Original ContributionDifferential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4
Introduction
Endothelial dysfunction in diabetes mellitus was shown to be secondary to increased superoxide production within the endothelium and/or smooth muscle cells [1]. Subsequent studies with vascular tissue from diabetic animals [1] and patients [2] pointed toward NADPH oxidase and an uncoupled nitric oxide synthase (NOS) [3] as potential superoxide sources. Furthermore, in nonvascular tissues mitochondria [4], [5] and in plasma xanthine oxidase [6], [7] were identified as sources of increased superoxide formation in the diabetic state. The vascular NADPH oxidase shares several characteristics with the multicomponent enzyme complex described in neutrophils (for review see [8]). The endothelial, the adventitial, and the neutrophil NADPH oxidase consist of the flavocytochrome b558 subunits gp91phox and p22phox as well as the cytosolic factors p47phox and p67phox and the small molecular weight G protein rac-1 [8]. These subunits were also found to exist in rat cardiac myocytes where they are thought to play a role in α-adrenoceptor stimulation-induced hypertrophy [9], [10]. Furthermore, p47phox protein was increased in rat ventricular myocytes cultured in high glucose medium [11]. However, smooth muscle cells from large arteries lack gp91phox, but instead express the recently identified gp91phox homologues nox1 and nox4 [12], [13]. Both nox1 and nox4 mRNA were also detected in endothelial cells and fibroblasts, whereas a high level of nox2 mRNA, much less nox1 and no nox4, was detectable in inflammatory cells [14]. Overexpression of nox1 in fibroblasts and nox4 expression in the kidney increased superoxide production [12], [15] while nox4 overexpression in adipose cells increased insulin-induced hydrogen peroxide production [16], indicating that these NADPH oxidase subunits are functionally important. Recent studies have provided evidence for increased mRNA and/or protein levels of gp91phox, p22phox, p47phox, and p67phox in blood vessels from diabetic animals [1] and diabetic patients [2]. Gp91phox, nox1, and nox4 harbor the electron-transfer moieties of the enzyme complex and thus serve as the catalytic component; hence, regulation of these subunits is considered to be of utmost importance to the ultimate functioning of the enzyme. Because multiple homologues are expressed, it is unclear which ones play a role in specific vascular diseases. Based on previous studies from our group [1] and others [17] on the role of nox2 (gp91phox) in diabetes, the effect of streptozotocin-induced type I diabetes on the expression of the NADPH oxidase subunits nox1 and nox4 and on the vascular NADPH oxidase activity was analyzed.
Section snippets
Materials
Streptozotocin, lucigenin, protease inhibitor cocktail for general use, NADH, NADPH, hypoxanthine, rotenone, DPI, and dithiothreitol (DTT) were all purchased from Sigma. The monoclonal anti-eNOS antibody was from Transduction Laboratories (Heidelberg, Germany), nox1 antibody from Santa Cruz Biotechnology (CA), anti-VASP, phospho-specific, and anti-sGCβ1 (rabbit) from Calbiochem (Darmstadt, Germany), anti-cGK-I from Chemicon (Temecula, CA) and the labeled (Alexa Fluor 594) goat anti-mouse
Animal body weights and blood glucose levels
In order to verify a diabetic state in STZ-treated rats we assessed blood glucose levels and body weights. All rats having received STZ had blood glucose levels >400 mg/dl 24 h after injection and >500 mg/dl at the time of sacrifice. This was significantly higher than the blood glucose levels of control animals at the time of sacrifice (170 ± 3.8 mg/dl). Furthermore, the body weights of the diabetic animals were significantly lower than those of the control group (Table 1). The diabetic animals
Discussion
In the present study, we provide the first evidence for upregulation of the gp91phox homologue nox1 in the aorta from type I diabetic animals. Our data indicate that 8 week STZ treatment increases vascular superoxide production throughout all layers of the vessel wall, as well as the expression of the nox1, but not nox4. Increased expression of nox1 was associated with an increase in NADPH oxidase activity in aortic membrane fractions, and similarly increased NADPH oxidase activity was also
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (Mu 1071/3–1 and 4–1). We greatly appreciate the technical assistance of Claudia Kuper and Hartwig Wieboldt.
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