Oxidative modification of rat sulfotransferase 1A1 activity in hepatic tissue slices correlates with effects on the purified enzyme.

Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1'-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 µM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 µM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme.

Genetic interleukin-10 deficiency causes vascular remodeling via the upregulation of Nox1.

BACKGROUND AND HYPOTHESIS: Interleukin (IL)-10 is an anti-inflammatory cytokine. Nox1 is a mitogenic oxidase (p65-mox). The objective of this study was to test a hypothesis that IL-10 deficiency would cause vascular remodeling via the upregulation of Nox1. METHODS AND RESULTS: Recombinant adeno-associated virus (AAV) carrying short hairpin small interference RNA for Nox1 (AAV.Nox1shRNA) was constructed for in-vivo-specific inhibition of Nox1. Three groups of IL-10 gene knockout (IL-10KO) mice and three groups of wild-type mice were used. Three groups of each strain received intravenous delivery of AAV.Nox1shRNA, AAV with scrambled shRNA, and PBS, respectively. Animals were euthanized at 3 weeks after gene delivery. IL-10KO increased Nox1 protein expression, NADPH oxidase activity, and superoxide production in aortas. IL-10KO also resulted in a significant decrease in aortic medial thickness, a loss of smooth muscle cells (SMCs), and an increase in vascular collagen deposition, indicating vascular remodeling. The IL-10KO induced increases in NADPH oxidase activity and superoxide production, and vascular remodeling were abolished by silencing of Nox1 (p65-mox), suggesting that these effects may be mediated by the upregulation of Nox1. In addition, IL-10KO increased endothelin-1 levels in plasma and aortas, and this effect was partially blocked by silencing of Nox1. RNA interference silencing of Nox1 obliterated the IL-10KO-induced increases in IL-6 expression in aortas, superoxide production, and matrix metalloproteinase-9 activity in aortic SMCs, and SMC migration. CONCLUSION: IL-10 is essential for the maintenance of normal vasculature, as IL-10 deficiency resulted in vascular damage and remodeling. The IL-10KO-induced vascular structure damage may be mediated by the upregulation of Nox1.

Endothelin (ET)-1 inhibits nicotinamide adenine dinucleotide phosphate oxidase activity in human abdominal aortic endothelial cells: a novel function of ETB1 receptors.

Endothelin (ET)-1 stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and increases superoxide production in some cells such as vascular smooth muscle cells. Here, we reported that ET1 inhibited NADPH oxidase activity, superoxide generation, and cell proliferation in human abdominal aortic endothelial cells (HAAECs) via the ETB1-Pyk2-Rac1-Nox1 pathway. Superoxide production was determined by assessing ethidium fluorescence using flow cytometry in HAAECs exposed to ET1 (10-30 nm) at different time intervals. ET1 significantly decreased superoxide production in HAAECs in the presence of NG-nitro-L-arginine methyl ester, indicating that ET1 suppressed superoxide generation independent of nitric oxide synthase. ET1 significantly attenuated NADPH oxidase activity and cell proliferation, which could be abolished by silence of Nox1 gene, suggesting that ET1-induced inhibition of NADPH oxidase activity was mediated by Nox1. Furthermore, RNA interference silence of ETB1 receptors significantly increased NADPH oxidase activity, and blocked the inhibitory effect of ET1 on NADPH oxidase activity. Activation of ETB1 receptors by ET1 suppressed protein phosphorylation of pyk2 (Y402) and Rac1, suggesting that ET1 inhibited NADPH oxidase activity via ETB1-Pyk2-Rac1 pathway. Indeed, inhibition of Pyk2 by AG-17 abolished ET1-induced suppression of NADPH oxidase activity. ET1 also attenuated angiotensin II-induced activation of NADPH oxidase and cell proliferation. This study demonstrated, for the first time, that ET1, via ETB1, inhibited NADPH oxidase activity in HAAECs by suppressing the Pyk2-Rac1-Nox1 pathway. This finding reveals a novel function of ETB1 receptors in regulating endothelial NADPH oxidase activity, superoxide production, and cell proliferation, opening a new avenue for understanding the role of ETB1 receptors in protecting endothelial cells.