Hans Löw

NADH oxidase of liver plasma membrane stimulated by diferric transferrin and neoplastic transformation induced by the carcinogen 2-acetylaminofluorene

NADH oxidase of purified plasma membranes (electron transfer from NADH to oxygen) was stimulated by the growth factor diferric transferrin. This stimulation was of an activity not inhibited by cyanide and was not seen in plasma membranes prepared from hyperplastic nodules from liver of animals fed the hepatocarcinogen, 2-acetylaminofluorene, nor was it due to reduction of iron associated with diferric transferrin. With plasma membranes from nodules, the activity was already elevated and the added transferrin was without effect. The stimulation by diferric transferrin did not correlate with the absence of transferrin receptors which were increased at the nodule plasma membranes. With liver plasma membranes, the stimulation by diferric transferrin raised the plasma membrane NADH oxidase specific activity to approximately that of the nodule plasma membranes. In contrast to NADH oxidase, which was markedly stimulated by the diferric transferrin, NADH ferricyanide oxidoreductase or reduction of ferric ammonium citrate by liver plasma membranes was approximately equal to or slightly greater than that of the nodule plasma membrane and unaffected by diferric transferrin. The results suggest the possibility of coupling of NADH oxidase activity to a growth factor response in mammalian cells as observed previously for this enzyme in another system.

Dehydrogenases of the Plasma Membrane

Oxidation-reduction reactions are widespread in biological systems and are basic to life processes and cellular metabolism. Nicotinamide-nucleotide-linked electron transport is generally part of a complex chain or array of carriers linked both structurally and functionally to cellular membranes. Some of the carriers may be loosely bound to the membrane or even “soluble” in the cytoplasm; others are structured as integral proteins of the membrane. The most widely studied are those of the electron-transport system found in and restricted to the inner mitochondrial membrane. Here, a sequence of components is organized mostly on the inner mitochondrial membrane with three sites of potential energy coupling to ATP formation (DePierre and Ernster, 1977).

α-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the alpha-adrenergic control of Ca2+ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca2+ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 microM for each agonist. Activation of the rate of ferricyanide reduction by both 10 microM methoxamine and 10 microM phenylephrine was blocked by the alpha-adrenergic antagonist, phenoxybenzamine, but not by the beta-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the alpha-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the alpha-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca2+ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl2 significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca2+ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by alpha-adrenergic agonists resulted from a change in the apparent Vmax, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that alpha-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca2+. The data suggest that alpha-agonist-mediated changes in electron efflux may lead to Ca2+ influx.

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent Km = 22 microM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 microM), dibutyryl cyclic AMP (0.1 mM) and the beta-adrenergic agonist ritodrine (10 microM) also had no effect, however, the alpha 1-adrenergic agonist, methoxamine (10 microM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the alpha-adrenergic agonist methoxamine.

Sirtuin Activation: A Role for Plasma Membrane in the Cell Growth Puzzle

For more than 20 years, the observation that impermeable oxidants can stimulate cell growth has not been satisfactorily explained. The discovery of sirtuins provides a logical answer to the puzzle. The NADH-dependent transplasma membrane electron transport system, which is stimulated by growth factors and interventions such as calorie restriction, can transfer electrons to external acceptors and protect against stress-induced apoptosis. We hypothesize that the activation of plasma membrane electron transport contributes to the cytosolic NAD(+) pool required for sirtuin to activate transcription factors necessary for cell growth and survival.

The oxidative function of diferric transferrin.

There is evidence for an unexpected role of diferric transferrin as a terminal oxidase for the transplasma membrane oxidation of cytosolic NADH. In the original studies which showed the reduction of iron in transferrin by the plasma membranes NADH oxidase, the possible role of the reduction on iron uptake was emphasized. The rapid reoxidation of transferrin iron under aerobic conditions precludes a role for surface reduction at neutral pH for release of iron for uptake at the plasma membrane. The stimulation of cytosolic NADH oxidation by diferric transferrin indicates that the transferrin can act as a terminal oxidase for the transplasma membrane NADH oxidase or can bind to a site which activates the oxidase. Since plasma membrane NADH oxidases clearly play a role in cell signaling, the relation of ferric transferrin stimulation of NADH oxidase to cell control should be considered, especially in relation to the growth promotion by transferrin not related to iron uptake. The oxidase can also contribute to control of cytosolic NAD concentration, and thereby can activate sirtuins for control of ageing and growth.

