The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria

The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca²⁺ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca²⁺. This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed.     

NAD(P)H氧化酶在心衰犬心肌组织中的表达

目的观察扩张型心肌病心力衰竭(dilated cardiomyopathy-chronic heart failure, DCM-CHF)犬模型心肌中超氧化物来源之一的NAD(P)H氧化酶亚组分p47phox的表达.方法通过快速右心室起搏来建立扩张型心肌病心力衰竭(DCM-CHF)犬的模型,模型建立成功后,取犬心做病理检查(大体观测及组织切片HE染色)和免疫组化法检测p47phox的蛋白表达,并利用图像分析系统测量p47phox的蛋白阳性表达区域平均光密度值,进行定量分析.结果模型犬除有充血性心力衰竭的症状、体征外,心脏大体标本观测示DCM-CHF犬模型的心脏外形较正常对照犬增大,同时有左室腔扩大、室壁变薄;HE染色示DCM-CHF犬模型心肌细胞肿胀,大小不一;免疫组化结果显示DCM-CHF犬模型的心肌表达p47phox蛋白平均光密度值为0.3672±0.0214,而对照组平均光密度值为0.0954±0.0344,示DCM-CHF犬p47phox蛋白表达显著增多(P<0.01).结论 DCM-CHF犬模型的心肌中p47phox蛋白表达较正常对照犬显著增加,提示NAD(P)H氧化酶可能参与心力衰竭的病理生理过程.     

Cyclic AMP Enhancement of Depolarization-Induced NAD(P)H Changes in Brain Cortical Slices

The increased levels of NAD(P)H effected by electrical depolarization are markedly augmented in the presence of cyclic AMP, isoproterenol, or RO 20-1724, agents known to elevate cyclic AMP in rat brain slices. The data presented indicate that the cyclic AMP effect on an important component of intermediate metabolism is not an enhancement of a basal response but a separate response that is activated by depolarization, is Ca2+-dependent, regulates cytochrome a-a3 independently of its effects on NAD(P)H levels, and is dependent on a substrate other than glucose.     

集胞蓝藻PCC6803含疏水亚基的NAD（P）H脱氢酶亚复合体的分离

利用离子交换与凝胶过滤层析 ,从n dodecylβ D maltoside(DM)处理的集胞蓝藻SynechocystisPCC6 80 3细胞粗提液中 ,首次分离到两个包含NDH疏水亚基NdhA的亚复合体。酶活性分析表明 ,分离到的NDH亚复合体具有NADPH 氮蓝四唑 (NBT)氧化还原酶活性 ,以NADPH为电子供体可以还原铁氰化钾、二溴百里香醌 (DBMIB)、二氯酚靛酚 (DCPIP)、duroquinone以及UQ 0等质醌类电子受体。     

Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiae

Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole‐cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed‐batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95 mM adenine and 9.5 mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed‐batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95 mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl‐4‐chloro acetoacetate (CAAE) to the corresponding (S)‐4‐chloro‐3‐hydroxybutanoate (S‐CHBE) was evaluated with S. cerevisiae FasB His6 as an example. An intracellular threshold concentration above 0.07 mM NADP(H) was sufficient to increase the biocatalytic S‐CHBE productivity by 25 % compared to lower intracellular NADP(H) concentrations.     

Metabolic monitoring by using the rate of change of NAD(P)H fluorescene

The amino acid fermentation by Corynebacterium glutamicum was monitored with an new technique that uses the first derivative of the NAD(P)H fluorescene signal. The rate of change of NAD(P)H pools is indicative of intracellular redox balance variations that correspond to metabolic changes. The profile of this signal showed several characteristics that coincided with major metabolic events during fermentation. We show here that the derivative fluorescence signal can accurately estimate points of threonine depletion, viable cell count, and the end of amino acid formation. Furthermore, on-line optimization strategies can be developed by using the derivative fluorescene signal. (c) 1994 John Wiley & Sons, Inc.     

NAD(P)H: quinone oxidoreductase 1 inducer activity of novel 4-aminoquinazoline derivatives

Fourteen novel 4-aminoquinazoline derivatives 2–15 were designed and synthesized. The structure of the newly synthesized compounds was established on the basis of elemental analyses, IR, ¹H-NMR, ¹³C-NMR, and mass spectral data. The compounds were evaluated for their potential cytoprotective activity in murine Hepa1c1c7 cells. All of the synthesized compounds showed concentration-dependent ability to induce the cytoprotective enzyme NAD(P)H: quinone oxidoreductase (NQO1) with potencies in the low- to sub-micromolar range. This approach offers an encouraging framework which may lead to the discovery of potent cytoprotective agents.     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.     

NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats

Increased bioavailability of reactive oxygen species (ROS) has been implicated in the pathogenesis of mineralocorticoid hypertension. To find out the source of ROS, we evaluated the role of NAD(P)H oxidase in blood pressure (BP) elevation, cardiovascular hypertrophy, and fibrosis in aldosterone-salt rats. Aldosterone infusion (0.75 microg/h) significantly increased BP, which is attenuated by apocynin (1.5 mmol/L). Cardiac hypertrophy developed by aldosterone infusion was also normalized with apocynin. Greater mRNA for p22phox and NAD(P)H oxidase activity (more than twofold) in aorta of aldosterone-infused rats was reduced in apocynin-treated rats. Aldosterone infusion increased marginally procollagen I and III expression in LV compared to controls and apocynin decreased procollagen. Masson's Trichrome stain showed increased cardiac perivascular fibrosis, which was reduced by apocynin. These results suggest that NAD(P)H oxidase plays an important role in cardiovascular damage associated with mineralocorticoid hypertension.     

Vanadate-stimulated NADH oxidation in plasma membrane

The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane. When vanadate is added, this rate is stimulated 10- to 20-fold. The absorption spectrum of vanadate does not change with the disappearance of NADH. The reaction is inhibited by superoxide dismutase, and there is no activity under an argon atmosphere. This indicates that oxygen is the electron acceptor and the reaction is mediated by superoxide. The vanadate stimulation is not limited to plasma membrane. Golgi apparatus and endoplasmic reticulum show similar increase in NADH oxidase activity when vanadate is added. The endomembranes have significant vanadate-stimulated activity with both NADH and NADPH. The vanadate-stimulated NADH oxidase in plasma membrane is inhibited by compounds, which inhibit NADH dehydrogenase activity: catechols, anthracycline drugs and manganese. This activity is stimulated by high phosphate and sulfate anion concentrations.     

Metformin induces suppression of NAD(P)H oxidase activity in podocytes

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms.The experiments were performed in normal (NG, 5.6 mM) and high (HG, 30 mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-³H]-glucose.ROS production increased by about 27% (187 ± 8 vs. 238 ± 9 arbitrary units AU, P < 0.01) in HG. Metformin (2 mM, 2 h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58 ± 0.07 to. 0.99 ± 0.06, and from 0.53 ± 0.03 to 0.64 ± 0.03; P < 0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C.We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.     

Depletion of casein kinase I leads to a NAD(P)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

Background

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag⁻ phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.

Methods

A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.

Results

Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.

Conclusions

The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)⁺/NAD(P)H redox balance state.

General significance

The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O₂⁻). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O₂⁻ generation, besides complex III, stimulated NO degradation. Larger amounts of O₂⁻ were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O₂⁻ production were stimulated by free Ca²⁺ concentration. Together, these results indicate that Ca²⁺-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O₂⁻ production that favors NO degradation in potato tuber mitochondria.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

After incubation at 42°C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (ΔndhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of ΔndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42°C for 6 h, the photochemical activity declined more markedly in ΔndhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (ΔpH), while the consumption of ΔpH was highly inhibited in ΔndhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO₂ assimilation by providing extra ΔpH, thus reducing the photooxidative damage.     

Oscillation of NAD(P)H fluorescence in Escherichia coli culture performing dissimilative nitrate/nitrite reduction

When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.     

Pitfalls of using lucigenin in endothelial cells: Implications for NAD(P)H dependent superoxide formation

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (～ 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

NAD(P)H oscillation in relation to the rhythmic contraction in thePhysarum plasmodium

Summary ThePhysarum plasmodium shows rhythmic contractile activities with a period of a few min. Phases of the oscillation in the plasmodium migrating unindirectionally agreed sideways throughout at the frontal part. So, time course of an intracellular chemical component was determined by analyzing small pieces cut off successively from the frontal part of the large plasmodium. Intracellular NAD(P)H concentration oscillated with the same period as the rhythmic contraction but with a different phase advancing about 1/3 of the period. UV irradiation suppressed the rhythmic contraction without affecting the rhythmic variation of NAD(P)H. Thus, the NAD(P)H oscillator works independently of the rhythmic contractile system, but seems entraining with each other.Abbreviations UV ultraviolet - NADH nicotinamide adenine dinucleotide, reduced form - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - ATP adenosine 5-triphosphate - cAMP cyclic adenosine 3, 5-monophosphate - FMNH₂ flavin mononucleotide, reduced form - TCA tricarboxylic acid - BSA bovine serum albumin - DTT dithiothreitol