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions. Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2'-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated. Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 μM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress. In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

“…In contrast to classic NO donors used in pharmacotherapy, S-nitrosothiols could be promising substitutes as they do not induce the development of tolerance or cyanide poisoning (Al-Sa'doni and Ferro, 2000;Belcastro et al, 2017). S-nitrosothiols proved effective in a variety of cardiovascular disorders, including e.g.…”

“…Cellular responses to GSNO have in fact received great attention during the last few years (Broniowska et al, 2013). Indeed, GSNO does not induce any tolerance or oxidative stress (Al-Sa'doni and Ferro, 2000;Belcastro et al, 2017). To date, there have been nearly 20 clinical trials investigating the therapeutic efficacy of GSNO in multiple pathological contexts, and most of them have focused on its effects in cardiovascular diseases (Gaucher et al, 2013).…”

“…High levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were recently identified under oxidative stress conditions (Belcastro et al, 2017). In particular, the detailed pattern of S-nitrosated proteins indicates that most of S-nitrosylated proteins are of primary relevance for the performance of SMC functions being often altered in vascular diseases, such as cell communication, cytoskeletal organization, morphogenesis, contraction and movement.…”

“…NO showed an inhibitory action on inhibitory synaptic transmission in the spinal dorsal horn, implicating S-nitrosation in pain transmission via disinhibition of inhibitory neurons (Lu et al, 2009(Lu et al, , 2011. In rat aortic SMCs stressed in vitro with a free radical generator (2,2′-azobis(2-amidinopropane dihydrochloride, AAPH), actin was S-nitrosated following exposure to GSNO (Belcastro et al, 2017), but the real impact of this modification on SMCs functions remains to be elucidated.…”

Antioxidant Properties of S-Nitrosoglutathione and Nanotechnologies

“…Among RSNOs, the Snitrosoglutathione (GSNO), made by S-nitrosation of glutathione, was the main NO-donor investigated for its therapeutic potential as antiplatelet agent with arterioselective vasodilator effects and also with well-documented antithrombotic effects. The S-nitrosation process apart from its ability to modulate signaling pathways by post-translational modification of proteins (Pr-SNO) is also a protection of thiol functions from oxidation [2][3][4]. Indeed, GSNO was shown to be able to reduce disulfide bounds forming S-nitroso functions, which are a lower oxidation state of thiol functions than disulfide or sulfenic/sulfonic acid functions [2,5].…”