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PubMed1996

Different mechanisms of formation of glutathione-protein mixed disulfides of diamide and tert-butyl hydroperoxide in rat blood.

The mechanisms of glutathione-protein mixed disulfide (GSSP) formation caused by diamide and tert-butyl hydroperoxide were studied in rat blood after in vitro treatment in the 0.3-4 mM dose range. tert-Butyl hydroperoxide formed GSSP, via GSSG, according to the reaction, GSSG + PSH --> GSSP + GSH, whereas diamide reacted first with protein SH groups, giving PS-diamide adducts and then, after reaction with GSH, GSSP. Moreover, after diamide treatment, GSSP patterns were characterized by a much slower or irreversible dose-related return to basal levels in comparison with those observed with tert-butyl hydroperoxide, always reversible. Experiments with purified hemoglobin revealed the existence of a large fraction of protein SH groups which formed GSSP and had a higher reactivity than GSH. Experiments on glucose consumption and role of various erythrocyte enzymes, carried out to explain the inertness of GSSP to reduction after treatment of blood with diamide, were substantially negative. Other tests carried out to confirm the efficiency of the enzymatic machinery of blood samples successively treated with diamide and tert-butyl hydroperoxide, indicated that GSSP performed by diamide was difficult to reduce, whereas those generated by tert-butyl hydroperoxide were reversible as normal. Our results suggest that a fraction of GSSP generated by diamide is different and less susceptible to reduction than that obtained with tert-butyl hydroperoxide.

Animals↗
PubMed1994

Sensory neuropeptide-mediated bronchoconstriction of the guinea pig lung by diamide; a comparison to hydrogen peroxide.

The effect of the thiol oxidizing agent diamide on airway conductance, dynamic compliance and perfusion flow of isolated perfused and ventilated guinea pig lungs was investigated. When infused in the pulmonary circulation, diamide (100 microM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A2, the thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 did not affect the decrease in conductance and compliance induced by diamide. Diamide induced the release of the sensory neuropeptide calcitonin gene-related peptide. The bronchoconstriction and the release of calcitonin gene-related peptide induced by diamide were abolished by capsaicin pretreatment of the guinea pigs. Combined pretreatment with the NK1 and NK2 receptor antagonists, CP-96,345 and SR-48968, attenuated the effect of diamide. Hydrogen peroxide-induced vaso- and bronchoconstriction was not affected by capsaicin-pretreatment, nor did hydrogen peroxide induce detectable release of calcitonin gene-related peptide. The results indicate that diamide activates sensory nerves and induces neuropeptide release and neurokinin receptor-mediated bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

Animals↗
PubMed1998

The sulphydryl oxidizing reagent diamide affects phosphoinositide-mediated signal transduction: implications for the pathogenesis of Alzheimer's disease.

In fura-2-labelled human platelets, the thiol oxidising agent diamide decreases the intracellular calcium response to thrombin and serotonin without affecting the basal calcium levels. The effect of diamide on the thrombin response could be prevented by pre-treatment with dithiothreitol (DTT) and reduced when DTT was added 60 s after diamide. The effects of diamide and hydrogen peroxide on the thrombin response were additive. Hydrogen peroxide also produced a calcium response per se, but this response was not affected by diamide. Hydrogen peroxide increased rat brain phosphoinositide hydrolysis and reduced the response to carbachol and noradrenaline, whereas diamide was without effect. The binding of [3H]inositol-1,4,5-trisphosphate to human platelet membranes was inhibited by diamide but not by hydrogen peroxide. Thus diamide affects the phosphoinositide signal transduction pathway in a qualitatively different manner from that found with hydrogen peroxide. It is suggested that oxidative stress may contribute to the disturbances in the phosphoinositide transduction pathway that are found in Alzheimer's disease.

Alzheimer Disease↗
PubMed1988

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide.

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.

Animals↗
PubMed1991

Investigation of monovalent cation influxes of diamide-treated human erythrocytes in solutions of different ionic strength.

