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Reactive oxygen species generation has been implicated with diverse vascular and inflammatory diseases. NADPH oxidase is the major source of reactive oxygen species within phagocytes. It is activated by the phosphorylation and recruitment of cytosolic subunits p47phox, p67phox and rac2 to the membrane-bound cytochromeb558 formed by the p22phox and gp91phox subunits. In vascular cells, an analogous enzymatic complex is found within the membrane or cytosol but it has primarily signaling purposes. Both oxidases are inhibitable by thiol oxidants, not affected by the global redox cell state. Thiol oxidoreductases are effective modulators of the thiol redox state on cell membranes, in the ER and cytosol. Considering that spontaneous thiol–disulfide exchange reactions occur at rates too slow to be involved with regulatory pathways, we postulate that thiol oxidoreductases may control the redox state of important thiol sites on the NADPH oxidase, affecting its superoxide generating activity. Protein disulfide isomerase (PDI) is a ubiquitous multifunctional enzyme of the thiol oxidoreductase family, involved in regulation of diverse cellular mechanisms. Our objective is to investigate a possible interaction between PDI and the oxidases in vascular smooth muscle cells and neutrophils. Western blot analysis disclosed PDI in neutrophils within granules and the membrane, the same place where cytochrome b558 is found. In smooth muscle cells PDI was also found on the membrane fraction, and immunofluorescence disclosed a spatial colocalization between PDI and the oxidase. Superoxide generation was evaluated by superoxide dismutase-inhibitable cytochrome c reduction spectrophotometric assay using the cell free system (isolated membrane and cytosol from human neutrophils). Inhibition of PDI activity caused a 60% reduction of NADPH oxidase activity. With vascular smooth muscle cell homogenates, PDI inhibition caused similar oxidase activity. Angiotensin II, a known vascular NADPH oxidase agonist, induced in vascular cells a parallel increase in PDI activity. PDI modulation of thiol redox state on the vascular and phagocytic oxidases may thus represent a new regulatory mechanism of reactive oxygen species generation. |
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