Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.     

The effect of phenazine methosulphate on intermediary pathways of glucose metabolism in the lens at different glycaemic levels

In this study changes in alternative pathways of glucose metabolism are examined in the rat lens using radiolabelled glucose in a 1 hr in vitro incubation of 50 mM or 10 mM glucose with or without 0.1 mM phenazine methosulphate (PMS). PMS which reoxidizes NADPH ensures that the pentose phosphate pathway (PPP) is not limited by the supply of NADP+. The data shows that maximal activation of the PPP (with PMS) is 40% greater at high glucose concentrations than normal glucose. This difference in maximal stimulation may be explained by the increase glucose uptake in the hyperglycaemic incubation. In the high-glucose incubation with PMS, hexokinase activity and the glucose 6-phosphate pool is not limiting for the PPP. Under these conditions, PMS alter the NAD+/NADH and NADP+/NADPH ratio. The change in the redox state alters the flux through the polyol pathway, the glycerol 3-phosphate shuttle and the glycolytic control sites, glyceraldehyde 3-phosphate, pyruvate and lactate dehydrogenases. These results are discussed in relation to hyperglycaemia-induced oxidative stress.     

Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase

BACKGROUND: Lactate increases lucigenin chemiluminescence (CL)-detectable superoxide anion (O2.-) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by PO2 and cytosolic NAD(H) redox appears to be the detected source of O2.- production. Little is known about the importance of this O2.(-)-producing system in cardiac myocytes. METHODS AND RESULTS: In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2.- levels to approximately 1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2.-. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2.- production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2.- production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 micromol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)-elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2.- that was potentiated by lactate and inhibited by DPI. CONCLUSIONS: NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2.- production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in PO2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.     

Clinical meaning of increased alkaline phosphatase

Interleukin-1 (IL-1) is secreted by endothelial cells (ECs) and smooth-muscle cells (SMCs), which are two major component cells of vessels and detected in atherosclerotic lesions. To evaluate the effect of hyperglycemia on the secretion of IL-1 beta in endothelial cells in diabetic patients, we investigated the effects of high glucose and hyperosmolar conditions on the secretion of IL-1 beta from cultured human aortic endothelial cells (HAECs). HAECs were treated with high concentration of glucose or hyperosmolar condition for 3 days. IL-1 beta in the supernatant was measured by high sensitive enzyme-linked immunosorbent assay (ELISA). Under high concentration of glucose (16.6 mmol/L) and hyperosmolar condition (glucose 5.5 mmol/L + mannitol 11.1 mmol/L), the secretion of IL-1 beta was significantly increased (41.0 +/- 2.8 and 26.3 +/- 5.9% increase, respectively, compared with that of 5.5 mmol/L glucose). In conclusion, high glucose and hyperosmolar condition increase the secretion of IL-1 beta in HAECs. The results suggest that diabetic macroangiopathies might be accelerated partly through the increase of IL-1 beta secretion in HAECs.     

Biochemical characterization and localization of transglutaminase in wild-type and cell-death mutants of the nematode Caenorhabditis elegans

Glucose infusion into rats has been shown to sensitize/desensitize insulin secretion in response to glucose. In pancreatic islets from glucose-infused rats (GIR) (48 h, 50%, 2 ml/h) basal insulin release (2.8 mmol/l glucose) was more than fourfold compared with islets from saline-infused controls and the concentration-response curve for glucose was shifted to the left with a maximum at 11.1 mmol/l. The concentration-response curve for 45Ca2+ uptake was also shifted to the left in islets from GIR with a maximum at 11.1 mmol/l glucose. Starting from a high basal level at 2.8 mmol/l glucose KCl produced no insulin release or 45Ca2+ uptake in islets from GIR. Islets from GIR exhibited a higher ATP/ADP ratio in the presence of 2.8 mmol/l glucose and marked inhibition of 86Rb+ efflux occurred even at 3 mmol/l glucose. Moreover, in islets from GIR the redox ratios of pyridine nucleotides were increased. On the other hand insulin content was reduced to about 20%. The data suggest that a 48-h glucose infusion sensitizes glucose-induced insulin release in vitro in concentrations below 11.1 mmol/l. This may, at least in part, be due to enhanced glucose metabolism providing increased availability of critical metabolic factors including ATP which, in turn, decrease the threshold for depolarization and therefore calcium uptake. Calcium uptake may then be further augmented by elevation of the redox state of pyridine nucleotides.     

