Molecular characterization and localization of the NAD(P)H oxidase components gp91-phox and p22-phox in endothelial cells

The production of reactive oxygen species (ROS) within endothelial cells ma y have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies in dicate that a phagocyte-type NAD(P)H oxidase is a major source of endotheli al ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specif icity (NADH>NADPH). In the present study, we (1) cloned and characterized t he cDNA and predicted amino acid structures of the 2 major subunits of rat coronary microvascular endothelial cell NAD(P)H oxidase, gp91-phox and p22- phox; (2) undertook a detailed comparison with phagocytic NADPH oxidase seq uences, and (3) studied the subcellular location of these subunits in endot helial cells. Although these studies revealed an overall high degree of hom ology (>90%) between the endothelial and phagocytic oxidase subunits, the e ndothelial gp91-phox sequence has potentially important differences in a pu tative NADPH-binding domain and in putative glycosylation sites. In additio n, the subcellular location of the endothelial gp91-phox and p22-phox subun its is significantly different from that reported for the neutrophil oxidas e, in that they are predominantly intracellular and collocated in the vicin ity of the endoplasmic reticulum. This first detailed characterization of g p91-phox and p22-phox structure and location in endothelial cells provides new data that may account, in part, for the differences in function between the phagocytic and endothelial NAD(P)H oxidases.