Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Abstract

Superoxide anion (O 2 ؊ ) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O 2 ؊ is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O 2 ؊ production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O 2 ؊ -generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22 phox , gp91 phox , p47 phox , and p67 phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O 2 ؊ production. Furthermore, immunodepletion of p67 phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67 phox . Further study into the regulation of this adventitial source of O 2 ؊ is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.In recent years, reactive oxygen species (ROS) such as superoxide anion (O 2 Ϫ ) have been shown to play a number of roles in the body. Phagocytic cells produce ROS as a primary host defense mechanism (1), whereas other cells utilize ROS as intracellular second messengers for a wide range of cellular functions. For example, ROS participate in Ras-mediated mitogenic signaling in fibroblasts as well as in leukocyte apoptosis (2, 3).The phagocyte NADPH oxidase or respiratory burst oxidase is the best characterized ROS-generating system and is a multicomponent enzyme complex that catalyzes the oneelectron reduction of oxygen to O 2 Ϫ . Its components include the two membrane-spanning polypeptides, p22 phox and gp91 phox , which comprise flavocytochrome b 558 , and three cytoplasmic polypeptides, p40 phox , p47 phox , and p67 phox (4-6). Additionally, the cytosolic guanine nucleotide-binding protein Rac2 is required for oxidase activation (7). Exposure of the cell to a variety of agonists induces the association of the cytosolic with the membrane-associated components and causes activation of the normally dormant oxidase (4-6).Several groups have shown that NAD(P)H oxidase(s) exist in nonphagocytic cells including carotid body (8), mesangial cells (9), vascular smooth muscle cells (10, 11), endothelial cells (12, 13), and fibroblasts (14). The NAD(P)H oxidase systems in these cells have not yet been well characterized, and even the substrate specificity of these oxidase(s) with regard to NADH and NADPH is still not clear (10)(11)(12)15). In vascular smooth muscle cells of the bovine pulmonary artery and rat aorta, an NADH oxidase has been described, and in the rat aorta these cells express...

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…Nox1 in vascular smooth muscle participates in cell proliferation [46], while Nox4 in these cells participates in maintenance of the differentiated phenotype [47,48]. Nox2 and associated regulatory proteins are also expressed in adventitia, where they contribute to constitutive ROS generation [49] and to Angiotensin II-induced vascular tone in part through inactivation of NO by superoxide [50]. In addition, Nox5 is expressed in vascular smooth muscle [51] and [Petumnetcha and Lambeth, unpublished].…”

Redox Control of Renal Function and Hypertension

“…In the vasculature, the endothelial as well as adventitial NAD(P)H oxidase is composed of gp91 phox (Nox2) and p22 phox , as well as p47 phox and p67 phox and the G protein Rac1 (88,245). It is now established that NAD(P)H oxidase expressed in endothelial cells is an important source of free oxygen radical generation in the arterial wall (147).…”

Nitric Oxide–Induced Increase in p21 Sdi1/Cip1/Waf1 Expression During the Cell Cycle in Aortic Adventitial Fibroblasts

“…10,11 Recent in vivo and in vitro studies have implicated the adventitial fibroblast as a major site for superoxide production in response to angiotensin II administration, 12,13 and we have recently shown that adventitial fibroblasts are a major source of the inducible form of NO synthase in aortic tissue after the in vivo administration of endotoxin to produce an inflammatory response. 14 To begin to evaluate the possible role of NO in modulating vascular remodeling during hypertension, we have used procedures for obtaining adult rat adventitial fibroblasts in culture and have studied the effects of NO on cell proliferation under standardized conditions in which quiescent cells were stimulated with FCS.…”