Oxidation Of Pyridine Nucleotides Research Articles

Oxidation of pyridine nucleotides is an early event in the lethality of allyl alcohol

The involvement of altered pyridine nucleotide concentrations in the cytolethality of allyl alcohol was studied in isolated rat hepatocytes. NAD +, NADH, NADP +, NADPH and viability loss (leakage of lactate dehydrogenase into the medium) were measured in cells incubated with 0.5 mM allyl alcohol with or without the addition of 2 mM dithiothreitol at 30 min. Exposure to allyl alcohol increased NADH levels in the first 15 min of incubation. A sharp drop in NADH and NADPH with an accumulation of NADP + occurred between 30 and 60 min of incubation with allyl alcohol, indicating an oxidation and interconversion of pyridine nucleotides. Dithiothreitol prevented the oxidation of pyridine nucleotides, but not their reduction or interconversion, and protected against cell killing by allyl alcohol. The results suggest that pyridine nucleotide oxidation might be important for allyl alcohol-induced cytotoxicity; however, a causal relationship between pyridine nucleotide oxidation and cell killing is yet to be demonstrated.

Peroxynitrite stimulates the pyridine nucleotide-linked Ca2+ release from intact rat liver mitochondria.

Rat liver mitochondria contain a specific Ca2+ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed in a Ca2+-dependent manner to ADP-ribose and nicotinamide. We have previously shown that NAD+ hydrolysis is inhibited by cyclosporin A and is possible only when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxynitrite (ONOO-), which can form from nitric oxide (nitrogen monoxide, NO.) and superoxide anion (O2-), at low concentrations stimulates the specific Ca2+ release pathway. Both peroxynitrite-induced pyridine nucleotide hydrolysis and Ca2+ release are inhibited by cyclosporin A, and peroxynitrite is ineffective when pyridine nucleotides are kept reduced. Ca2+ release induced by peroxynitrite occurs with maintenance of the mitochondrial membrane potential and is not accompanied by entry of sucrose into mitochondria. The results suggest that peroxynitrite stimulates the specific Ca2+ release from intact mitochondria by modifying critical mitochondrial thiols other than glutathione in such a way that hydrolysis of oxidized pyridine nucleotides is achieved. These findings provide further insight into the regulation of Ca2+ release from mitochondria by nitric oxide and its congeners.

Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status

Treatment of isolated mitochondria with Ca 2+ and inorganic phosphate (P i) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca 2+-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca 2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca 2+ and P i abolished large amplitude swelling of mitochondria. The GSH status during a Ca 2+- and P i-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca 2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.

Oxidants in mitochondria: from physiology to diseases

Reactive oxygen species (ROS: superoxide radical, O 2 −; hydrogen peroxide, H 2O 2; hydroxyl radical, OH), which arise from the univalent reduction of dioxygen are formed in mitochondria. We summarize here results which indicate that ROS, and also the radical nitrogen monoxide (‘nitric oxide’, NO), act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Hydrogen peroxide, which originates in mitochondria predominantly from the dismutation of superoxide, causes oxidation of mitochondrial pyridine nucleotides and thereby stimulates a specific Ca 2+ release from intact mitochondria. This release is prevented by cyclosporin A (CSA). Hydrogen peroxide thus contributes to the maintenance of cellular Ca 2+ homeostasis. A stimulation of mitochondrial ROS production followed by an enhanced Ca 2+ release and re-uptake (Ca 2+ ‘cycling’) by mitochondria causes apoptosis and necrosis, and contributes to hypoxia/reperfusion injury. These kinds of cell injury can be attenuated at the mitochondrial level by CSA. When ROS are produced in excessive amounts in mitochondria nucleic acids, proteins, and lipids are extensively modified by oxidation. Physiological (sub-micromolar) concentrations of NO potently and reversibly deenergize mitochondria at oxygen tensions that prevail in cells by transiently binding to cytochrome oxidase. This is paralleled by mitochondrial Ca 2+ release and uptake. Higher NO concentrations or prolonged exposure of cells to NO causes their death. It is concluded that ROS and NO are important physiological reactants in mitochondria and become toxic only when present in excessive amounts.

