Reduced Activity of the Aortic Gamma-Glutamyltransferase Does Not Decrease S-Nitrosoglutathione Induced Vasorelaxation of Rat Aortic Rings

Aims: Gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, is involved in the release of nitric oxide (NO) from S-nitrosoglutathione (GSNO) and in the GSNO-induced vasodilation. Endogenous GSNO is a physiological storage form of NO in tissues while exogenous GSNO is an interesting candidate for compensating for the decreased NO bioavailability occurring during cardiovascular diseases. We investigated in a rat model of human hypertension, the spontaneous hypertensive rat (SHR), submitted or not to high salt diet, whether a decreased vascular GGT activity modifies the vasorelaxant effect of GSNO. Methods: Thoracic aortic rings isolated from male SHR and Wistar Kyoto rats (WKY) aged 20–22 weeks—submitted or not for 8 weeks to a high salt diet (1% w/v NaCl in drinking water) were pre-constricted with phenylephrine then submitted to concentration-vasorelaxant response curves (maximal response: Emax; pD2) to carbachol or sodium nitroprusside to evaluate endothelial dependent or independent NO-induced vasodilation, or GSNO (exogenous NO vasodilation depending from the endothelial GGT activity). GGT activity was measured using a chromogenic substrate in aortic homogenates. Its role in GSNO-induced relaxation was assessed following inhibition of the enzyme activity (serine-borate complex). That of protein disulfide isomerase (PDI), another redox sensitive enzyme involved in GSNO metabolism, was assessed following inhibition with bacitracin. Results: Aortic GGT activity (18–23 μmol/min/mg of tissue in adult WKY) decreased by 33% in SHR and 45% in SHR with high salt diet. Emax and pD2 for sodium nitroprusside were similar in all groups. Emax for carbachol decreased by −14%, reflecting slight endothelial NO-dependent dysfunction. The GSNO curve was slightly shifted to the left in SHR and in SHR with high salt diet, showing a small enhanced sensitivity to GSNO. Involvements of GGT, as that of PDI, in the GSNO effects were similar in all groups (pD2 for GSNO −0.5 to −1.5 following enzymatic inhibition). Conclusion: Hypertension is associated with a decreased aortic GGT activity without decreasing the vasorelaxant effects of GSNO, whose bioactivity may be supplemented through the alternative enzymatic activity of PDI

Synthesis and evaluation of new designed multiple ligands directed towards both peroxisome proliferator-activated receptor-γ and angiotensin II type 1 receptor

Because of the complex biological networks, many pathologic disorders fail to be treated with a molecule directed towards a single target. Thus, combination therapies are often necessary, but they have many drawbacks. An alternative consists in building molecules intended to interact with multiple targets, called designed multiple ligands. We followed such a strategy in order to treat metabolic syndrome, by setting up molecules directed towards both type 1 angiotensin II (AT1) receptor and peroxisome proliferator-activated receptor-γ (PPAR-γ). For this purpose, many molecules were prepared by merging both pharmacophores following three different strategies. Their ability to activate PPAR-γ and to block AT1 receptors were evaluated in vitro. This strategy led to the preparation of many new PPAR-γ activating and AT1 blocking molecules. Among them, some exhibited both activities, highlighting the convenience of this approach

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

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

International audienceAmong 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

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

Cardiovascular diseases are associated with oxidative stress and reduced nitric oxide (NO) bioavailability. The ability of NO donors like S-nitrosoglutathione (GSNO) to regulate NO bioavailability under oxidative stress is poorly studied. Here, we monitored protein S-nitrosation (Pr-SNO), a post-translational protein modification in smooth muscle cells exposed to GSNO under oxidative stress. Intracellular thiol redox status in relation with the extent and distribution of GSNO-induced intracellular Pr-SNO (LC-MALDI MS) were assessed. The role of the gammaglutamyl transferase (GGT), a redox enzyme metabolizing GSNO, in Pr-SNO formation was also studied. GSNO prevented the oxidation of proteins SH groups. Concomitantly, a 2-fold increase of GSNO-dependent Pr-SNO formation still depending on GGT activity was observed. Mass spectrometry identified 51 proteins S-nitrosated by GSNO under oxidative stress (vs 32 in basal condition), including a higher number of cytoskeletal proteins (17 vs 8 in basal condition) related to cell morphogenesis and movement. Furthermore, additional proteins belong to cell adhesion and protein trafficking were S-nitrosated under oxidative stress. Oxidative stress modifies the extent and distribution of GSNO induced Pr-SNO formation, a NO storage form in tissue. Further studies will likely elucidate the pathophysiological significance of these observations

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

Cardiovascular diseases are associated with oxidative stress and reduced nitric oxide (NO) bioavailability. The ability of NO donors like S-nitrosoglutathione (GSNO) to regulate NO bioavailability under oxidative stress is poorly studied. Here, we monitored protein S-nitrosation (Pr-SNO), a post-translational protein modification in smooth muscle cells exposed to GSNO under oxidative stress. Intracellular thiol redox status in relation with the extent and distribution of GSNO-induced intracellular Pr-SNO (LC-MALDI MS) were assessed. The role of the gammaglutamyl transferase (GGT), a redox enzyme metabolizing GSNO, in Pr-SNO formation was also studied. GSNO prevented the oxidation of proteins SH groups. Concomitantly, a 2-fold increase of GSNO-dependent Pr-SNO formation still depending on GGT activity was observed. Mass spectrometry identified 51 proteins S-nitrosated by GSNO under oxidative stress (vs 32 in basal condition), including a higher number of cytoskeletal proteins (17 vs 8 in basal condition) related to cell morphogenesis and movement. Furthermore, additional proteins belong to cell adhesion and protein trafficking were S-nitrosated under oxidative stress. Oxidative stress modifies the extent and distribution of GSNO induced Pr-SNO formation, a NO storage form in tissue. Further studies will likely elucidate the pathophysiological significance of these observations