Copper increases superoxide dismutase activity in rat liver

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT²⁺ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT²⁺ photoreduction in the presence of 10^?4m EDTA; (iii) anaerobic NBT²⁺ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO₄ solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O₂^? as it has been shown in in vitro experiments [E. M. Russanov S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys.215, 220–229]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

Acute exercise increases expression of extracellular superoxide dismutase in skeletal muscle and the aorta

Abstract

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise.     

Acute exercise increases expression of extracellular superoxide dismutase in skeletal muscle and the aorta

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise.     

Oxidative stress,NO* and smooth muscle cell extracellular superoxide dismutase expression

Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors.Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation (n-acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO*-releasing compounds.In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.     

Hypercholesterolemia increases manganese superoxide dismutase immunoreactive macrophages in myocardium

The effect of hypercholesterolemia on manganese superoxide dismutase (MnSOD)-containing macrophages was investigated in male New Zealand white rabbits. Macrophages from control animals, which were marked with the RAM-11 antibody, demonstrated co-localization with MnSOD immunoreactivity, e.g. in the peri- and paravascular space within the myocardium, but not in the bone marrow. In rabbits fed a 0.5% cholesterol-enriched diet for 42 days, a significant increase (P<0.01) of MnSOD-immunoreactive macrophages within the myocardium was found concomitant to the drastic elevation of serum cholesterol level. In the bone marrow, MnSOD immunoreactivity did not change after cholesterol feeding. Thus in cholesterol-fed rabbits, the increase of MnSOD-containing macrophages seems to parallel that of lipoproteins. MnSOD is considered as being protective against the cytotoxic effects of those superoxide anions, possibly generated in macrophages, which are involved in the metabolism of modified lipoproteins.     

Inhibition of gene expression of heparin-binding epidermal growth factor-like growth factor by extracellular superoxide dismutase in rat aortic smooth muscle cells

Both extracellular superoxide dismsutase (EC-SOD) and heparin binding EGF like growth factor (HB-EGF) are produced in smooth muscle cells of the arterial wall, and are thought to play pathological roles in atherosclerosis with heparin binding characteristics. EC-SOD treatment clearly reduced the H₂O₂ induced expression of HB-EGF in rat aortic smooth muscle cells (RASMC). EC-SOD also inhibited the induction of HB-EGF by 12-O-tetradecanoylphorbol-13-acetate (TPA) in RASMC by 60%. Both H₂O₂ and TPA increased intracellular ROS levels, and EC-SOD inhibited ROS generation only for the case of H₂O₂ but not TPA. Treatment of the cells with heparin alone decreased HB-EGF expression by 20%, whereas EC-SOD alone and a co-incubation with EC-SOD and heparin suppressed the induction by 60 and 70%, respectively. These results suggest that EC-SOD is related to the EGF signaling in two ways, competition for HSPG with HB-EGF and as an ROS scavenger.     

Nitric oxide releasing reagent,S-nitroso-N-acetylpenicillamine,enhances the expression of manganese superoxide dismutase mRNA in rat vascular smooth muscle cells

Cultured rat vascular smooth muscle cells (VSMCs) produce nitric oxide (NO) under stimulation by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). NO synthase (NOS) and manganese superoxide dismutase (Mn-SOD) mRNA expressions are simultaneously induced by these stimulants in rat VSMCs. In VSMCs, S-nitroso-N-acetyl penicillamine (SNAP), one of the NO releasing reagents, induces Mn-SOD mRNA which may protect the VSMCs themselves. This suggests that NO itself may enhance the expression of Mn-SOD to protect the VSMC themselves against NO radicals in cultured rat VSMCs. © 1996 Wiley-Liss, Inc.     

Studies on cytosolic superoxide dismutase from intestinal mucosa

CuZn superoxide dismutase from monkey (Macaca radiata) intestinal mucosa was purified to homogenity. The enzyme showed a subunit molecular weight of 16000. The enzyme preparation from intestinal mucosa of rat, rabbit, guinea-pig and monkey was distinctly different in electrophoretic mobility and in elution profile on ion-exchange chromatography, possibly due to their difference in charge. The difference may not be due to glycosylation, since the enzyme was not stained for glycoprotein. Polyclonal antibody against purified monkey enzyme inhibited the activity of intestinal CuZn superoxide dismutase from rat, rabbit and guinea-pig. Thus it appears that intestinal CuZn superoxide dismutases from different sources, despite being similar in immunological and other properties, differ in certain amino acids and hence in charge.     