Chapter 6 Oxidase control of plasma membrane proton transport

Publisher Summary Plasma membranes contain electron transport systems. Some of these systems, such as the superoxide-generating NADPH oxidase of neutrophils, are well-known. Others, such as the NADH oxidase found in all cells, are not fully defined . Still others, such as glutathione oxidase, have barely been recognized. Electron transport across the plasma membrane results in proton release from cells. It is clear that some of this release in some cells is based on activation of the Na + /H + exchanger by electron flow. Activation of other channels or the proton-exporting ATPase may also be involved. Direct proton transport through the electron transport components may also account for a small part of the proton movement in plasma membranes. The activation of the proton movement by plasma membrane electron transport shows interaction with hormones or growth factors. It can be controlled by oncogene products associated with the plasma membrane and are susceptible to inhibition by known antitumor agents.

The requirement for transmembranal electron flow for cell proliferation

Diferric transferrin or other iron complexes have long been recognized as essential for cellular growth1–5. This requirement is primarily based on the essential role of iron in heme and DNA synthesis6,7. Removal of iron from cells inhibits growth8. The iron monement into the cell may depend on endocytosis of ferric transferrin attached to transferrin receptor in the plasma membrane9 or ferric iron uptake directly through the membrane by undefined transport systems5,10. Ferric chelates have also been used in place of transferrin to stimulate growth4,11. The discovery of an electron transport system in the plasma membrane which could reduce ferric chelates as well as other impermeable oxidants12,13 introduced a mechanism for ferric iron reduction outside the membrane before transport as ferrous iron. The discovery that impermeable ferricyanide stimulated cell growth under serum deficient conditions introduced a possible new role for ferric compounds as external oxidants to stimulate cell proliferation13,14. Since it has been shown that ferricyanide cannot stimulate growth of L1210 cells after iron depletion by desferrioxamine and cannot act as an iron supply for these cells5 we examined its effect on growth of cells which had not been depleted of iron.

Plasma membrane coenzyme Q: evidence for a role in autism

Background The Voltage Dependent Anion Channel (VDAC) is involved in control of autism. Treatments, including coenzyme Q, have had some success on autism control. Data sources Correlation of porin redox activity and expression of autism is based on extensive literature, especially studies of antibodies, identification of cytosolic nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase activity in the VDAC, and evidence for extreme sensitivity of the dehydrogenase to a mercurial. Evidence for a coenzyme Q requirement came from extraction and analog inhibition of NADH ferricyanide reductase in the erythrocyte plasma membrane, done in 1994, and reinterpreted when it was identified in VDAC in 2004. The effects of ubiquinol (the QH2 – reduced form of coenzyme Q) in children with autism were studied. Results A new role for coenzyme Q in the porin channels has implications on autism. Ubiquinol, the more active form of coenzyme Q, produces favorable response in children with autism. Agents which affected electron transport in porin show parallel effects in autism. Conclusion We propose a hypothesis that autism is controlled by a coenzyme Q-dependent redox system in the porin channels; this conclusion is based on the effects of agents that positively or negatively affect electron transport and the symptoms of autism. The full understanding of the mechanism of their control needs to be established.

Transplasma Membrane Electron Transport Functions as a Ferric Reductase

We have previously presented evidence that ferric ions and iron in diferric transferric can be reduced at the cell surface by the transplasma membrane electron transport system acting in conjuction with the transferrin receptor 1,2. In this paper we examine the reaction further especially in relation to the inhibition of both ferric citrate and diferric transferrin reduction by apotransferrin. We also consider the difference in activity found with different diferric transferrin preparations containing different amounts of loosely bound ferric iron. We will also consider the effects of argon, and cell transformation on the rate of ferric iron reduction in the presence and absence of diferric transferrin. The objective is to evaluate the extent to which loosely bound iron on transferrin can contribute to the formation of ferrous bathophenanthroline disulfonate (Fe(BPS)3) through the transmembrane electron transport system. We also show that iron reduction at acidic pH by the transmembrane enzyme is not inhibited by apotransferrin which would favor its role for ferric reduction in acidic endosomes.