Hydroperoxides selectively inhibit human erythrocyte membrane enzymes

doi:10.1016/0003-9861(89)90512-2

Archives of Biochemistry and Biophysics

Volume 273, Issue 2, September 1989, Pages 527-534

https://doi.org/10.1016/0003-9861(89)90512-2 Get rights and content

Abstract

Treatment of washed erythrocytes with tert-butyl hydroperoxide (0.5 mm, 10 min) inhibited basal Ca²⁺ + Mg²⁺-ATPase activity by 40% and calmodulin-stimulated activity by 54%. The inhibition was accompanied by the formation of methemoglobin and the aggregation of some membrane proteins into a high-molecular-weight polymer. Membranes, isolated from washed erythrocytes, showed a similar pattern of inhibition. Basal Ca²⁺ + Mg²⁺-ATPase activity was inhibited 50% at 10 min and 70% at 30 min while calmodulin-stimulated activity was inhibited 70% at 10 min and 84% at 30 min. Thiobarbituric acid-reactive products formed slowly during the first 10 min and then increased sharply between 10 and 30 min. The polymerization of membrane proteins was also observed during the tert-butyl hydroperoxide exposure. Inhibition of erythrocyte membrane enzymes was selective. The Na⁺ + K⁺-stimulated Mg²⁺ ATPase, like the Ca²⁺ + Mg²⁺-ATPase, was sensitive to membrane oxidation but the activities of Mg²⁺-ATPase and acetylcholinesterase were less inhibited by tert-butyl hydroperoxide. Acetylcholinterase was found to be very resistant to hydroperoxide treatment with less than 10% loss of activity. The effects of two other hyproperoxides on enzyme inhibition were studied also. Cumene hydroperoxide (0.5 mm) was found to be as potent as tert-butyl hydroperoxide but hydrogen peroxide at 10 mm did not produce thiobarbituric acid-reactive products or inhibit Ca²⁺ + Mg²⁺-ATPase activity until after 20 min. The selective effects of peroxides on these enzyme activities are discussed.

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Tertiary butyl hydroperoxide induced oxidative damage in mice erythrocytes: Protection by taurine
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When cells or tissues are exposed to external toxin like TBHP, membrane permeability occurs [1,6] at the same time the membrane is hyperpolarized and water pores are formed. Reports [7] also suggest that TBHP selectively inhibits membrane bound enzymes, increases the formation of methemoglobin and causes aggregation of some membrane proteins into high-molecular-weight polymers. In the red cell cytosol TBHP is either reduced by glutathione, Gpx reducing system or by hemoglobin.
The present study was undertaken to investigate the protective role of taurine, against t-butyl hydroperoxide (TBHP) induced oxidative stress in murine erythrocytes. Erythrocytes were treated either with TBHP alone or with taurine, followed by TBHP exposure. TBHP-induced oxidative stress increased methemoglobin formation, lipid peroxidation and protein carbonylation in erythrocytes. The same exposure, however, depleted cellular GSH content and altered the activities of the antioxidant enzymes as well as of methemoglobin reductase; reduced activities of Ca⁺ and Na⁺/K⁺ ATPase and intracellular ATP levels. Taurine transport inhibitor, β-alanine, treated erythrocytes showed increased phosphatidylserine externalization and ROS formation on TBHP exposure and taurine could not revert the effect. TBHP exposure increased intracellular calcium and upregulated the level of calpain. Administration of taurine could, however, prevent the TBHP induced oxidative imbalance. Electron micrographs of erythrocytes showed changed morphology with an increase in the number of echinocytes. Taurine treatment could restore the normal levels of the antioxidant enzymes and metabolites of the erythrocytes. Results suggest that the oxidative insult introduced in erythrocytes by TBHP administration is prevented by taurine mainly via membrane stabilization.
Erythrocytic enzymes and antioxidant status in people with type 2 diabetes: Beneficial effect of Syzygium cumini leaf extract in vitro
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All samples were run in duplicate or triplicate and the enzyme activity was expressed in μmol AcSCh/h/hemoglobin. The analysis of the production of TBARS was performed essentially by the method of Moore et al. [30], with minor modifications. The samples of ghosts were incubated with ASc at 200 μg/mL for 15 min.
The aim of the present study was to investigate the effects of Syzygium cumini leaf extract (ASc), on Adenosine deaminase (ADA) and Acetylcholinesterase (AChE) activities, and also on oxidative stress parameters in erythrocytes hemolysates (RBCs) and erythrocytes membranes (ghosts) from type 2 diabetics patients (Type 2 DM) under in vitro conditions. Non protein thiol groups (NP-SH), AChE, Catalase (CAT) and Superoxide Dismutase (SOD) activities were measure in RBCs. Further, ADA activity, Thiobarbituric Acid-Reactive Substances (TBARS) levels and protein thiol groups (P-SH) were estimated in ghosts. Also, P-SH and Vitamin C (VIT C) were measure in plasma sample. The results demonstrated that ADA and AChE activities, besides TBARS levels were higher in erythrocytes of Type 2 DM, while SOD activity and NP-SH levels were decreased when compared to control group. ASc, in vitro, reduced ADA and AChE activities and some parameters of oxidative stress. Furthermore, we observed correlations between VIT C and P-SH levels, ADA activity and P-SH levels, as well as NP-SH and TBARS levels in diabetics. The results suggest that ASc in vitro is able to promote the reduction of inflammation and oxidative stress parameters, and act against biochemical changes occurring in Diabetes mellitus (DM).
Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear
2002, Life Sciences
During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca²⁺/Mg²⁺-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na⁺/K⁺-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca²⁺/Mg²⁺-ATPase and Na⁺/K⁺-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.
Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels
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Using isolated bovine brain microvessels as an in vitro model of the blood–brain barrier (BBB) we have evaluated the role of free radical generating solutions on some amino acid transport systems operating on the endothelial cell membrane. Fe²⁺/ascorbate, phenylhydrazine and CuSO₄ did not affect any of the transport system tested, while exposure of bovine brain microvessels to tert-butylhydroperoxide (t-BHP) caused a reduced capacity to take up small neutral amino acids via the Na⁺-dependent A-system. The presence of glucose during t-BHP treatment did not prevent this inhibition, which was partially counteracted when the isolated microvessels were incubated with 5 mM inosine before the oxidative stress. Incubation of the isolated capillaries with 5 mM dithiothreitol, after exposure to t-BHP, resulted in a 50% recovery of the α-methylaminoisobutyrate (MeAIB) uptake by the A-system. Treatment with t-BHP, which had no effect on the l-system of neutral amino acid transport, caused a significant decrease of the intracellular levels of ATP, of glutathione (GSH), and of γ-glutamyltranspeptidase (GGT) activity, while no significant modification of hexokinase (HK) or of alkaline phosphatase (ALKP) activities were observed. Oxidative damage of the BBB appears therefore to impair essentially the metabolic pathways which ensure the energy requirement for the endothelial cells, thus inhibiting the energy-dependent amino acid transport system “A”.
Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction in high glucose-treated human erythrocytes
2001, Free Radical Biology and Medicine
Vitamin B₆ (pyridoxine) supplementation has been found beneficial in preventing diabetic neuropathy and retinopathy, and the glycosylation of proteins. Oxygen radicals and oxidative damage have been implicated in the cellular dysfunction and complications of diabetes. This study was undertaken to test the hypothesis that pyridoxine (P) and pyridoxamine (PM) inhibit superoxide radical production, reduce lipid peroxidation and glycosylation, and increase the (Na⁺ + K⁺)-ATPase activity in high glucose-exposed red blood cells (RBC). Superoxide radical production was assessed by the reduction of cytochrome C by glucose in the presence and absence of P or PM in a cell-free buffered solution. To examine cellular effects, washed normal human RBC were treated with control and high glucose concentrations with and without P or PM. Both P and PM significantly lowered lipid peroxidation and glycated hemoglobin (HbA₁) formation in high glucose-exposed RBC. P and PM significantly prevented the reduction in (Na⁺ + K⁺- ATPase activity in high glucose-treated RBC. Thus, P or PM can inhibit oxygen radical production, which in turn prevents the lipid peroxidation, protein glycosylation, and (Na⁺ + K⁺)-ATPase activity reduction induced by the hyperglycemia. This study describes a new biochemical mechanism by which P or PM supplementation may delay or inhibit the development of complications in diabetes.
Lipoic acid decreases lipid peroxidation and protein glycosylation and increases (Na+ + K+)- and Ca++-ATPase activities in high glucose-treated human erythrocytes
2000, Free Radical Biology and Medicine
Lipoic acid supplementation has been found to be beneficial in preventing neurovascular abnormalities in diabetic neuropathy. Insufficient (Na⁺ + K⁺)-ATPase activity has been suggested as a contributing factor in the development of diabetic neuropathy. This study was undertaken to test the hypothesis that lipoic acid reduces lipid peroxidation and glycosylation and can increase the (Na⁺ + K⁺)- and Ca⁺⁺-ATPase activities in high glucose-exposed red blood cells (RBC). Washed normal human RBC were treated with normal (6 mM) and high glucose concentrations (45 mM) with 0–0.2 mM lipoic acid (mixture of S and R sterioisomers) in a shaking water bath at 37°C for 24 h. There was a significant stimulation of glucose consumption by RBC in the presence of lipoic acid both in normal and high glucose-treated RBC. Lipoic acid significantly lowered the level of glycated hemoglobin (GHb) and lipid peroxidation in RBC exposed to high glucose concentrations. High glucose treatment significantly lowered the activities of (Na⁺ + K⁺)- and Ca⁺⁺-ATPases of RBC membranes. Lipoic acid addition significantly blocked the reduction in activities of (Na⁺ + K⁺)- and Ca⁺⁺-ATPases in high glucose- treated RBC. There were no differences in lipid peroxidation, GHb and (Na⁺ + K⁺)- and Ca⁺⁺-ATPase activity levels in normal glucose-treated RBC with and without lipoic acid. Thus, lipoic acid can lower lipid peroxidation and protein glycosylation, and increase (Na⁺ + K⁺)- and Ca⁺⁺-ATPase activities in high-glucose exposed RBC, which provides a potential mechanism by which lipoic acid may delay or inhibit the development of neuropathy in diabetes.

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Archives of Biochemistry and Biophysics

Abstract

References (31)

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Trends Pharmacol. Sci

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Cited by (34)

Tertiary butyl hydroperoxide induced oxidative damage in mice erythrocytes: Protection by taurine

Erythrocytic enzymes and antioxidant status in people with type 2 diabetes: Beneficial effect of Syzygium cumini leaf extract in vitro

Increased oxidative stress and decreased activities of Ca<sup>2+</sup>/Mg<sup>2+</sup>-ATPase and Na<sup>+</sup>/K<sup>+</sup>-ATPase in the red blood cells of the hibernating black bear

Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels

Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na<sup>+</sup> + K<sup>+</sup>)-ATPase activity reduction in high glucose-treated human erythrocytes

Lipoic acid decreases lipid peroxidation and protein glycosylation and increases (Na<sup>+</sup> + K<sup>+</sup>)- and Ca<sup>++</sup>-ATPase activities in high glucose-treated human erythrocytes