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PubMed2005

Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin.

Co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli resulted in the production of cobyrinic acid a,c-diamide. A cbiD deletion mutant of this strain produced 1-desmethylcobyrinic acid a,c-diamide, indicating that CbiD is involved in C-1 methylation in the anaerobic pathway to cobalamin. Strains that did not have the cbiP gene also produced 1-desmethylcobyrinic acid a,c-diamide, and strains that had neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity.

Amides↗

PubMed1976

Diamide acts intracellularly to enhance transmitter release: the differential permeation of diamide, DIP, DIP+1 and DIP+2 across the nerve terminal membrane.

The actions of the new potent thiol oxidizing agents, diazene dicarboxylic acid bis (N'-methyl piperazide) (DIP) and the N'-methyl iodide (DIP + 1) and the bis-N'-methyl iodide (DIP + 2) salts of DIP, were tested at the frog neuromuscular junction. At 20 degrees C, DIP was as fast as the thiol oxidizing agent, diamide, in evoking transmitter release but was appreciably less effective at 6 degrees C. DIP + 1 and DIP + 2 did not increase transmitter release. Since the three agents are potent oxidizers of glutathione and since the effectiveness of the compounds appears to depend on their ability to exist, at least in part, in a neutral form at physiological pH, it is concluded that their action as promoters of transmitter release depends on their ability to permeate nerve terminal membranes. Thus, both diamide and DIP act to increase transmitter release by the intracellular oxidation of glutathione. The two charged agents, DIP + 1 and DIP + 2, are potent muscular depolarizing agents. It is probable that the quaternary nitrogen groups of these compounds render them cholinomimetics.

Acetylcholine↗

PubMed1980

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes.

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

Adaptation, Physiological↗

PubMed1993

Glutathione oxidation and embryotoxicity elicited by diamide in the developing rat conceptus in vitro.

This study was performed in the rat whole-embryo culture system to investigate the effects of glutathione oxidation by diamide, a thiol oxidant, in developing rat conceptuses during early organogenesis. The effects of diamide on reduced glutathione (GSH), glutathione disulfide (GSSG), and embryotoxicity were found to be concentration and time dependent. Diamide at concentrations of 75 and 100 microM produced abnormal axial rotation (62-89%), decreased viability (to 69% by 100 microM diamide), and reduced protein and DNA content in the embryo and visceral yolk sac (VYS) when evaluated on Day 11. High concentrations of diamide (250-500 microM) resulted in 100% mortality. GSH and GSSG levels in the conceptuses were not significantly affected during 2 hr following diamide addition at concentrations of 50 to 100 microM. At concentrations of 250 and 500 microM, rapid GSH depletion (50% of control) was seen within 5 min of exposure and was followed at 5-30 min by a significant increase in GSSG relative to control values. Diamide (500 microM) exposure for only 15 min on Gestational Day 10 was sufficient to elicit malformations (53% of exposed conceptuses with abnormal axial rotation) without significant loss of viability. After 30 min of exposure to the high concentration (500 microM), viability was decreased to 71% and defects of axial rotation increased to 87% in surviving conceptuses. This indicates that events associated with initial exposure are critical for expression of toxicity. Inhibition of glutathione disulfide reductase (GSSG reductase) activities in embryo and VYS with 1,3-bis(2-chloroethyl)-1-nitro-sourea prior to diamide addition potentiated the embryotoxicity of diamide (75 microM) and resulted in corresponding reductions in GSH/GSSG ratios as determined during the first 2 hr of exposure. Inhibition of new GSH synthesis with L-buthionine-[S,R]-sulfoximine during diamide (75 microM) exposure also exacerbated toxicity compared to diamide treatment alone. These results implicate the involvement of GSH synthesis and GSSG reductase activity in mediating the embryotoxicity of diamide.

Animals↗

PubMed1995

Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro.

Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in increased glutathione disulfide (GSSG) and protein-glutathione mixed disulfide (protein-S-SG) formation, which is accompanied by embryotoxicity and reductions in amniotic fluid volume. The altered functions of critical proteins or enzymes caused by the formation of protein-S-SG perturb cellular metabolism and may be involved in the embryotoxicity produced by GSH oxidation. The present study investigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by diamide exposure in organogenesis-stage rat conceptus (gestational day 10) in vitro. The metabolism of glucose via the PPP, measured as amounts of CO2 production from D-[1-14C]-glucose, was significantly increased in the conceptus exposed to 100-500 microM diamide to levels 2.5-3-fold those of controls. It was found that these substantial increases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway. Changes in glycolysis due to diamide treatment were also determined by measurements of lactate production from D-[U-14C]-glucose. Production of lactate by the conceptus exposed to 250-500 microM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis. Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yolk sac (VYS) compared to those from the embryo. Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when determined in the supernatants from the VYS of the conceptus exposed to 500 microM diamide for 60 min. In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N-ethylmaleimide (NEM, 100 microM) within 15 min of the exposure. In contrast to diamide and NEM, no alterations in lactate production were seen in the conceptus treated with the GSH depletor L-buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media. Further experiments demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 microM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent. These results indicated the involvement of thiol/disulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and protein-S-SG formation could be a critical event associated with mechanisms of embryotoxicity elicited by oxidative stress. It was suggested in this study that, under these experimental conditions, embryotoxicity induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis).

Animals↗

PubMed1978

The reduction of diamide by rat liver mitochondria and the role of glutathione.

Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a) thiol-blocking reagents (N-ethylmaleimide, p-hydroxymercuribenzoate, mersalyl and 2,6-dichlorophenol-indophenol);(b) respiratory inhibitors (arsenicals, malonate and antimycin, but not cyanide or oligomycin; inhibition by antimycin is reversed by ATP); (c) uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol and valinomycin with K+; inhibition by the first of these uncouplers is not reversed by cyanide); (d) reagents affecting energy conservation (Ca2+, increasing pH, phosphate; phosphate inhibition is augmented by catalytic ADP or ATP and augmentation is abolished by respiratory inhibitors). Concentrations of mitochondrial glutathione are high when diamide reduction is uninhibited, but low after adding one of the above inhibitors such that the reduction rate is roughly proportional to the glutathione concentration. Endogenous ATP concentrations are lower in the presence of diamide than without, but the difference is abolished by respiratory inhibitors. With oligomycin added, however, ATP concentrations are higher in the presence of diamide and this positive increment is decreased by antimycin, N-ethylmaleimide and malonate. In the presence of diamide and an uncoupler, the mitochondrial glutathione content does not fall if various reducible substrates are present, although the inhibition of diamide reduction is not relieved. Some of these substrates prevent the fall in reduced glutathione concentration found with diamide and phosphate. They also relieve the inhibition of diamide reduction and the relief is sensitive to butylmalonate. The inhibition of diamide reduction by N-ethylmaleimide, mersalyl or p-hydroxymercuribenzoate is not relieved by reducible substrates, but the latter mitigate the fall in the concentration of glutathione. Inhibitors of carriers of tricarboxylic acid-cycle intermediates also inhibit reduction of diamide. The reduced glutathione concentration remains high when they are added singly, but falls when two of them are combined. It is proposed that diamide may enter the matrix as a protonated adduct formed with the thiol groups of mitochondrial carriers and then be reduced in the matrix by glutathione, which is regenerated via NADH, energy-dependent transhydrogenase and NADP+-specific glutathione reductase. Some of the high-energy equivalents required for the transhydrogeneration may be generated by the substrate phosphorylation step of the tricarboxylic acid cycle.

Adenosine Triphosphate↗

PubMed2002

[The effect of diamide on the expression of macrophage inflammatory protein-1 alpha in endothelial cells].

OBJECTIVE: To study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells. METHODS: After exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers. RESULTS: Incubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group. CONCLUSIONS: Diamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Arteriosclerosis↗

PubMed2004

Investigation on the effects of diamide on NO production in vascular endothelial cells (VEC).

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. The study on the effect of diamide (N(2)H(4).H(2)O) on NO production in vascular endothelial cells (VEC) may provide significant reference for VEC's modeling in studying cardiovascular diseases. The objective of this study was to elucidate how high concentration diamide (V(diamide)/V(culture miedium) = 5 ml/l) and low concentration diamide (V(diamide)/V(culture miedium) = 0.5 ml/l) affect NO production in a human endothelial cell line (ECV304). After cells were incubated with diamide (5 or 0.5 ml/l) for 4, 6, 8 or 10h, respectively, the amounts of NO metabolites released by the cells were quantitated and the degree of damage of VEC was observed using microscope. The results showed that NO production in VEC tended to decrease with the lapse of time in the 0.5 ml/l diamide group. In the 5 ml/l diamide group, on the contrary, NO production in VEC tended to increase with the lapse of time. At the same time, from the morphologic observation, the VEC were damaged severely after treated with 5 ml/l diamide. So it could be concluded that the severe damage induced by high concentration diamide would have triggered the express of inducible nitric oxide synthases (iNOS). Just for the expresssion of iNOS, NO production in VEC treated with high concentration diamide occurred abnormally in contrast to the 0.5 ml/l group.

Cells, Cultured↗

PubMed1992

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide.

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10-200 microM) stimulation of 5-HT transport that was rapid and sustained over 0-10 minutes of incubation. When platelets were incubated with diamide (10-200 microM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 microM, while washed platelets incubated with diamide (10-200 microM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (Vmax control = 28.4 +/- 1.4 vs. Vmax diamide = 60.9 +/- 4.1 pM/10(8) platelets/4 min) but did not significantly alter the Km values. Ouabain, an inhibitor of platelet Na(+)-K+ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.

Animals↗

PubMed1985

Inhibition of beta-amino acid transport by diamide does not involve the brush border membrane surface.

Diamide (dicarboxylic acid bis-(N,N-dimethylamide) has been shown in previous studies to block the uptake of the beta-amino acid taurine at its high affinity transport site in rat renal cortex slices. Diamide may act by increasing the efflux of taurine from the slice. Studies performed in rat slices again indicate enhanced efflux over 8-12 minutes. The time course of reduced glutathione (GSH) depletion from renal cortex is similar, indicating a potential interaction between GSH depletion and inhibition of taurine accumulation. Diamide further blocks the uptake of taurine by collagenase-isolated renal tubules in a dose-dependent fashion with greater inhibition at 20 minutes than at 5 minutes. The effect of 9 mM diamide on the Na+ -dependent accumulation of taurine (10 and 250 microM) by brush border membrane vesicles was examined, and the taurine uptake value both initially and at equilibrium was the same in the presence and absence of diamide. That the effect in tubules is greater at 20 minutes than at 5 minutes is consistent with the idea that diamide enhances efflux of taurine immediately after exposure of tubules to taurine, or that diamide influences some intracellular process, requiring a time interval before this action is observed. Isolation of the brush border surface and subsequent transport studies of taurine are not influenced by diamide. Thus, diamide inhibition of taurine uptake does not involve physiochemical alteration of the membrane surface where active amino acid transport occurs, despite the thiol-oxidizing properties of this agent. Further, these studies suggest that diamide either acts at the basolateral surface, rather than the brush border surface of rat renal cortex or requires the presence of an intact tubule, capable of metabolism, prior to its inhibitory action.

Animals↗

PubMed1976

The thiol-oxidizing agent diamide increases transmitter release by decreasing calcium requirements for neuromuscular transmission in the frog.

Diamide, which in concentrations of 10(-5) M and higher oxidizes glutathione intracellularly, produces a dose-related increase in the frequency of miniature end-plate potentials (MEPPs). With high enough doses, quantal release is blocked, apparently through exhaustion. The early phase of MEPP frequency increase is accompanied by an increase in EPP amplitude that may reach more than 10-fold and is therefore not produced by depolarization of axon terminals. Subsequently, EPP amplitude is reduced and falls to zero, associated with failure of invasion of the nerve action into the terminals while the MEPP frequency remains elevated. Both facilitation and PTP follow the time course of change in EPP amplitude. The increase in MEPP frequency with diamide does not require external Ca2+ but raising external Ca2+ increases the MEPP rate in the presence of diamide. External Ca2+ is necessary for EPP appearance and also potentiates the diamide effects. Conversely diamide reduces the requirements for Ca2+ in releasing ACh. Diamide substitutes for external Ca2+ in K+ evoked MEPP release and in the absence of external Ca2+, diamide-evoked MEPP release is increased by raising external Mg2+ levels. The action of diamide may be dependent on the actual release of Ca2+ from intracellular stores or it may work through mimicking some of the actions of Ca2+. The action of diamide bears close resemblance to the effects of prolonged stimulation of the motor axon at 10 Hz.

Acetylcholine↗

PubMed1987

Changes in the distribution and organization of platelet actin induced by diamide and its functional consequences.

Exposure of blood platelets to diamide (azodicarboxylic acid-bis-dimethylamide) results in oxidation of sulphydryl groups present in the cytoskeleton and other proteins. This results in dramatic changes in functional behaviour of the cells. The distribution and level of organization of the major cytoskeletal protein actin has been studied analytically by the DNase-I inhibition assay and morphologically by electron microscopy (EM) of Triton X-100 treated platelets adherent to EM grids. Exposure to diamide results in a redistribution of actin within the cell reflected in an increase in cytoskeletal F-actin and a concomitant decrease in cytosolic actin. The magnitude of these changes depends upon the concentration of diamide and the time of exposure. Diamide also alters platelet aggregatory functions in response to certain stimuli. Treatment of normal human platelets with 0.1 mM diamide proceeds via disaggregation (5 min exposure to diamide), inhibition of aggregation (30 min exposure), to finally a normalization of the aggregation response after 60-120 min incubation with diamide. In parallel with the return to full functional response the distribution of F-actin between the cytoskeleton and cytoplasmic compartments returns to the control pattern. Incubation of the platelets with 0.5 mM diamide for 60 or more minutes leads to total inhibition of the aggregatory ability. In these cells the cytoskeleton associated F-actin remains significantly elevated and the structural organization of the cytoskeleton is markedly altered. In contrast to the network of filaments subadjacent to the surface membrane seen in unstimulated platelets, the cytoskeleton now shows electron dense zones in the more central parts of the cytoplasm. This diamide-induced structural reorganization of platelet cytoskeletal elements, associated with the inhibition of functional responses, emphasizes the dynamic nature of the membrane-cytoskeletal axis and its importance in the expression of shape changes and aggregatory phenomena in response to surface stimuli.

Actins↗

PubMed1989

Modulation of diamide toxicity in thermotolerant cells by inhibition of protein synthesis.

Chinese hamster ovary cells were exposed in vitro to various concentrations of diamide for 1 h at 37 degrees C. This treatment resulted in a dose dependent increase in cytotoxicity. Cells were also heated at 43 degrees C for 15 min, incubated at 37 degrees C for 3 h, and then exposed to various concentrations of diamide. This heat shock has been shown previously to trigger the synthesis of heat shock proteins and the development of thermotolerance. Further, under these experimental conditions both were inhibited if protein synthesis was inhibited by exposure to cycloheximide (M. L. Freeman et al., Radiat. Res., 112: 195-203, 1987). Diamide toxicity was diminished in cells made thermotolerant by the 43 degrees C/15-min heat shock. For example, at the highest dose used, 0.8 mM, survival increased from 0.93% to 6.1%. However, diamide toxicity was unaffected if the cells were exposed to diamide 3 h after a 43 degrees C/60 min heat shock. This latter heat shock produced significant inhibition of protein synthesis whereas the 15-min heat shock did not (M. L. Freeman et al., Cancer Res., 48: 7033-7037, 1988). Further, a 43 degrees C/15-min heat shock did not confer protection against diamide toxicity if the cells were simultaneously exposed to cycloheximide. Exposure to 0.8 mM diamide was shown to oxidize specific cellular proteins as measured by 2-dimensional thiol blotting. However, the degree of protein thiol modification was not affected by a prior heat shock. Nor did the heat shock increase the intracellular concentration of glutathione or the activity of glutathione reductase. The diamide treatment caused specific, as opposed to general, protein thiol oxidation and heat shock did not prevent this. It is hypothesized that it was the oxidation of protein thiols which led to cellular toxicity. Protein synthesis, triggered by heat shock, protected cells from the diamide toxicity without preventing protein thiol modification. These results suggest that the proteins synthesized after heat shock can provide protection against the consequences of aberrant proteins produced by thiol oxidation.