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.     

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.     

Ageing transition of superoxide dismutase in rat retina

To examine the relationship between retinal ageing and superoxide dismutase, the distribution and expression of the dismutase was studied in the retina of 2-year-old Sprague--Dawley albino rats with immunohistochemistry and immunochemical quantitative analysis. Eight-week-old Sprague--Dawley albino rats were used as controls. In 2-year-old rats, manganese superoxide dismutase (Mn-SOD) immunoreactivities in the photoreceptor inner segments, the outer nuclear layer and the inner plexiform layer were stronger than those in 8-week-old rats. Copper--zinc superoxide dismutase (CuZn-SOD) immunoreactivities in the outer nuclear layer and inner plexiform layer of 2-year-old rats were stronger than those in 8-week-old rats. Faint CuZn-SOD immunoreactivity became visible in the photoreceptor inner segments of 2-year-old rats, whereas no CuZn-SOD immunoreactivity was observed in 8-week-old rats. Our immunochemical quantitative analysis also showed an increase in the immunoreactivities of superoxide dismutases in the sensory retina with age. The transition of the dismutases may have some relationship with retinal ageing. © 1998 Chapman & Hall     

Effect of lignocaine on intestinal smooth muscle of rat ileum

The effect of lignocaine on tone and contractility of intestinal smooth muscle, and on contractures produced by ACh or TEA, was studied in isolated ileum of the rat. Lignocaine (0.1-100 microM) produced concentration-dependent contractures in the rat ileum. In low concentrations, lignocaine increased the amplitude of spontaneous contractions and contractions produced by transmural stimulation. High concentrations of lignocaine abolished all contractile responses and produced a marked contracture in rat ileum. Lignocaine (10 microM) also reduced the contractures produced by ACh (0.01-10 microM). In contrast, the contractures produced by TEA (0.1-10 mM) were markedly increased by lignocaine. Furthermore, the contracture produced by lignocaine was reduced by lowering the external calcium from 2.5 mM to 1.5 mM. It was concluded that lignocaine in moderate and high concentrations produces a contracture in rat intestinal smooth muscle. Whereas lignocaine reduces the ACh-induced contracture, it increases that produced by TEA in the same preparation. The results further suggest that lignocaine modifies cholinergic responses and affects excitation-contraction coupling in rat intestinal smooth muscle.     

Hyperglycemia increases endothelial superoxide that impairs smooth muscle cell Na+-K+-ATPase activity

Nitric oxide (NO) plays an important role in the control of numerous vascular functions including basal Na+-K+-ATPase activity in arterial tissue. Hyperglycemia inhibits Na+-K+-ATPase activity in rabbit aorta, in part, through diminished bioactivity of NO. The precise mechanism(s) for such observations, however, are not yet clear. The purpose of this study was to examine the role of superoxide in modulating NO-mediated control of Na+-K+-ATPase in response to hyperglycemia. Rabbit aorta incubated with hyperglycemic glucose concentrations (44 mM) demonstrated a 50% reduction in Na+-K+-ATPase activity that was abrogated by superoxide dismutase. Hyperglycemia also produced a 50% increase in steady-state vascular superoxide measured by lucigenin-enhanced chemiluminescence that was closely associated with reduced Na+-K+-ATPase activity. Specifically, the hyperglycemia-induced increase in vascular superoxide was endothelium dependent, inhibited by L-arginine, and stimulated by N(omega)-nitro-L-arginine. Aldose reductase inhibition with zopolrestat also inhibited the hyperglycemia-induced increase in vascular superoxide. In each manipulation of vascular superoxide, a reciprocal change in Na+-K+-ATPase activity was observed. Finally, a commercially available preparation of Na+-K+-ATPase was inhibited by pyrogallol, a superoxide generator. These data suggest that hyperglycemia induces an increase in endothelial superoxide that inhibits the stimulatory effect of NO on vascular Na+-K+-ATPase activity.     

Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.