Interactions of Adriamycin aglycones with mitochondria may mediate Adriamycin cardiotoxicity

Adriamycin and related anthracyclines are potent oncolytic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone metabolites of Adriamycin (5–20 μM) induce a Ca 2+-dependent increase in the permeability of the inner mitochondrial membrane of both heart and liver mitochondria to small (< 1500 Da) solutes; this phenomenon is accompanied by release of mitochondrial Ca 2+, mitochondrial swelling, collapse of the membrane potential, oxidation of mitochondrial pyridine nucleotides [NAD(P)H], uncoupling, and a transition from the condensed to the orthodox conformation and is inhibited by ATP, dithiothreitol, the immunosuppressant cyclosporin A, and the ubiquitous polyamine spermine. Aglycones also modify mitochondrial sulfhydryl groups and induce a Ca 2+ independent oxidation of mitochondrial NAD(P)H which appears to reflect electron transport from NADH to oxygen, mediated by the aglycones and resulting in the production of Superoxide (O 2 −). Selenium deficiency and butylated hydroxytoluene inhibit aglycone-induced Ca 2+ release from liver, but not heart, mitochondria, suggesting that the interactions of the aglycones with mitochondria diner in these two tissues. It can be proposed that the effects of Adriamycin aglycones on heart mitochondria are responsible for the cardiotoxicity of the parent drug.

Extraction and Assay of Creatine Phosphate, Purine, and Pyridine Nucleotides in Cardiac Tissue by Reversed-Phase High-Performance Liquid Chromatography

The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellular states. Nevertheless, technical, theoretical, and methodological difficulties in extraction and quantification procedures have so far limited our understanding of the exact role that these substances play in metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure with HClO 4 for purine and oxidized pyridine nucleotides and then developed a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tissue were frozen and ground at −180°C using the appropriate extraction procedure. The separation and quantification of the metabolites were performed using a reversed-phase 3-μm Supelchem C18 column, with the addition of tetrabutylammonium as an ion-pair agent to the buffer solution, by ultraviolet detection. The recovery of the external and internal standards always exceeded 90%. The autooxidation or interconversion processes were almost insignificant for each reduced form. This technique allowed us to avoid complex enzymatic procedures and difficulties in the selective assay of pyridine nucleotides with chemiluminescence and surface spectroscopy.

Ca 2+-dependent permeabilization of the inner mitochondrial membrane by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca 2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955–19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and Ca 2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca 2+ release. The presence of DTT prevented but did not reverse the elimination of Δψ induced by DIDS. EGTA prevented Δψ elimination and decreased the amount of protein aggregates, suggesting that the binding of CA 2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed Δψ by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca 2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca 2+-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

Superoxide generation by lipoxygenase in the presence of NADH and NADPH

The ability of soybean lipoxygenase to mediate NAD(P)H oxidation and concomitant Superoxide generation in the presence of linoleic acid was examined. At optimum pH of 8.3, lipoxygenase oxidized both NADH and NADPH in the presence of 700 μM linoleic acid. The oxidation of NAD(P)H was biphasic. The initial rates of NADH and NADPH oxidation were 130 and 140 nmoles/min/nmole of enzyme respectively and the corresponding final rates were 344 and 350 nmoles/min/nmole of enzyme. The apparent K m values calculated for NADH and NADPH oxidation were 13 ju.m and 117 μM respectively. NAD(P)H oxidation was accompanied by the reduction of either ferricytochrome c or nitroblue tetrazolium (NBT) which can be abolished (approx. 85%) by Superoxide dismutase (SOD) suggesting the generation of Superoxide anion radicals. Under optimal conditions, the rates of Superoxide generation, measured as the SOD-inhibitable reduction of ferricytochrome c, were 325 and 235 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Under identical experimental conditions, the SOD-inhibitable NBT reduction rates were 308 and 210 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Both NADH and NADPH could be regenerated after oxidation using the appropriate dehydrogenases. These results strongly suggest that lipoxygenase not only generates lipid hydroperoxides but can also generate Superoxide via oxidation of pyridine nucleotides and may, therefore, significantly contribute to oxidative stress in cells.