Animals↗

PubMed1992

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation.

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithiothreitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux. Maximum 3H-NEM incorporation into the DTT-reduced thiols occurred at 50 microM DTT. Saturation labelling by 3H-NEM of about 2 x 10(4) diamide-protected thiols/cell occurred at 25 microM NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.

Animals↗

PubMed1982

Effect of diamide and reduced glutathione on the elevated levels of cyclic AMP in rat pancreatic islets exposed to glucose, p-chloromercuribenzoate and aminophylline.

In rat pancreatic islets the effects of diamide, which has been shown to decrease islet levels of reduced glutathione (GSH), and of exogenous GSH were investigated on cyclic AMP as increased by glucose, p-chloromercuribenzoate, and aminophylline. In addition the effect of diamide on islet ATP level, low Km and high Km phosphodiesterases was studied. Diamide (0.1 mM) inhibited the increase of cyclic AMP (cAMP) in response to glucose (16.7 mM), and p-chloro-mercuribenzoate (1 mM) in the presence of 5.6 mM glucose. No inhibitory effect of diamide could be demonstrated when cAMP was raised by 10 mM aminophylline in the presence of 5.6 mM glucose. The glucose (27.7 mM) stimulated increase of cAMP was further augmented by GSH (0.4 mM) whereas GSH in the presence of 5.6 mM glucose had no such effect. Diamide neither affected islet high Km nor low Km cAMP-phosphodiesterases. Diamide (0.1 mM) as used in this study did not affect islet AMP levels. A concentration dependent decrease of ATP was observed, however, with higher concentrations of diamide (0.25, 0.5 and 1.0 mM). It is suggested that the accumulation of islet cAMP in response to glucose and para-chloromercuribenzoate depends on the redox state of islet thiols. Since thiol oxidant diamide neither affected cAMP-phosphodiesterase activities nor inhibited aminophylline induced accumulation of cAMP in the presence of low glucose the possibility is raised that in pancreatic islets the formation of cAMP rather than its degradation depends on the redox state of islet thiols.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Oxidative activation of K-Cl cotransport by diamide in erythrocytes from humans with red cell disorders, and from several other mammalian species.

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0-1.0 mm) in K-free Cl or NO3 media of variable osmolalities (200-450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO3) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) approximately rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0. 00001) and in NO3 (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity.

Anemia, Sickle Cell↗

PubMed1981

The effect of diamide on transmitter release and on synaptic vesicle population at vertebrate synapses.

Diamide, a sulfhydryl-oxidizing agent, has previously been shown to cause acetylcholine release in two preparations in the absence of added Ca2+. Similarities in action between diamide and alpha-latrotoxin, a component of black widow spider venom which causes transmitter release with no added Ca2+, and which seems to require a disulfide bond for its action, led us to study further the transmitter-releasing properties of diamide. In rat cerebral cortical slices we show that diamide, like alpha-latrotoxin, released all transmitters studied; GABA, acetylcholine, norepinephrine and dopamine. The response reached a peak after a delay (5-15 min), in contrast to the much faster release evoked by high K+ (within 3 min). Diamide-induced GABA release was found to occur equally well in the absence of added Ca2+, and was blocked when diamide was reduced prior to addition. Our ultrastructural studies of the frog neuromuscular junction showed that whereas alpha-latrotoxin caused the elimination of synaptic vesicles, diamide did not. Dithiothreitol, a disulfide-reducing agent, also caused GABA release, but this effect was Ca2+-dependent, blocked by high Mg2+, and occurred without delay. These observations comparing the 3 transmitter-releasing agents have further delineated the sulfhydryl/disulfide-group involvement in transmitter release and have demonstrated that dithiothreitol is operating at a different site from either alpha-latrotoxin or diamide.

Acetylcholine↗