Total and residual i.e. (ouabain + bumetanide + EGTA)-insensitive K+ as well as Na+ influxes were investigated in human erythrocytes before and after treatment with diamide (5 mM). In physiological and in low ionic strength solution these influxes were increased after diamide treatment. Diamide-treated cells do not exhibit significant differences between the total and residual influxes for both Na+ and K+. The diamide-induced cation fluxes in low ionic strength solution are significantly higher compared with the fluxes in physiological ionic strength solution. The diamide-induced K+ influx is not chloride-dependent, and replacement of NaCl by sodium methylsulfate does not significantly reduce this flux. A subsequent incubation of diamide-treated erythrocytes with dithioerythritol which restores the cellular glutathione level to its original value only partly decreases the enhanced K+ influx. From these results it can be concluded that electrodiffusion and K/Cl cotransport are not involved in the diamide-induced stimulation of the residual K+ influx of human erythrocytes.

Biological Transport↗
PubMed1990

Diamide induced shift in protein and glutathione thiol: disulfide status delays DNA rejoining after X-irradiation of human cancer cells.

By treating a human tumor cell line with various concentrations of diamide, we explored the relationship between extent and duration of protein and nonprotein thiol oxidation, initiation of DNA double-strand break rejoining after X-rays, and the degree of radiosensitization. We also examined the relationship between protein thiol status and the non-protein thiol, glutathione (GSH). A549 cells were irradiated and incubated postirradiation with 0, 100, 300 or 500 microM diamide for 1 h. The dose of radiation required to give 10% survival decreased from 4.8 Gy to 3.2 Gy with 300 microM and to 2.7 Gy with 500 microM diamide (enhancement ratios of 1.5 and 1.8, respectively) but was not significantly affected by 100 microM diamide. The time of initiation of double-stranded DNA rejoining after X-irradiation (DNA repair) was delayed by 300 and 500 microM diamide. Furthermore, DNA rejoining began only after total cellular protein thiol content recovered to 55% of pretreatment levels for both concentrations. Intracellular GSH/GSSG ratios decreased immediately after diamide addition to less than 1. Large decreases in GSH/GSSG ratio preceded significant loss of protein thiols, but protein-glutathione mixed disulfides accounted for a minor percentage of the total protein thiol oxidized (up to 20%). We believe that diamide-induced protein thiol loss, and not GSH oxidation, is related to the cessation of DNA strand rejoining after X-irradiation, thereby affecting survival.

Azo Compounds↗
PubMed2005

Diamide stress induces a metallothionein BmtA through a repressor BxmR and is modulated by Zn-inducible BmtA in the cyanobacterium Oscillatoria brevis.

A cysteine-rich metal binding protein MT (metallothionein) (named BmtA) is induced upon exposure to multiple heavy metal ions in the freshwater cyanobacterium Oscillatoria brevis. The SmtB/ArsR family repressor BxmR from O. brevis represses the expression of an operon encoding bmtA and bxmR. In the present study, the expression of bmtA was induced in vivo by diamide, a specific thiol oxidant, in O. brevis cells. In vitro electrophoretic gel mobility shift experiments revealed that the incubation with diamide induces disassembly of the BxmR-bxmR/bmtA operator (O)/promoter (P) complex [multiple resolvable complexes of BxmR with oligonucleotide (named P5) containing a single 12-2-12 inverted repeat derived from the O/P region of bxmR/bmtA]. Thus, the exposure to diamide induces MT mRNA in O. brevis, and this induction is associated with diamide-mediated inhibition of BxmR-P5 complex. BxmR is more sensitive to diamide than to H(2)O(2). Furthermore, pretreatment of O. brevis with Zn decreased intracellular peroxidation products caused by diamide. Thus, these results imply that MT induced by Zn-pretreatment functions to protect O. brevis cells against diamide stress.

Cyanobacteria↗
PubMed1983

Effect of diamide on isolated guinea pig atria.