Further characterization of the events involved in mitochondrial Ca 2+ release and pore formation by prooxidants

Addition of the prooxidant 3,5-dimethyl- N-acetyl- ρ-benzoquinone imine (3,5(Me) 2NAPQI) to Ca 2+-loaded mitochondria caused a rapid and extensive release of the sequestered Ca 2+. Ca 2+ release was accompanied by irreversible NAD(P)H oxidation and was followed by the release of adenine and pyridine nucleotides into the extramitochondrial medium; this is evidence of the opening of the pore in the inner mitochondrial membrane. Preincubation of the mitochondria with ADP, cyclosporin A (CSA), m-iodobenzylguanidine (MIBG) or Mg 2+ inhibited the prooxidant-induced Ca 2+ release and prevented pore-opening. When mitochondria were preincubated with ruthenium red, Ca 2+ release was only minimally stimulated by 3,5(Me) 2NAPQI. However, increasing the concentration of the prooxidant caused release of an increasing fraction of the sequestered Ca 2+. Alternatively, increasing the intra-mitochondrial Ca 2+ load resulted in a lowering of the concentration of 3,5(Me) 2NAPQI required for near complete Ca 2+ release to occur. In the presence of ruthenium red, 3,5(Me) 2NAPQI-induced Ca 2+ release was accompanied by irreversible pyridine nucleotide oxidation and followed by the release of nucleotides into the extramitochondrial medium, events which were prevented on preincubation with CSA. Similarly, the addition of CSA, ADP or MIBG during 3,5(Me) 2+NAPQI-induced Ca 2+ release arrested further Ca 2+ release. In addition to their inhibitory effect on the 3,5(Me) 2NAPQI-induced Ca 2+ release, CSA, ADP or MIBG also decreased the rate of the basal, ruthenium red-induced mitochondrial Ca 2+ release by 45–70%. It is proposed that the basal, ruthenium red-induced and the prooxidant-induced mitochondrial Ca 2+ release occur through a common component that is sensitive to inhibition by CSA, ADP and MIBG and that is involved in mitochondrial pore formation. Furthermore, 3,5(Me) 2NAPQI-induced pore opening does not involve Ca 2+-cycling, but rather involves a site(s) that is (are) synergistically activated by Ca 2+ and the prooxidant.

Oxidation of Pyridine Nucleotides and Depletion of ATP and ADP during Calcium- and Inorganic Phosphate-Induced Mitochondrial Permeability Transition

We have examined the pyridine and adenine nucleotide status during calcium- and inorganic phosphate-induced permeability transition. Calcium- and inorganic phosphate-induced permeability transition is accompanied by the rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP. The addition of cyclosporin A, a potent inhibitor of the permeability transition prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP.

Control of oxidative metabolism in volume-overloaded rat hearts: effects of different lipid substrates

The relationship between intracellular energy parameters and myocardial O2 consumption (VO2) was studied in control and volume-overloaded hearts perfused with different lipid substrates and over a range of left ventricular work loads. In control hearts, a unique linear relationship between log of cytosolic [ATP]/[ADPf].[Pi] (where [ADPf] is concentration of free ADP) and myocardial VO2 was observed between low and high work loads for both fatty acids studied. In volume-overloaded hearts perfused in the presence of exogenous palmitate, the slope of the relationship between log [ATP]/[ADPf].[Pi] and myocardial VO2 was considerably depressed. It would seem that, under these conditions, much of the thermodynamic control of respiratory chain function has been lost. When myocardial VO2 was expressed as a function of cytosolic ADPf, the cytosolic ADPf was not regulatory. This may be related to a substrate limitation of the respiratory chain, as suggested by an excessive oxidation of pyridine nucleotides. When octanoate, instead of palmitate, was used, most of the above limitation of the respiration disappeared. With this substrate, the reduction of mitochondrial pyridine nucleotides in volume-overloaded hearts was similar to that in controls, and the linear relationship between log [ATP]/[ADPf].[Pi] and myocardial VO2 reappeared over the range of work loads studied. The above failure of cytosolic phosphate potential and ADPf to drive respiration when mitochondrial NADH is low fits well with the integrated model of kinetic regulation, as proposed by recent nuclear magnetic resonance studies. our results also indicate that, even at high respiratory rates, free-energy change of ATP synthesis of volume-overloaded hearts can be protected by use of an appropriate substrate. This, in turn, prevents contractile failure.