Sulfur reduction by human erythrocytes

Washed human erythrocytes incubated with glucose and S8 and purged with N2 produced H2S at a nearly constant rate of 170 mumol (L cells)-1 min-1, which continued for several hours. In sealed vials up to 25 mM HS- accumulated. Glucose caused the fastest H2S production, although either lactate or glycerol could support slower rates. When glucose was added without S8, anoxic H2S production nonetheless occurred at about 1.5% of the maximum rate, after 24 hr totaling 0.5 mmol H2S (L cells)-1, suggesting the presence of endogenous reducible sulfur. Anaerobic conditions were not required, since oxygenated cells produced H2S from S8 at 80% of the anoxic rate. Using cell lysates, production of H2S occurred after addition of either glutathione, NADH, or NADPH. The observations suggest possible physiological roles for H2S as an electron carrier, and are consistent with an evolutionary relationship between eukaryotic cytoplasm and sulfur-reducing Archaea.     

Neurotrophin regulation of energy homeostasis in the central nervous system

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimer's, and Parkinson's diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.     

Regulation of glucose transporter (GLUT 3) and aldose reductase mRNA inbovine retinal endothelial cells and retinal pericytes in high glucose and high galactose culture

The regulation of GLUT-3 and aldose reductase mRNA in retinal endothelial cells and retinal pericytes was studied in response to variations in the extracellular concentration of hexoses. In physiological concentrations of glucose (5 mmol/l), an increase in the level of GLUT-3 mRNA was observed in cultured cells compared to the level of mRNA found in the absence of glucose. In contrast, there was little change in the level of GLUT-3 mRNA when the cells were cultured in the presence of 5 mmol/l galactose. In high concentrations of glucose, there was a decline in GLUT-3 mRNA indicating that the GLUT-3 mRNA is regulated by the extracellular concentration of glucose. In contrast, at both 5 mmol/l and 25 mmol/l glucose, the level of aldose reductase mRNA was increased. Furthermore, there were differences in the magnitude of the increase of aldose reductase mRNA between bovine retinal pericytes and bovine retinal endothelial cells with a greater increase being observed in the pericytes. We propose that this demonstration of a facilitative glucose transporter system within retinal cells, and in particular the specific response to different hexoses and the known distinct kinetic parameters of the transporter system in specific cell types, highlights the heterogeneity of hexose transport mechanisms in retinal cells. Thus, hypergalactosaemia as a model system for the study of diabetic retinopathy should be used with caution.     

The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy and this may include abnormalities in growth characteristics and the extracellular matrix. As the actual mechanisms by which glucose induces VSMC dysfunction remain unclear, the aim of this study was to assess the potential role of glucose-induced oxidative stress. Porcine aortic VSMCs were cultured for 10 days in either 5 mmol/l normal glucose or 25 mmol/l D-glucose (high glucose). There was evidence of oxidative stress as indicated by a 50% increase in intracellular malondialdehyde (p < 0.05), increased mRNA expression of CuZn superoxide dismutase and Mn superoxide dismutase (by 51% and 37% respectively, p < 0.01) and a 50% decrease in glutathione in 25 mmol/l D-glucose (p < 0.001). Growth was increased by 25.0% (p < 0.01). mRNA expression of extracellular matrix proteins (collagens I, III, IV and fibronectin) was not altered by high glucose in these experimental conditions. Repletion of glutathione with N-acetyl L-cysteine (1 mmol/l) in VSMC grown in high glucose was associated with reduction in malondialdehyde and restored growth to that of normal glucose. The water soluble analogue of vitamin E, Trolox (200 mumol/l), reduced malondialdehyde concentrations, but had no effect on glutathione depletion or the increased growth rate seen with high glucose. The addition of buthionine sulphoximine (10 mumol/l) to VSMC cultured in normal glucose reduced glutathione, increased malondialdehyde and increased growth to a similar extent as that found in high glucose alone. These results suggest that thiol status, rather than lipid peroxides, is a key factor in modulating VSMC growth and that mRNA expression of extracellular matrix proteins is not increased in VSMC under conditions of glucose-induced oxidative stress.     