Diamide [(diazinedicarboxylic acid-bis (dimethylamide)] 50 micrograms/ml induced a positive inotropic and chronotropic effect on spontaneously beating atria from normal and reserpine pretreated guinea pigs and a positive inotropic action on electrically driven left atria. Cumulative addition of diamide 600 micrograms/ml caused a systolic standstill of atria. D(-) INPEA 2 micrograms/ml, a beta-adrenergic blocking agent, antagonized the positive chronotropic action of diamide 50 micrograms/ml while it did not affect the positive inotropic action caused by diamide. In isolated atria perfused with Ca2+ free medium and recovered by stepwise addition of calcium chloride, the calcium level required to restore normal activity was 1.04 mmol/1 while it was 0.52 mmol/l in the presence of diamide. Ruthenium red 10 micrograms/ml, a specific inhibitor of calcium transport in mitochondria, antagonized diamide-induced recovery of the cardiac activity in calcium poor medium. Two different mechanisms, that involve calcium movements and beta-receptor, have been proposed for diamide action on heart muscle.

Animals↗
PubMed1990

Reversible elimination of myofibrillar Ca2+ sensitivity by diamide and other sulfhydryl reagents: comparison with reversible contracture produced in intact cells.

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.

Adenosine Triphosphatases↗
PubMed1982

Effects of a thioreactive agent, diamide, on neuromuscular transmission in lobster.

Diamide[diazine-dicarboxylic acid-bis(dimethylamide)], a thiol-oxidizing agent, has both pre- and postsynaptic actions on the glutaminergic neuromuscular junction of the lobster walking leg. Postsynaptically, diamide produced an increase in the response to exogenously applied glutamate, whereas the effect of diamide on presynaptic transmitter release involved two major changes: 1) a decrease in excitatory junction potential amplitude and 2) an increase in miniature junction potential frequency. Short-term facilitation also decreased. Equilibration with 1,4-dithiothreitol (a sulfhydryl-reducing agent) reversed the decline in excitatory junction potential frequency, and the fall in short-term facilitation. The miniature junction potential frequency increase induced by diamide was independent of external Ca2+, as diamide in a Ca2+-free solution produced a similar response to that in a Ca2+-containing solution. We propose that the action of diamide on transmitter release is similar to the action of polyvalent cations, i.e., diamide has two sites of action, a blockade of inward Ca2+ flux and an increased release of Ca2+ inside the terminal.

Action Potentials↗
PubMed1990

Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli. Effects of diazenedicarboxylic acid bis(N,N'-dimethylamide) (diamide).

Anaerobically grown Escherichia coli accumulate active manganese-containing superoxide dismutase (MnSOD) upon exposure to diamide. This induction requires de novo biosynthesis of MnSOD. Catalase, glutathione disulfide reductase, and glucose-6-phosphate dehydrogenase were also induced by diamide in anaerobic E. coli. A GSH-negative strain of E. coli did not produce MnSOD under anaerobic conditions and was as responsive to diamide as was the wild type strain. Diamide which had been prereduced, by incubation with GSH, was ineffective. NO3- plus paraquat, which elicits increased anaerobic biosynthesis of the MnSOD polypeptide, but not of active MnSOD, synergized with diamide in the induction of active MnSOD. A similar increase in the ability of diamide to cause anaerobic biosynthesis of active MnSOD was seen when the production of the MnSOD polypeptide was increased by isopropyl-beta-D-thiogalactopyranoside, in a strain bearing the MnSOD gene under the control of the tac promoter. These results are explained in terms of a dual action of diamide, i.e. at both the transcriptional and the maturational levels of biosynthesis of MnSOD. Oxidative inactivation of an Fe(II)-containing repressor and oxidative facilitation of insertion of manganese, in place of iron, into the nascent MnSOD polypeptide, are the postulated bases of this dual action.

Anaerobiosis↗
PubMed2003

Expression of macrophage inflammatory protein 1 alpha in the endothelial cells exposed to diamide.

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.

Cells, Cultured↗
PubMed1978

Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide.

This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.

Adenosine Triphosphate↗
PubMed1983

Spindle disturbances in mammalian cells. I. Changes in the quantity of free sulfhydryl groups in relation to survival and C-mitosis in V79 Chinese hamster cells after treatment with colcemid, diamide, carbaryl and methyl mercury.

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30-40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.

Animals↗
PubMed1991

Effect of diamide on nucleoside and glucose transport in Plasmodium falciparum and Babesia bovis infected erythrocytes.

