Thiol-oxidizing Agent Diamide Research Articles

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

The contraction and relaxation of the heart is controlled by stimulation of the β1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of β1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

URM1-Mediated Ubiquitin-Like Modification Is Required for Oxidative Stress Adaptation During Infection of the Rice Blast Fungus.

Ubiquitin is a small modifier protein which is usually conjugated to substrate proteins for degradation. In recent years, a number of ubiquitin-like proteins have been identified; however, their roles in eukaryotes are largely unknown. Here, we describe a ubiquitin-like protein URM1, and found it plays important roles in the development and infection process of the rice blast fungus, Magnaporthe oryzae. Targeted deletion of URM1 in M. oryzae resulted in slight reduction in vegetative growth and significant decrease in conidiation. More importantly, the Δurm1 mutant also showed evident reduction in virulence to host plants. Infection process observation demonstrated that the mutant was arrested in invasive growth and resulted in accumulation of massive host reactive oxygen species (ROS). Further, we found the Δurm1 mutant was sensitive to the cell wall disturbing reagents, thiol oxidizing agent diamide and rapamycin. We also showed that URM1-mediated modification was responsive to oxidative stresses, and the thioredoxin peroxidase Ahp1 was one of the important urmylation targets. These results suggested that URM1-mediated urmylation plays important roles in detoxification of host oxidative stress to facilitate invasive growth in M. oryzae.

Arabidopsis thaliana alcohol dehydrogenase is differently affected by several redox modifications.

In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.

GPx1 knockdown suppresses chondrogenic differentiation of ATDC5 cells through induction of reductive stress.

Glutathione peroxidase 1 (GPx1) is a selenium (Se)-containing protein and is induced in cartilage formation. GPx1 eliminates reactive oxygen species (ROS), which are required for chondrogenic induction. The physiological properties of GPx1 in cartilage and the redox mechanisms involved are not known. The effects of GPx1 on chondrogenic differentiation of ATDC5 cells were examined through short hairpin RNA-mediated gene silencing. The results demonstrated that GPx1 knockdown impaired gene expression of sex determining region Y-box 9, collagen II (Col II), and aggrecan. GPx1 knockdown suppressed the accumulation of cartilage glycosaminoglycans (GAGs) and the proliferation of chondrocyte. GPx1 knockdown also induced cell apoptosis. However, cell sensitivity toward exogenous oxidative stress was not increased after GPx1 knockdown. Unexpectedly, GPx1 knockdown not only induced oxidative stress characterized by the increased production of ROS but also caused reductive stress indicated by an elevation of glutathione (GSH)/oxidized GSH (GSSG) ratio. Furthermore, GPx1 knockdown-mediated reductive and oxidative stress could be antagonized by a thiol-oxidizing agent diamide and a thiol-containing compound N-acetylcysteine (NAC), respectively. Moreover, NAC attenuated GPx1 knockdown-induced cell apoptosis, while diamide prevented GPx1 knockdown-suppressed chondrocyte proliferation. Finally, diamide but not NAC could rescue GPx1 knockdown-mediated impaired chondrogenic differentiation. In summary, GPx1 is essential for chondrogenic induction in ATDC5 cells mainly through modulation of intracellular GSH/GSSG ratio, rather than an antioxidant enzyme to detoxify ROS. In addition, GPx1 knockdown-induced impaired chondrogenesis may participate in the pathogenesis of the endemic osteoarthropathy due to Se deficiency. These observations offer novel insights for the development of therapeutic target during cartilage degeneration.

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

The role of thiol redox systems in the response of Escherichia coli to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV254) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.

The thiol-oxidizing agent diamide reduces isoproterenol-stimulated amylase release in rat parotid acinar cells

In parotid acinar cells, activation of beta-adrenergic receptors provokes exocytotic amylase release via the accumulation of intracellular cAMP. Cellular redox status plays a pivotal role in the regulation of various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damages. In this study, we examined effect of diamide, a thiol-oxidizing reagent, on amylase release in rat parotid acinar cells. In the presence of diamide, isoproterenol (IPR)-induced cAMP formation and amylase release were partially reduced. Diamide had no effect on amylase release induced by forskolin and mastoparan, an adenylate cyclase activator and heterotrimeric GTP binding protein activator, respectively. In the cells pretreated with diamide, the binding affinity of [(3)H]dihydroalprenolol to beta-receptors was reduced. These results suggest that oxidative stress results in reduction of binding affinity of ligand on beta-receptor and consequently reduces protein secretory function in rat parotid acinar cells.

Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts

Depolarization of the mitochondrial inner membrane potential (ΔΨ m) associated with oxidative stress is thought to be a critical factor in cardiac dysfunction and cell injury following ischemia–reperfusion or exposure to cardiotoxic agents. In isolated cardiomyocytes, mitochondrially-generated reactive oxygen species (ROS) can readily trigger cell-wide collapse or oscillations of ΔΨ m but it is not known whether these phenomena scale to the level of the whole heart. Here we utilize two-photon laser scanning fluorescence microscopy to track ΔΨ m, ROS, and reduced glutathione (GSH) levels in intact perfused guinea-pig hearts subjected to simulated ischemia reperfusion or GSH depletion with the thiol oxidizing agent diamide. Exposure to oxidative stress by either method provoked heterogeneous ΔΨ m depolarization and occasional oscillation in clusters of myocytes in the epicardium in association with increased mitochondrial ROS production. Furthermore, the whole-heart oxidative stress dramatically increased the sensitivity of seemingly quiescent cells to ΔΨ m depolarization induced by a localized laser flash. These effects were directly correlated with depletion of the intracellular GSH pool. Unexpectedly, hearts perfused with nominally Ca 2+-free solution or those switched from 0.5 mM Ca 2+ to nominally Ca 2+-free solution also displayed heterogeneous ΔΨ m depolarization and oscillation, in parallel with net oxidation of the GSH pool. The findings demonstrate that metabolic heterogeneity initiated by mitochondrial ROS-induced ROS release is present in the intact heart, and that the redox state of the glutathione pool is a key determinant of loss of ΔΨ m.

Oxidative Stress Inactivates Cobalamin-Independent Methionine Synthase (MetE) in Escherichia coli

In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.

Ybp1 Is Required for the Hydrogen Peroxide-induced Oxidation of the Yap1 Transcription Factor

We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides. Ybp1 is required for H2O2-induced expression of the antioxidant encoding gene TRX2. Our data indicate that the effects of Ybp1 are mediated through the Yap1 transcription factor. Indeed, Ybp1 forms a stress-induced complex with Yap1 in vivo and stimulates the nuclear accumulation of Yap1 in response to H2O2 but not in response to the thiol-oxidizing agent diamide. The H2O2-induced nuclear accumulation of Yap1 is regulated by the oxidation of specific cysteine residues and is dependent on the thiol peroxidase Gpx3. Our data suggest that Ybp1 is required for the H2O2-induced oxidation of Yap1 and acts in the same pathway as Gpx3. Consequently, Ybp1 represents a novel class of stress regulator of Yap1. These data have important implications for the regulation of protein oxidation and stress responses in eukaryotes.

A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis.

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA. We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.

Import and metabolism of glutathione by Streptococcus mutans.

Glutathione (gamma-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including Streptococcus mutans ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by S. mutans 33402 in 7H9 medium was shown to require glucose and to occur with an apparent Km of 18+/-5 microM. GSSG, GSH, S-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of gamma-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the gamma-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and gamma-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of D/L-homocysteine, as well as GSH and gamma-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when S. mutans was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by S. mutans ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (S. agalactiae ATCC 12927, S. pyogenes ATCC 8668, and Enterococcus faecalis ATCC 29212) imported glutathione at negligible or markedly lower rates.

Glutathione depletion exacerbates impairment by oxidative stress of phosphoinositide hydrolysis, AP-1, and NF-κB activation by cholinergic stimulation

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-κB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H 2O 2 or diamide, with or without prior depletion of cellular glutathione. H 2O 2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-κB activation was unaffected by H 2O 2 unless glutathione was depleted, in which case there was a H 2O 2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H 2O 2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H 2O 2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-κB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H 2O 2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H 2O 2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.

Erythrocyte thiol status regulates band 3 phosphotyrosine level via oxidation/reduction of band 3-associated phosphotyrosine phosphatase

Oxidative stress-induced tyrosine phosphorylation has been ascribed to activation of phosphotyrosine kinase or to inhibition of phosphotyrosine phosphatase (PTP). We have previously identified a PTP associated with band 3 in the human erythrocyte membrane, a PTP that is normally highly active and prevents the appearance of band 3 phosphotyrosine. Here we show that treatment of erythrocytes with the thiol-oxidizing agent diamide leads to the formation of PTP disulfides (PTP-band 3 mixed disulfides) and inhibition of dephosphorylation, allowing the accumulation of band 3 phosphotyrosine. Upon reduction of the disulfides, the band 3 phosphotyrosine is dephosphorylated. Erythrocyte thiol alkylation by N-ethylmaleimide results in irreversible PTP inhibition and irreversible phosphorylation. The results are consistent with the notion that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP.