Identification of fast spurts of pyridine nucleotide oxidation evoked by light stimulation in the isolated perfused vertebrate retina

Transitory increases of ultraviolet transmission on stimulation with light were recorded simultaneously with electroretinogram on and off effects from isolated vertebrate retina. The spectral distribution of the optical light responses coincided with that of NADH reduction. The correlation of the optical, or respiratory, responses and the electrical responses were very close within a wide range of stimulus parameters, suggesting an interpretation in terms of supply and demand of energy with a tight coupling between the two kinds of evoked activity. Prerequisite to the response behaviour was the preservation of synaptic signal transmission from first- to higher-order retinal neurons.

Cyclosporine A protects mitochondria in an in vitro model of hypoxia/reperfusion injury

Hypoxia/reperfusion injury is a major clinical problem. One of its hallmarks is an increased cytosolic Ca 2+ content and an increased generation of reactive oxygen species in the cytosol and in mitochondria. In the present study of an in vitro model of hypoxia/reperfusion injury, mitochondria are exposed to Ca 2+ in combination with extra- and intramitochondrially acting prooxidants. In this model mitochondria are damaged in a Ca 2+-dependent manner. The extent and the site(s) of damage depend on both the kind of respiratory substrate and prooxidant used. The major damage occurs specifically at site I of the respiratory chain, and is due to hydrolysis of oxidized pyridine nucleotides and Ca 2+ release followed by Ca 2+ re-uptake (Ca 2+ ‘cycling’). Cyclosporine A completely protects against this damage. The protection is due to inhibition of pyridine nucleotide hydrolysis, an obligatory step in the sequence of events that links prooxidants to Ca 2+ release from intact mitochondria.

In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate‐deficient sunflower and maize leaves

ABSTRACTThe effects of extreme phosphate (Pi) deficiency during growth on the contents of adenylates and pyridine nucleotides and the in vivo photochemical activity of photosystem II (PSII) were determined in leaves of Helianthus annuus and Zea mays grown under controlled environmental conditions. Phosphate deficiency decreased the amounts of ATP and ADP per unit leaf area and the adenylate energy charge of leaves. The amounts of oxidized pyridine nucleotides per unit leaf area decreased with Pi deficiency, but not those of reduced pyridine nucleotides. This resulted in an increase in the ratio of reduced to oxidized pyridine nucleotides in Pi‐deficient leaves. Analysis of chlorophyll a fluorescence at room temperature showed that Pi deficiency decreased the efficiency of excitation capture by open PSII reaction centres (φe), the in vivo quantum yield of PSII photochemistry (φPSII) and the photochemical quenching co‐efficient (qP), and increased the non‐photochemical quenching co‐efficient (qN) indicating possible photoinhibitory damage to PSII. Supplying Pi to Pi‐deficient sunflower leaves reversed the long‐term effects of Pi‐deficiency on PSII photochemistry. Feeding Pi‐sufficient sunflower leaves with mannose or FCCP rapidly produced effects on chlorophyll a fluorescence similar to long‐term Pi‐deficiency. Our results suggest a direct role of Pi and photophosphorylation on PSII photochemistry in both long‐and short‐term responses of photosynthetic machinery to Pi deficiency. The relationship between φPSII and the apparent quantum yield of CO2 assimilation determined at varying light intensity and 21 kPa O2 and 35 Pa CO2 partial pressures in the ambient air was linear in Pi‐sufficient and Pi‐deficient leaves of sunflower and maize. Calculations show that there was relatively more PSII activity per mole of CO2 assimilated by the Pi‐deficient leaves. This indicates that in these leaves a greater proportion of photosynthetic electrons transported across PSII was used for processes other than CO2 reduction. Therefore, we conclude that in vivo photosynthetic electron transport through PSII did not limit photosynthesis in Pi‐deficient leaves of sunflower and maize and that the decreased CO2 assimilation was a consequence of a smaller ATP content and lower energy charge which restricted production of ribulose, 1‐5, bisphosphate, the acceptor for CO2.