Normal human erythrocytes, preincubated with the oxidizing agent diamide, did not demonstrate any increased permeability, but showed a significant decrease in their ability to transport the nucleoside adenosine. Diamide appeared to have little effect on glucose permeation in uninfected and Plasmodium falciparum infected cells. The inhibition of adenosine transport in human erythrocytes by diamide pretreatment appeared to be unrelated to the inhibition by the established nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). An ID50 for diamide of 0.3 mM was determined for 1 microM adenosine transport in human erythrocytes after preincubation for 45 min at 37 degrees C. However, preincubation of diamide (20 mM, 60 min at 37 degrees C) with Babesia bovis-infected bovine erythrocytes resulted in complete inhibition of the capacity of the parasitised cell to transport adenosine and partial inhibition of glucose permeation. By contrast, diamide was shown to have little or no effect on the new or induced nucleoside permeation site in P. falciparum (trophozoite) infected erythrocytes nor on the glucose transporter in these cells. The results further indicate the differences between the normal human erythrocyte nucleoside and glucose transporters and those new or altered transporters in the membrane of P. falciparum or B. bovis-infected red blood cells.

Animals↗
PubMed1979

The effect of oxidant stress on diamide-treated human granulocytes.

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xamthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.

Azo Compounds↗
PubMed1990

Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction.

Small tissue fragments excised from the electric organ of Torpedo marmorata were treated with diamide, a penetrating thiol oxidizing agent, until synaptic transmission was blocked. At this stage, we found an unexpected number of exo-endocytotic images in the presynaptic plasmalemma. Omega-shaped profiles, some of them coated, were seen in thin sections of fixed tissue and pits opened in the P-face of the presynaptic membrane in freeze-fracture replicas from rapidly-frozen preparations. Diamide-treated specimens were frozen at 1 ms time intervals before, during and after a single electrical stimulus. This stimulation did not result in a further increase in the density of presynaptic pits, not in any change affecting the density or size distribution of intramembrane particles. This result is in contrast with what is observed in untreated specimens where transmission of a nerve impulse is accompanied by a momentary rise in the number of large particles. The density of synaptic vesicles--especially that of a subpopulation of small size vesicles--transiently increased within the first 2 h of diamide treatment. During the first stages of intoxication, diamide prolonged the time course of postsynaptic potentials--both spontaneous and evoked--probably by altering the gating properties of receptors (acetyl-cholinesterase activity was not impaired). Later on, all evoked responses were blocked. The spontaneous transmitter release greatly increased, first in the form of quantal miniature potentials. These then subsided whereas a class of very small potentials was generated at a high frequency. Also under the action of diamide, calcium progressively accumulated in the tissue but the number of synaptic vesicles containing calcium deposits was reduced. It is concluded that diamide causes a marked increase in the number of exo-endocytotic images in the presynaptic membrane, suppresses quantal but not subquantal release, and interferes with calcium sequestration in and extrusion from terminals.

Animals↗
PubMed1984

Radiosensitization by diamide at low radiation doses.

In vitro studies in this laboratory indicated that at low radiation doses (0-3 Gy), sensitization by misonidazole diminishes. We have suggested that this results from a failure of misonidazole to enhance effectively the single-hit or alpha component of cell inactivation. Our low dose assay uses a partially automated microscopic analysis procedure which scores both surviving and killed cells. If enough cells are scored, this assay provides much better accuracy at low doses than the conventional assay. Recently, we have applied our low dose assay to a study of sensitization by diamide. This was done, in part, as a test of our assay system, since many investigators have shown that diamide substantially reduces the shoulder of the hypoxic cell survival curve. At high doses (D = 6.5 to 10 Gy, S = 0.1 to 0.01) we obtained for 150 microM diamide an enhancement ratio (ER) of 2.6 for hypoxic cells and 1.1 for aerobic cells. At low doses, 0-2.5 Gy, diamide still sensitized hypoxic cells well; with 150 microM diamide, the ER at 1 Gy (S = 0.8) was 2.0, about equal to the oxygen enhancement ratio (OER) at this dose. The fact that diamide shows good sensitization at low doses with our assay adds to the credibility of the assay and reduces the likelihood that our observations with misonidazole are an artifact of our system.

Anaerobiosis↗