NADH Oxidase Activity Of Plasma Membranes Research Articles

P-363 - Cytochrome c stimulates the superoxide anion production by cytochrome b5 reductase in neuronal synaptic plasma membrane vesicles

The plasma membrane NADH oxidase activity of synaptosomes and cerebellar granule neurons releases superoxide anion (O2•-) as a collateral product. In this work, we have measured the effect of cytochrome c (Cyt c) on the NADH-dependent O2•- production by rat neuronal synaptic plasma membrane vesicles. In these membranes, the Cyt c stimulated O2•- production was inhibited by antibodies against cytochrome b5 reductase (Cb5R). O2•- production by Cb5R was confirmed using purified soluble and membrane recombinant enzymes. Addition of Cyt c to the assay induced a burst of O2•- concomitant to the reduction of Cyt c by Cb5R. The low dissociation constant measured for Cyt c/Cb5R complex (0.40 ±0.05 and 0.38 ±0.02 μM for soluble and membrane Cb5R, respectively), support that Cb5R is a main molecular partner of Cyt c upon its release from mitochondria during apoptosis. O2•- production and Cyt c reduction are consequences of the stimulated NADH consumption by Cb5R upon complex formation with Cyt c. Participation of Cb5R in the apoptosis signalling network is suggested.

NADH-linked metabolic plasticity of MCF-7 breast cancer cells surviving in a nutrient-deprived microenvironment.

Characteristic of the tumor microenvironment are fluctuating gradients of reduced nutrient levels and released lactate. A fundamental issue is how tumor cells modulate their metabolic activity when both glucose and glutamine levels become limiting in the presence of high exogenous lactate. For functional analyses, the activities of pyruvate kinase, lactate dehydrogenase (LDH) and plasma membrane NADH oxidase (NOX) as well as cell growth were measured in breast cancer MCF-7 cells cultured in medium containing various concentrations of these metabolites. After 3 days at glucose concentrations below 2.5 mM, cell number was higher with 0.1 mM than with 1.0 mM glutamine, indicating that the glucose/glutamine balance is important for growth. On the other hand, NOX activity increased with increasing glucose >2.5 mM, but only with low glutamine (0.1 mM). Pyruvate kinase activity also increased, with LDH activity remaining 2-3-fold lower. Here NOX could have a complementary role in reoxidizing NADH for glycolysis. Exogenous lactate supported cell survival at limiting concentrations of glucose and glutamine while increasing NOX and pyruvate kinase activities as well as NADH levels. It is proposed that lactate supports cell survival by fuelling gluconeogenesis and/or the TCA cycle in mitochondria, from where NADH could be shuttled to the cytosol and reoxidized by NOX. Cell survival and the metabolic phenotype are thus interrelated to the dynamics of NADH and plasma membrane NOX activity, which are regulated by the balance of glucose/glutamine levels, in conjunction with lactate in a precarious tumor microenvironment.

Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells

Event Abstract Back to Event Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells Angela M. Otto1* and Cornelia Janzon2 1 Technische Universitaet Muenchen, Institute of Medical Engineering (IMETUM), Germany 2 Klinikum Leverkusen, Abteilung Onkologie/Haematologie, Germany Background and aim. The microenvironment of tumor cells is characterized by a gradual deprivation of nutrients and oxygen, an accumulation of lactate, and increased acidification. The question is how variable combinations of these components change tumor cell growth and metabolic activity in quantitative terms. The aim here is to investigate how extracellular lactate and pH affect this metabolic activity under limiting nutrient conditions. Methods. The breast cancer cell line MCF-7 was cultivated in “low growth” medium (DMEM with reduced FCS, containing tumor-relevant concentrations of glucose, glutamine as well as lactate, and set at different pH (6.6 – 7.4) for up to 4 days. Growth was determined by cell count. The WST-assay was used as a measure of plasma membrane NADH-oxidase activity (1). The activities of pyruvate kinase and lactate dehydrogenase (LDH) in cell lysates were measured by the kinetics of NADH-based changes in absorption. Lactate production was determined in cell culture supernatants using LDH activity as a read out. Results. At pH 7.4 with combinations of variable glucose concentrations and 0.1 mM glutamine, tumor cell growth was low, but the activity of NADH oxidase activity markedly increased at 2.5. mM glucose. Pyruvate kinase activity was also increased, while LDH activity remained 2-3-fold lower than that of pyruvate kinase. The inhibition of mitochondrial respiration by rotenone transiently increased NADH oxidase activity further. Lactate (25 mM) further increased NADH oxidase activity, but enhanced growth only at limiting conditions. At an acidic pH (6.6), these tumor cells become virtually independent of glucose and glutamine levels for both growth and the metabolic parameters tested. The culture medium of such cells contained only 7% of the lactic acid measured for cultures at pH 7.4, suggesting attenuated lactate release. Conclusions / Discussion. Extracellular pH has a differential effect on the replenishment of NAD+ from NADH produced by glycolysis. This is not solely accomplished by LDH, but may also involve plasma membrane NADH oxidase activity. Furthermore, lactate may be converted to pyruvate and support the TCA-cycle, thereby enhancing mitochondrial NADH levels. The resistance of NADH oxidase activity to rotenone suggests that reduction equivalents, along with the respective protons, are shuttled from the mitochondria to the cytosol. A hypothesis is proposed to explain the changes in plasma membrane NADH oxidase activity with respect to glycolysis and the tricarbonic acid cycle. Acknowledgements Part of this work was supported by the German Research Council (DFG). References (1) Berridge, M.V. and Tan, A.S. (1998) Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1. Protoplasma, 205:74-82. Keywords: Tumor Cells, Cultured, nutrient deprivation, Glucose, Glutamine, extracellular pH, tumor cell metabolism, Pyruvate Kinase, Lactate Dehydrogenases, extracellular lactate, NADH-oxidase, cell growth analysis Conference: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013. Presentation Type: Abstract Topic: 4. pH and energy metabolism Citation: Otto AM and Janzon C (2014). Extracellular pH, lactate, and a hypothesis for NADH-associated proton dynamics in tumor cells. Front. Pharmacol. Conference Abstract: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer. doi: 10.3389/conf.fphar.2014.61.00031 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Dec 2013; Published Online: 07 Feb 2014. * Correspondence: Dr. Angela M Otto, Technische Universitaet Muenchen, Institute of Medical Engineering (IMETUM), Garching, 85748, Germany, otto@tum.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Angela M Otto Cornelia Janzon Google Angela M Otto Cornelia Janzon Google Scholar Angela M Otto Cornelia Janzon PubMed Angela M Otto Cornelia Janzon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Plasma membrane NADH‐oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (ρ0)

The mammalian plasma membrane (PM) NADH-oxidoreductase (PMOR) system is a multi-enzyme complex located in the plasma membrane of all eukaryotic cells, harboring at least two distinct activities, the plasma membrane NADH-ferricyanide reductase and the NADH-oxidase. To assess the behaviour of the two activities of the PMOR system, we measured the NADH-ferricyanide reductase and NADH-oxidase activities in fibroblast cell lines derived from patients carrying a mitochondrial DNA (mtDNA) G11778A mutation. We also measured the two activities in other cell lines, the HL-60 and HeLa (S3) lines, as well as in rho0 cells (cells devoid of mtDNA) generated from those lines and the fibroblast cells. These rho0 cells consequently lack oxidative phosphorylation and rely on anaerobic glycolysis for their ATP need. We have proposed that in rho0 cells, at least in part, up-regulation of the PMOR is a necessity to maintain the NAD+/NADH ratio, and a pre-requisite for cell growth and viability. We show here that the PM NADH-ferricyanide reductase activity was up-regulated in HL-AV2 (HL-60 rho0) cell lines, but not in the other rho0 and mtDNA mutant lines. The plasma membrane NADH oxidase activity was found to be up-regulated in both HL-AV2 and HeLa rho0 cell lines, but not significantly in the fibroblast rho0 and G11778A lines.

Decomposition Analyses Applied to a Complex Ultradian Biorhythm: The Oscillating NADH Oxidase Activity of Plasma Membranes Having a Potential Time-Keeping (Clock) Function.

