S-nitrosoglutathione potentiates protein S-nitrosation under oxidative stress, a potential improvement of NO storage into smooth muscle cells

Introduction To counteract NO deficiency occurring with oxidative stress (OS) in cardiovascular diseases, administration of S-nitrosothiols (RSNO) like S-nitrosoglutathione (GSNO), the main storage form of NO in tissues [1], represents an alternative to other NO-donors, with no tolerance nor OS induction. However, their ability to regulate NO bioavailability under OS is unknown. As S-nitrosation of proteins, the formation of high molecular weight RSNOs, is also considered as a form of NO storage in tissues [2], we evaluated whether an administration of GSNO will regulate protein S-nitrosation in an OS model of rat smooth muscle cells. Material and methods A rat smooth muscle cell line (SMC A-10) was stimulated by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH; 50 mM, 2 h, 37°C) to mimic OS. Intracellular thiol status as content of reduced glutathione (GSH) (2,3-naphthalene dicarboxaldehyde assay) and reduced thiol containing proteins (Ellman’s method) were monitored. The activity of γ-glutamyl transpeptidase (GGT), specifically implied in GSNO catabolism, was measured using L-γ-glutamyl-p-nitroanilide as chromogenic substrate. Then, the thiol status modifications and intracellular peptides/proteins S-nitrosation (2,3-diaminonaphthalene/Hg2+assay) were monitored in stressed SMC incubated for 1 h with 50 µM GSNO. S-nitrosated proteins were purified (biotin switch technique) and identified by mass spectrometry. Results Under OS, the intracellular content of reduced thiols was greatly decreased for GSH (59±4 to 29 ± 5 nmol/mg proteins, n = 3) compared to proteins (148 ± 6 to 125 ± 4 nmol/mg proteins, n = 3), with no impact of GSNO. However, GSNO increased the global content of intracellular S-nitrosated peptides/proteins upon OS (0.53 ± 0.04 to 1.07±0.09 nmol/mg proteins, n = 3). Although the GGT activity decreased (1.35 ± 0.20 to 0.39±0.14 nmol/min/mg proteins) under OS, it was still implied at 38±5% (using serine borate complex, a GGT specific inhibitor) into the intracellular peptides/proteins S-nitrosation. The final mass spectrometry identification revealed that 71 proteins were S-nitrosated under control condition and this rose to 93 under OS. Discussion/conclusion The increase in intracellular S-nitrosated proteins in smooth muscle cells submitted to OS and treated with GSNO can be the starting point for GSNO to restore the NO pool. How and when this NO pool can be released has to be further evaluated. References 1. Maron BA et al. Antioxid Redox Signal (2013) 18, 270-287. 2. Rayner BS et al. J Biol Chem (2005) 280, 9985-9993.