S-glutathionylation is a redox-regulated modification that uncouples eNOS, switching its function from NO synthesis to •O2− generation, and serves to regulate vascular function. While in vitro or in vivo eNOS S-glutathionylation with modification of Cys689 and Cys908 of its reductase domain is triggered by high levels of GSSG or oxidative thiyl radical formation, it remains unclear how this process may be reversed. Glutaredoxin-1 (Grx1), a cytosolic/glutathione-dependent enzyme, can reverse protein S-glutathionylation; however, its role in regulating eNOS S-glutathionylation remains unknown. We demonstrate that Grx1 in the presence of GSH (1 mM) reverses GSSG-mediated eNOS S-glutathionylation with restoration of NO synthase activity. Since Grx1 also catalyzes protein S-glutathionylation with increased [GSSG]/[GSH], we measured its effect on eNOS-S-glutathionylation when [GSSG]/[GSH] was > 0.2, as can occur in cells and tissues under oxidative stress, and observed increased eNOS S-glutathionylation with a marked decrease in eNOS activity without uncoupling. This eNOS S-glutathionylation was reversed with decrease in [GSSG]/[GSH] to < 0.1. LC/MS/MS identified a new site of eNOS S-glutathionylation by Grx1 at Cys382, on the surface of the oxygenase domain, without modification of Cys689 or Cys908 that are buried within the reductase. Furthermore, Grx1 was demonstrated to be a protein partner of eNOS in vitro and in normal endothelial cells, supporting its role in eNOS redox-regulation. In endothelial cells, Grx1 inhibition or gene silencing increased eNOS S-glutathionylation and decreased cellular NO generation. Thus, Grx1 can exert an important role in the redox-regulation of eNOS in cells.

Over-activation of p38 is implicated in many cardiovascular diseases (CVDs), including myocardial infarction, hypertrophy, heart failure, and ischemic heart disease. Numerous therapeutic interventions for CVDs have been directed towards the inhibition of the p38 mitogen-activated protein kinase activation that contributes to the detrimental effect after ischemia/reperfusion (I/R) injuries. However, the efficacy of these treatments is far from ideal as they lack specificity and are associated with high toxicity. Previously, we demonstrated that N-acetyl cysteine (NAC) pre-treatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 on the modulation of the p38 pathway. In rat endothelial cells, DUSP4 is time-dependently degraded with H/R (0.25 ± 0.07 fold change of control after 2 h H/R). Its degradation is closely associated with hyper-phosphorylation of p38 (2.1 ± 0.36 fold change) and cell apoptosis, as indicated by the increase in cells immunopositive for cleaved caspase-3 (12.59% ± 3.38%) or TUNEL labeling (29.46% ± 3.75%). The inhibition of p38 kinase activity with 20 µM SB203580 during H/R prevents H/R-induced apoptosis, assessed via TUNEL (12.99% ± 1.89%). Conversely, DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81% ± 5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47% ± 1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4−/− hearts had significantly larger infarct size than WT. The increase in I/R-induced infarct in DUSP4−/− mice significantly correlates with reduced functional recovery (assessed by: RPP%, LVDP%, HR%, and dP/dtmax) as well as lower CF% and a higher initial LVEDP. From immunoblotting analysis, it is evident that p38 is significantly over-activated in DUSP4−/− mice after I/R injury. The activation of cleaved caspase-3 is seen in both WT and DUSP4−/− I/R hearts. Infusion of a p38 inhibitor prior to ischemia and during the reperfusion improves both WT and DUSP4−/− cardiac function. Therefore, the identification of p38 kinase modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.

Redox imbalance is a primary cause for endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of GSH, the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and BH4 levels. Immunoblotting of eNOS and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are up-regulated by NAC. EPR spin-trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd2+ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd2+-induced apoptosis. Moreover, the increased DUSP4 expression can redox regulate p38 and ERK1/2 pathways from hyper-activation, providing a survival mechanism against the toxicity of Cd2+. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS translation, and thus protects against Cd2+-induced stress. Depletion of intracellular GSH by BSO makes cells more susceptible to Cd2+-induced apoptosis. Pre-treatment with NAC prevents p38 over-activation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.

No abstract

No abstract