Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91- phox and p22- phox in Endothelial Cells

Abstract

Abstract-The production of reactive oxygen species (ROS) within endothelial cells may have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies indicate that a phagocyte-type NAD(P)H oxidase is a major source of endothelial ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specificity (NADHϾNADPH). In the present study, we (1) cloned and characterized the 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 sequences; and (3) studied the subcellular location of these subunits in endothelial cells. Although these studies revealed an overall high degree of homology (Ͼ90%) between the endothelial and phagocytic oxidase subunits, the endothelial gp91-phox sequence has potentially important differences in a putative NADPH-binding domain and in putative glycosylation sites. In addition, the subcellular location of the endothelial gp91-phox and p22-phox subunits is significantly different from that reported for the neutrophil oxidase, in that they are predominantly intracellular and collocated in the vicinity of the endoplasmic reticulum. This first detailed characterization of gp91-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. (Arterioscler Thromb Vasc

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

“…Indeed, increases in overall NAD(P)H oxidase activity and O 2 -production as well as mRNA and protein expressions of p22-phox, a membrane-bound component of NAD(P)H oxidase system, have been demonstrated in proliferating compared to resting CMEC. NAD(P)H oxidase is a multicomponent enzyme system that consists of several cytsosolic subunits including p47-phox and p67-phox as well as two membrane-bound components namely p22-phox and gp91-phox that make up cytochrome b 558 [11,12].…”

“…Since cytochrome b 558 accounts for enzyme activity and stability as a whole, it is assumed that the alteration(s) occur in the expression of one subunit is/are reflective of similar changes in the other [12]. Hence, the current study solely concentrated on the changes in mRNA and protein expression of p22-phox subunit that is expressed in abundance in CMEC [12].…”

“…Hence, the current study solely concentrated on the changes in mRNA and protein expression of p22-phox subunit that is expressed in abundance in CMEC [12].…”

“…The membrane-bound components of (p22-phox and gp91-phox that make up cytochrome b 558 which is responsible for enzyme activity and stability as a whole) a phagocyte-like NAD(P)H oxidase have also been characterized in endothelial cells [12]. Although low level production of ROS in non-phagocytic cells seems to be beneficial and is involved in several regulatory cellular processes including gene expression, higher quantities of ROS are implicated in the pathogenesis of atherosclerosis through activating VSMC proliferation and migration [1,13,14].…”

Signaling by the Respiratory Burst in Macrophages

“…The assembled oxidase can then use cytosolic NADPH to reduce extracellular O 2 to O :¡ 2 . Several studies showed that the mitogenic response of nonphagocytic cells requires the production of ROS by an NADPH oxidase (7,16,17 ) and similar or identical phox components have been found in a variety of cell types, including smooth muscle cells, broblasts, endothelial cells, and others, either at the RNA or protein levels (18)(19)(20). Homologues of gp91 phox , the catalytic moiety of the phagocyte NADPH oxidase, have been identi ed and are referred to as NOX.…”