Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells

Abstract

The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91phox, p22phox, p47phox, and p67phox, in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91phox and p22phox. After subcellular protein fractionation, ∼50% of the gp91phox (91-kDa band), p22phox, p67phox, and p40phox pools and ∼30% of the p47phox were present in the 1475 × g ("nucleus-rich") fraction. Likewise, ∼50% of total NADPH-dependent O·2 production (assessed by lucigenin (5 μM) chemiluminescence) was found in the 1475 × g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22phox, gp91phox, p47phox, and p40phox existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton.