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.

Keywords: Gamma-glutamyltransferase; Mass spectrometry; Oxidative stress; Protein S-nitrosation; Protein disulfide isomerase; S-nitrosoglutathione.