Our laboratory recently described a tight interaction between the major reactive oxygen species source NAD(P)H oxidase and the endoplasmic reticulum (ER) redox chaperone Protein Disulfide Isomerase (PDI) in vascular smooth muscle cells (VSMC). Such interaction is likely a pathway for bridging ER stress and oxidative stress. Neointimal vascular cells after injury, e.g., share the occurrence of oxidative stress, NAD(P)H oxidase expression, severe ER stress and in particular decreased SOD activity. Thus, we postulated that ER stress might also regulate SOD activity. VSMC were challenged, for different time periods, with the ER stress inducer Tunicamycin (Tn, 5m g/ml), or the hypertrophic agent Angiotensin II (AII, 250nM). Both Tn and AII triggered NAD(P)H oxidase activity and ROS production to comparable levels, but AII did not induced ER stress markers. SOD activity, determined through inhibition of cytochrome-C reduction, was assessed at 30min and 2, 4 and 14 h after stimulus. With Tn, there was sustained 25-40% decrease in SOD activity, which was already pronounced at 30min (41± 9% decrease). In contrast, with AII there were minor (0-24%) non-sustained decreases. Western blotting analysis of VSMC exposed to ER stressors for 14h showed no consistent change in CuZnSOD expression, while extracellular SOD, a major SOD isoform in vascular cells, exhibited a complex pattern consistent with changes in post-translational modification and/or degradation. Indeed, another study (Nonaka et al, 2001) suggests decreased ecSOD glycosylation due to ER stress. Also, ER stressors promoted increased PDI translocation to VSMC membranes (western blotting), with parallel increase in PDI activity (insulin reduction assay). Since PDI is known to bind copper, we investigated in vitro whether PDI might compete with SOD for copper. To accomplish these studies, we purified recombinant PDI without histidine tag in E. coli. We confirmed by DTNB assay that PDI is able to bind 4 copper ions per monomer. Preliminary assays using cytochrome-C reduction inhibition suggest that PDI does not work as a copper chaperone for apoSOD or anti-chaperone for SOD, but cannot exclude that the 2 enzymes compete for this ion in the cell. Thus, ER stress induces early sustained decrease in SOD activity, strengthening the association between that ER stress and oxidative stress in vascular cells.

 

(Supported by FAPESP and CNPQ Milênio Redoxoma)