Free radicals in diabetic endothelial cell dysfunction

Several studies have shown impairment of endothelium-dependent relaxations as well as increased release of vasoconstrictor prostanoids in arteries from diabetic animals and humans. This impairment is restored towards normal by prostaglandin (PG) H2/thromboxane A2 receptor blockade or superoxide dismutase, indicating that the PGH2 and/or superoxide anion (O2-.) generated contributes to the abnormality. Of particular note is that PGH2 impairs endothelium-dependent relaxations and causes contractions by a mechanism that involves generation of O2-. in the endothelium. The effects of elevated glucose are exacerbated by increased aldose reductase activity leading to depletion of NADPH and generation of reactive oxidants. Because NADPH is required for generation of nitric oxide from L-arginine, the depletion of NADPH leads to reduced nitric oxide formation. In a manner similar to that observed with elevated glucose, oxygen-derived free radicals or activation of protein kinase C also cause impairment of endothelium-dependent relaxations, smooth muscle contractions, and release constrictor prostanoids, indicating that a common mechanism for the impairment of endothelial cell function may be operative in diabetes. In this review the cumulative effects of oxidative stress on diabetic endothelial cell dysfunction, together with the complex interrelationship of cyclooxygenase catalysis, protein kinase C activity, and flux through the polyol pathway, are considered.     

Cold-hardiness-specific glutathione reductase isozymes in red spruce. Thermal dependence of kinetic parameters and possible regulatory mechanisms

The thermal dependence of kinetic parameters has been determined in purified or partially purified preparations of cold-hardiness-specific glutathione reductase isozymes from red spruce (Picea rubens Sarg.) needles to investigate a possible functional adaptation of these isozymes to environmental temperature. We have previously purified glutathione reductase isozymes specific for nonhardened (GR-1NH) or hardened (GR-1H) needles. Isozymes that were distinct from GR-1NH and GR-1H, but appeared to be very similar to each other, were also purified from nonhardened (GR-2NH) or hardened (GR-2H) needles (A. Hausladen, R.G. Alscher [1994] Plant Physiol 105: 205-213). GR-1NH had 2-fold higher Km values for NADPH and 2- to 4-fold lower Km values for oxidized glutathione (GSSG) than GR-2NH, and a similar difference was found between GR-1H and GR-2H. However, no differences in Km values were found between the hardiness-specific isozymes GR-1NH and GR-1H. There was only a small effect of temperature on the Km(GSSG) of GR-1H and GR-2H, and no significant temperature effect on Km(NADPH) or Km(GSSG) could be found for the other isozymes. These results are discussed with respect to "thermal kinetic windows," and it is proposed that the relative independence of Km values to temperature ensures adequate enzyme function in a species that is exposed to extreme temperature differences in its natural habitat. A variety of substrates has been tested to characterize any further differences among the isozymes, but all isozymes are highly specific for their substrates, NADPH and GSSG. The reversible reductive inactivation by NADPH (redox interconversion) is more pronounced in GR-1H than in GR-2H. Reduced, partially inactive GR-1H is further deactivated by H2O2, whereas GR-2H is fully reactivated by the same treatment. Both isozymes are reactivated by GSSG or reduced glutathione. It is proposed that this property of GR-2H ensures enzyme function under oxidative conditions, and that in vivo the enzyme may exist in its partially inactive form and be activated in the presence of increased levels of GSSG or oxidants.     

Hormonal and reproductive influences and risk of melanoma in women

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast, <13% of beta-cells responded to the addition of alanine (5-10 mmol/l) or arginine (5-10 mmol/l) to 3 mol/l glucose with an increase in [Ca2+]c. More than one-fourth of alpha-cells responded with an increase in [Ca2+]c when glucose concentration in perifusion solution was reduced from 11 to 0 mmol/l. These results indicate that [Ca2+]c changes in islet cells stimulated by glucose or amino acid were characteristic of the cell species, at least in the alpha- and beta-cell. This technique provides a useful tool to investigate not only the intracellular signal transduction but also the intercellular signal transmission in the intact islet.     