Modification of ionic currents underlying action potentials in mouse nerve terminals by the thioloxidizing agent diamide

The effect of diamide, a thiol-oxidizing agent, was tested using electrophysiological techniques to determine whether its ability to alter neuromuscular transmission in vitro could be attributed to alterations of ion channels controlling neuronal excitability and/or acetylcholine release. In mouse triangularis sterni preparations, diamide transiently increased the evoked release of acetylcholine and then blocked release. Extracellular recording of perineural waveforms associated with neuronal action potentials at motor nerve terminals showed that diamide reduced the waveforms associated with the delayed rectifier K + current, a Ca 2+ current and a Ca 2+-activated K + current ( i k, ca ). Inhibition of quantal transmitter release was not associated with failure of action potentials to invade nerve terminals. Thus, diamide modifies the ionic currents underlying the nerve terminal action potential, some of these changes probably account for the complex effects of diamide on quantal transmission.

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 μM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A 2, 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 NK 1 and NK 2 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.

Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase. Sensitivity towards agents that are thought to induce oxidative stress.

Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanothione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over-expression of enzyme activity (10-14-fold) in transformed cells. Following treatment of L. donovani cells with the thiol-oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over-expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate-limiting step in the metabolism of hydrogen peroxide.

Effect of the thiol-oxidizing agent diamide on NH2Cl-induced rat colonic electrolyte secretion.

The granulocyte-derived oxidant, monochloramine (NH2Cl), is known to stimulate chloride ion secretion in rat distal colonic mucosa mounted in Ussing chambers, through mechanisms that are sensitive and insensitive to tetrodotoxin (TTX). The possible role of intracellular thiols, in the mechanism of action of NH2Cl as a secretagogue, was evaluated with the thiol-oxidizing agent diamide and by measuring tissue sulfhydryl levels in response to NH2Cl. Serosal exposure to the antioxidant glutathione (0.25 mM), 5 min before NH2Cl (50 microM) addition, decreased the maximal effect of 50 microM NH2Cl on short-circuit current (Isc). The NH2Cl-stimulated increase in Isc was not affected by mucosal amiloride (5 microM). Pretreatment with 0.1 mM diamide shortened the lag period before the increase in Isc in response to NH2Cl, but it did not affect the maximal increase in Isc. Although TTX (0.5 microM) increased the lag time for achievement of the maximal Isc response to NH2Cl, the neurotoxin did not inhibit the effect of diamide, suggesting that diamide acts primarily on the nonneural component of NH2Cl-stimulated secretion. Incubation of colonic mucosa with NH2Cl, with or without diamide, decreased cellular acid-soluble sulfhydryl concentrations. Taken together, the results support a role for epithelial cell thiols in NH2Cl-stimulated electrolyte secretion by the rat colon.

Thiol modification in H2O2- and thromboxane-induced vaso- and bronchoconstriction in rat perfused lung

Hydrogen peroxide (H2O2), arachidonic acid (AA), and U-44069, a thromboxane analogue, all induced vaso- and bronchoconstriction in the isolated perfused rat lung. The role of protein sulfhydryl modifications in these processes was investigated. The thiol oxidizing agent diamide inhibited both vaso- and bronchoconstriction induced by H2O2, AA, or U-44069. Diamide had only a marginal effect on glutathione and protein thiol levels and no effect on lung mechanics. The diamide inhibition was reversible, and H2O2-induced vaso- and bronchoconstriction was almost maximal after 10 min of perfusion with buffer. The recovery was more rapid if dithiothreitol, a thiol reducing agent, was used in the buffer. H2O2- and AA-induced vaso- and bronchoconstriction is caused by thromboxane release. Diamide did not influence H2O2- or AA-dependent thromboxane formation, indicating that neither AA release nor AA metabolism to thromboxane is sensitive to thiol oxidation. Thus our results indicate that the site of diamide-induced thiol oxidation is the thromboxane receptor or its signal transduction.