NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nM, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26 -83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22 phox , Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22 phox , Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redoxsensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.Redox-dependent signal transduction, a central aspect of vascular physiology and pathophysiology, converges on enzyme systems generating reactive oxygen species, required to act as second messengers of cell stimulators such as angiotensin II (AII) 3 (1). Vascular isoforms of phagocyte NAD(P)H oxidase appear to be the most prominent source of basal as well as agonist-induced reactive oxygen species in the vessel (1-4). Yet, mechanisms that control activity of this multisubunit enzyme complex are incompletely understood. Most studies (reviewed in Refs. 3 and 4) have focused on issues related to subunit structure, undermining other aspects. We have previously shown that specific thiol oxidants/alkylators inhibit vascular NAD(P)H oxidase activity in a way that does not correlate with their induced decrease in intracellular glutathione levels (5). Moreover, recent evidence further suggests that oxidase activity is redox-regulated