Glucose-stimulated elevation of cytoplasmic calcium is defective in the diabetic Chinese hamster islet B cell

To characterize insulin release and cytoplasmic free Ca2+ ([Ca2+]i) levels in the diabetic Chinese hamster islet B cell, islets from genetically normal (subline M) and diabetic (subline L) hamsters were collagenase isolated. Insulin release and glucose utilization (conversion of D-[5-(3H)]glucose to 3H2O) were measured in whole islets; [Ca2+]i levels were measured in single islet cells using fura-2. The Ca2+ channel agonist, 12 mmol/l perchlorate, ClO4-, increased the subnormal insulin response during 20 mmol/l glucose perifusion, but did not normalize it. Glucose utilization measured over a 2-h period was normal. Glucose induced an initial decrease and then a rise in [Ca2+]i in 85% of the normal (presumably B) cells. In diabetic cells, the [Ca2+]i response was delayed, subnormal and only observed in 23% of the cells. When perchlorate or another Ca2+ channel agonist, 10 mumol/l CGP 28392, was added with glucose, a larger proportion of the diabetic cells (61-67%) showed increased [Ca2+]i and the mean [Ca2+]i response was not different from normal. However, neither perchlorate nor CGP 28392 could normalize glucose-stimulated insulin release, and K(+)-induced insulin release was decreased in diabetic islets. The K(+)-induced [Ca2+]i rise was essentially normal in all the diabetic islet cells. Therefore, the diabetic hamster islet appears to metabolize glucose normally, but has a diminished insulin response to glucose and K+. The Ca2+ channel agonists markedly improve the subnormal [Ca2+]i response but not the insulin response. Glucose-induced elevation of [Ca2+]i and exocytosis appear defective in the diabetic Chinese hamster B cell.     

Oxidant stress causes alteration in the attachment of mononuclear cells to collagen

The effect of oxidant stress on the attachment of blood mononuclear cells to collagen was investigated. Study of the kinetics of attachment of mononuclear cells pretreated with 0.2% H2O2 for 10' showed significantly lower attachment to collagen I substrata when compared to untreated controls. Reduced glutathione at 2.5mM concentration partially reversed the H2O2 induced alteration in attachment. Pretreatment with H2O2 caused a reduction in the number of free thiol groups both at the cell surface and intracellular sites. Changes in cell surface free thiol groups could be reversed by reduced glutathione. These results point to the importance of a stable redox status in the cellular environment for normal interaction of mononuclear cells with extracellular matrix components.     

Direct antagonism of ethanol''s effects on GABA(A) receptors by increased atmospheric pressure

We investigated hepatic blood flow, O2 exchange and metabolism in porcine endotoxic shock (Control, n = 8; Endotoxin, n = 10) with administration of hydroxyethylstarch to maintain arterial pressure (MAP)>60 mmHg. Before and 12, 18 and 24 h after starting continuous i.v. endotoxin we measured portal venous and hepatic arterial blood flow, intracapillary haemoglobin O2 saturation (Hb-O2%) of the liver surface and arterial, portal and hepatic venous lactate, pyruvate, glycerol and alanine concentrations. Glucose production rate was derived from the plasma isotope enrichment during infusion of [6,6-2H2]-glucose. Despite a sustained 50% increase in cardiac output endotoxin caused a progressive, significant fall in MAP. Liver blood flow significantly increased, but endotoxin affected neither hepatic O2 delivery and uptake nor mean intracapillary Hb-O2% and Hb-O2% frequency distributions. Endotoxin nearly doubled endogenous glucose production rate while hepatic lactate, alanine and glycerol uptake rates progressively decreased significantly. The lactate uptake rate even became negative (P<0.05 vs Control). Endotoxin caused portal and hepatic venous pH to fall significantly concomitant with significantly increased arterial, portal and hepatic venous lactate/pyruvate ratios. During endotoxic shock increased cardiac output achieved by colloid infusion maintained elevated liver blood flow and thereby macro- and microcirculatory O2 supply. Glucose production rate nearly doubled with complete dissociation of hepatic uptake of glucogenic precursors and glucose release. Despite well-preserved capillary oxygenation increased lactate/pyruvate ratios reflecting impaired cytosolic redox state suggested deranged liver energy balance, possibly due to the O2 requirements of gluconeogenesis.     

Esthetic problems in surgery of the male external genitalia: medico-legal aspects

A novel technique has been developed for semiquantitative detection of glutathione (GSH) in small volumes of liquid samples. GSH is detected via enzymatic linkage to the NADP/NADPH + H+ redox system through glutathione reductase. Accumulated NADPH is measured via the bioluminescent FMN oxidoreductase bacterial luciferase reaction. A linear correlation is obtained between bioluminescence intensity of the luciferase reaction and the GSH content of the liquid sample. Possible applications of this procedure are discussed.