Reduction of diferric transferrin by SV40 transformed pineal cells stimulates Na+/H+ antiport activity

Transplasmalemma electron transport by HeLa and pineal cells to reduce external ferricyanide is associated with proton release from the cells. Diferric transferrin also acts as an electron acceptor for the transmembrane oxidoreductase. We now show that reduction of external diferric transferrin by RPNA-209-1 SV40 transformed pineal cells is accompanied by proton release from the cells. The stoichiometry of proton release to electron transfer is much greater than would be expected from aniostropic electron flow across the membrane through protonated carriers. The proton release is not stimulated by apotransferrin and the diferric transferrin-stimulated activity is inhibited by apotransferrin. Apotransferrin also inhibits reduction of diferric transferrin by these cells. The proton release is dependent on external sodium ions and is inhibited by amiloride, which indicates that the proton release is mediated by the Na+/H+ antiport and that this antiport is activated by electron transport through the transmembrane dehydrogenase. Growth stimulation by diferric transferrin or other external oxidants can be based in part on activation of the Na+/H+ antiport.     

Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.

Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.     

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.     

Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.     

Transplasma membrane electron and proton transport is inhibited by chloroquine

Chloroquine is a weak base which has been shown to inhibit lysosomal acidification. Chloroquine inhibits iron uptake in reticulocytes at a concentration of 0.5 mM. It is also effective in the control of malaria and other parasitic diseases. We now report that chloroquine inhibits NADH diferric transferrin reductase as well as the proton release stimulated by diferric transferrin from liver and HeLa cells. Ammonium chloride which also inhibits endosome acidification does not significantly inhibit the NADH diferric transferrin reduction. NADH diferric transferrin reductase of isolated rat liver plasma membrane is inhibited by chloroquine at concentrations similar to those required for inhibition of diferric transferrin reduction by whole cells. Ferricyanide reduction by whole cells is also inhibited by chloroquine. These observations provide an alternative mechanism for chloroquine control of acidification of endosomes and suggests a new approach to control of protozoal parasites through inhibition of a transmembrane oxidoreductase which controls transmembrane proton movement.     

Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system

Intact cells are known to reduce external, impermeable electron acceptors. We now show that cells can reduce the iron in diferric transferrin at the cell surface and that this reduction reaction depends on the transferrin receptor as well as the transmembrane electron transport system. Reduction of external diferric transferrin is accompanied by oxidation of internal NADH which indicates that the transmembrane enzyme is an NADH diferric transferrin reductase. Highly purified liver plasma membranes have NADH diferric transferrin reductase activity which shows properties similar to the diferric transferrin reductases activity of intact cells. Cell growth stimulation by diferric transferrin and other impermeable oxidants which can react with the diferric transferrin reductase can be based on electron transport through he plasma membrane.     

Diferric transferrin reductase in the plasma membrane is inhibited by adriamycin

Diferric transferrin which is often necessary for growth of cells is reduced by the transplasma membrane electron transport system of HeLa cells with release of ferrous iron outside the cell. Reduction of external diferric transferrin is reflected in oxidation of internal NADH. Adriamycin, an antitumor drug, inhibits diferric transferrin reduction by the HeLa cells and inhibits concomittant oxidation of cytosolic NADH at concentrations, 10(-8)-10(-6)M, which inhibit cell growth. Isolated liver plasma membranes have an NADH diferric transferrin reductase activity which is inhibited by similar adriamycin concentrations. We propose that inhibition of cell growth by adriamycin can be based on inhibition of transplasmalemma diferric transferrin reductase.     

Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inK _m andV _max for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.     

Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of transmembrane electron transport activity in plasma membranes of simian virus 40 transformed pineal cells

Changes have been found in the plasma membrane enzyme system which carries out transmembrane electron transport and associated proton transport in Simian virus 40 (SV40) temperature-sensitive A (tsA) mutant-transformed rat pineal cell line, RPN209-1. This cell line was temperature-sensitive for the maintenance of transformation. RPN209-1 cells expressed the transformed phenotype (rapid growth, high cell density, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, and lower cloning efficiency in soft agar) at the nonpermissive temperature (40 degrees C). The reduction of external ferricyanide, hexaammine ruthenium and diferric transferrin was used to measure the transmembrane redox activity. The transformed RPN209-1 cells expressed a lower transmembrane redox activity, which is more sensitive to the antitumor drug adriamycin, when compared to the cells with a nontransformed phenotype. The lower transmembrane redox activity is associated with a decrease in the affinity for ferricyanide and a change in Vmax of the enzyme. Since the transformed cells have 25% lower concentration of NADH, the decrease in Vmax may be partly based on substrate limitation. Ionic strength variation in the assay media shows that the change in activity with transformation is not based on change in cell-surface change. Treatment with neuraminidase, however, indicates that sialic acid is important for enzyme activity, consistent with previous proposals that the transmembrane enzyme is a glycoprotein. The proton extrusion associated with transplasma membrane electron transport is increased in transformed cells relative to the rate of ferricyanide reduction. A relation between proton pumping transplasma membrane electron transport and growth stimulation by external oxidants is discussed.     

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

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.     

Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase

The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophenanthroline disulfonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.     

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.     

A Transplasma Membrane Redox System in Phycomyces blakesleeanus: Properties of a Ferricyanide Reductase Activity Regulated by Iron Level and Vitamin K3

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.