Seasonal decomposition analyses were applied to the statistical evaluation of an oscillating activity for a plasma membrane NADH oxidase activity with a temperature compensated period of 24 min. The decomposition fits were used to validate the cyclic oscillatory pattern. Three measured values, average percentage error (MAPE), a measure of the periodic oscillation, mean average deviation (MAD), a measure of the absolute average deviations from the fitted values, and mean standard deviation (MSD), the measure of standard deviation from the fitted values plus R-squared and the Henriksson-Merton p value were used to evaluate accuracy.Decomposition was carried out by fitting a trend line to the data, then detrending the data if necessary, by subtracting the trend component. The data, with or without detrending, were then smoothed by subtracting a centered moving average of length equal to the period length determined by Fourier analysis. Finally, the time series were decomposed into cyclic and error components. The findings not only validate the periodic nature of the major oscillations but suggest, as well, that the minor intervening fluctuations also recur within each period with a reproducible pattern of recurrence.

The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite.

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q 10 (Q 10H 2=ubiquinol) was oxidized at a rate of 15±6 nmol min −1 mg protein −1 depending on degree of purification. The apparent K m for Q 10H 2 oxidation was 33 μM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984). With coenzyme Q 0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q 0 and Q 0H 2 forms. With the mixture, a rate of Q 0H 2 oxidation of 8–17 nmol min −1 mg protein −1 was observed with an apparent K m of 0.22 mM. The rate of Q 10H 2 oxidation was not stimulated by addition of equal amounts of Q 10 and Q 10H 2. However, addition of Q 0 to the Q 10H 2 did stimulate. The oxidation of Q 10H 2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q 0H 2 may involve Q 0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids.

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10(-8) to 10(-5) M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 microM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 microM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture.

Chlorsulfuron blocks 2,4-D-induced cell enlargement and NADH oxidase in excised sections of soybean hypocotyls

Elongation of soybean hypocotyl sections induced by the auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was blocked by micromolar concentrations of the sulfonylurea herbicide chlorsulfuron (2-chloro- N-[[(4-methoxy-6-methyl-1,3,5-trizin-2yl)amino]carbonyl]benzenesulfonamide). The inhibition at equimolar concentrations of 2,4-D and chlorsulfuron was overcome by a 10- or 100-fold excess of 2,4-D. Stimulation by 2,4-D of an 2,4-D-responsive NADH-oxidase activity of the soybean plasma membrane also was blocked by the presence of concentrations of chlorsulfuron equimolar to the 2,4-D. Chlorsulfuron alone was largely without effect on either cell elongation or the auxin-stimulated NADH-oxidase activity over a range of chlorsulfuron concentrations. The results show a clear correlation between inhibition of auxin-stimulated cell elongation and the inhibition of an auxin-stimulated component of a plasma membrane NADH-oxidase activity.

Inhibition of the NADH oxidase activity of plasma membranes isolated from xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N?-(4-chlorophenyl)urea (LY 181984) correlates with drug susceptibility of growth

Inhibition of NADH oxidase activity of plasma membranes isolated from a series of human xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl) urea (LY 181984), correlated with the ability of the sulfonylurea to inhibit cell growth. Growth of rat kidney cells either untransformed or transformed with Kirsten-ras (K-ras) were unaffected by the sulfonylurea. Similarly, the NADH oxidase activity of isolated plasma membranes from K-ras transformed cells was unaffected by LY 181984. In contrast, when transformed with Harvey-ras (H-ras), both growth and NADH oxidase activity were inhibited. With the inactive but structurally related LY 181985 (N-4-methylphenyl-sulfonyl)-N′-(phenyl)urea), neither growth nor plasma membrane NADH oxidase activity of either sulfonylurea-susceptible or -resistant tissues or cell lines was inhibited. Both sulfonylureas were inactive with rat liver plasma membranes but NADH oxidase activity of plasma membranes and growth with HeLa cells was inhibited by the active (LY 181984) but not by the inactive (LY 181985) sulfonylurea. The findings suggest a possible correlation between inhibition of plasma membrane NADH oxidase activity by the antitumor sulfonylureas and their oncolytic action.

Cyclic AMP- plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes

Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the K m for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37°C with cyclic AMP (0.1–10 nM) and ATP (1–100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the K m for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of K m and V max as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1–2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4°C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4°C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37°C or after storage at 4°C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in K m , cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.

Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K(m) of the NADH oxidase after activation by lipids was four- to fivefold less than the K(m) before activation. The V(max) was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A(2) block the stimulation of the oxidase by auxins.