Studies on Hg(II)-induced H 2O 2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria

Studies were undertaken to investigate the principal actions underlying mercury-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl 2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H 2O 2) formation for up to 6 hr following Hg(II) treatment using succinate as the electron transport chain substrate. No increase in H 2O 2 formation was observed when NAD-linked substrates (malate/glutamate) were used, suggesting that Hg(II) affects H 2O 2 formation principally at the ubiquinone-cytochrome b region of the mitochondrial respiratory chain in vivo. Together with increased H 2O 2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobarbiturate reactive substances (TBARS), indicative of mitochondrial lipid peroxidation, was increased by 68%. Studies in vivo revealed a significant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated from kidneys of untreated rats. This effect was accompanied by significantly increased H 2O 2 formation, GSH depletion and TBARS formation linked to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytochrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mitochondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca 2+ in Hg(II)-induced mitochondrial oxidative stress was investigated. Ca 2+ alone (30–400 nmol/mg protein) produced no increase in H 2O 2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. However, Ca 2+ significantly increased H 2O 2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial electron transport chain, whereas TBARS formation was decreased significantly when the Ca 2+ uptake inhibitors, ruthenium red or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) causes depolarization of the mitochondrial inner membrane with consequent increased H 2O 2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron- dependent lipid peroxidation (TBARS formation). Since increased H 2O 2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreover, Hg(II)-induced alterations in mitochondrial Ca 2+ homeostasis may excerbate Hg(II)-induced oxidative stress in kidney cells.

Ca 2+-induced, phospholipase-independent injury during reoxygenation of anoxic mitochondria

Reoxygenation of rat-liver mitochondria after anoxic incubation induced release of matrix proteins. As assessed by release of a matrix enzyme, it was proportional to the rate of H 2O 2 production. The release was not observed with low concentrations of extramitochondrial free Ca 2+, indicating a Ca 2+-dependent pathway. Phospholipase A 2 was not involved in the reoxygenation injury, because non-esterified fatty acids did not increase on reoxygenation even when re-acylation was inhibited and because inhibitors of phospholipase A 2 had little effect on enzyme release. Cyclosporin A, ATP, ADP and inhibitors of pyridine nucleotide oxidation had a protective effect, strongly suggesting involvement of so-called Ca 2+-dependent permeability transition. Ca 2+ was also released from reoxygenated mitochondria and inhibition of reuptake of released Ca 2+ attenuated the enzyme release. Similar releases of aspartate aminotransferase and Ca 2+ were observed with mitochondria in an oxygen radical-generating system, hypoxanthine and xanthine oxidase. In this system, lecithin-cardiolipin liposomes also released entrapped Ca 2+ without disruption of the membrane. From these results, we conclude that during reoxygenation, Ca 2+ release and subsequent reuptake induced permeability transition of mitochondria, resulting in reoxygenation injury.

Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria

The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents ( t-butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca 2+ and inorganic phosphate concentrations), and was monitored via Ca 2+-release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C 50 of 0.38 ± 0.06 (SD) mM was measured for inhibition.

Events that precede and that follow S-(1,2-dichlorovinyl)- l-cysteine-induced release of mitochondrial Ca 2+ and their association with cytotoxicity to renal cells

Previous studies showed that S-(1,2-dichlorovinyl)- l-cysteine perturbs intracellular Ca 2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455–461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)- l-cysteine-induced mitochondrial Ca 2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)- l-cysteine-induced Ca 2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meto-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK 1 cells, elevation in the cytosolic Ca 2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca 2+- and Mg 2+-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)- l-cysteine-induced cytotoxicity in LLC-PKin1 cells is blocked by chelation of cytosolic Ca 2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that 5-(1,2-dichlorovinyl)- l-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca 2+ release. Elevated Ca 2+ concentrations may activate Ca 2+-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.

Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca 2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis

The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca 2+ release from rat liver mitochondria was studied. In Ca 2+-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD + hydrolysis or the formation of ADP-ribose from NAD +. Thus, whilst BHT prevented NAD + hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca 2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.