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...

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of Xdh , the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte Xdh substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte Xdh is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative.

Mitochondrial dysfunction and oxidative stress are strongly implicated in the pathogenesis of Parkinson disease (PD) and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2), which is a major enzymatic generator of superoxide. Although NOX2 has been examined in the context of PD, previous studies have focused on microglial function; the role of neuronal NOX2 in PD pathogenesis remains to be defined. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and 2 models thereof, neuronal NOX2 is highly active in substantia nigra dopamine neurons. Further, NOX2 activity is responsible for accumulation, post-translational modification and oligomerization of alpha-synuclein as well as activation of leucine-rich repeat kinase 2 (LRRK2). Administration of a brain-penetrant, specific NOX2 inhibitor prevented NOX2 activation and its downstream effects in vivo in a rat model of PD. We conclude that neuronal NOX2 is a major contributor to oxidative stress in PD, to alpha-synuclein pathology and to LRRK2 activation in idiopathic PD. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.