Transplasma membrane redox stimulates HeLa cell growth

Impermeable ferricyanide stimulates the growth of HeLa cells in absence of fetal bovine serum or other growth factors. A series of impermeable oxidants with redox potentials down to -125 mV stimulate equivalent growth. All of these oxidants are reduced by the transplasma membrane electron transport system. Oxidants with redox potentials below -175 mV are not reduced by the transmembrane electron transport and do not stimulate growth. Insulin which stimulates growth in absence of serum also stimulates transmembrane ferricyanide reduction. Ferricyanide increases growth in presence of insulin. Antitumor drugs, which inhibit HeLa cell growth, inhibit the transplasma membrane redox system. Transplasma membrane electron transport is accompanied by proton release from HeLa cells.     

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.     

Ceruloplasmin stimulates thymidine incorporation by CCL-39 cells in the absence of serum or growth factors

The incorporation of tritiated thymidine into CCL-39 cells grown in the absence of fetal calf serum or other growth factors is greatly increased by low concentrations of ceruloplasmin. The stimulation is greater than observed with serum or thrombin. Addition of serum decreases the thymidine incorporation with ceruloplasmin to the level with serum alone. As with serum, the response to ceruloplasmin is high at both 20% and 1% oxygen, which is consistent with the action of ceruloplasmin as an oxidant with a high affinity for oxygen. Since transplasma membrane electron transport increases cell growth and thymidine incorporation, ceruloplasmin may act as a terminal oxidase for ferrous iron or ascorbate to stimulate transplasma membrane electron transport. The four electron transfer from ceruloplasmin to oxygen to form water will prevent peroxide formation at the cell surface. Alternatively, superoxide formation inside the cell or membrane could employ the superoxide dismutase function of ceruloplasmin to produce peroxide. Either mechanism would be consistent with the previously described stimulation of growth by external oxidants.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Comparison of growth stimulation of HeLa cells,HL-60 cells,and mouse fibroblasts by coenzyme Q10

Summary The addition of coenzyme Q₁₀ to culture media stimulates the serum-free growth of HeLa, HL-60 cells, and mouse fibroblasts (Balb/3T3). With HeLa cells, the stimulation by coenzyme Q₁₀ is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport. This combined response to coenzyme Q₁₀ and ferricyanide is enhanced with insulin. -Tocopherylquinone can also stimulate the growth of HeLa cells, but vitamin K₁ is inactive. Specificity of quinone effects is indicated. Serum-free growth of Balb/3T3 and SV 40 transformed BaIb/3T3 (SV/T2) cells is also stimulated by coenzyme Qio with stimulation similar to HeLa cells. However, Balb/3T3 cells are not stimulated by ferricyanide, which does not increase the response to coenzyme Q₁₀. The transformed cells (SV/T2) respond better to ferricyanide alone, but the effects of coenzyme Qio and ferricyanide are not additive. Serum-free growth of HL-60 cells is stimulated dramatically by coenzyme Q₁₀. The extent of growth stimulation on HL-60 cells is almost six-fold that of HeLa or Balb/3T3 cells. The stimulation of NADH-ferricyanide reductase (a transmembrane redox enzyme) by coenzyme Q₁₀ with HL-60 cells is similar to their growth pattern in response to coenzyme Q₁₀. Unlike HL-60, HeLa and Balb/3T3 cells show little stimulation of ferricyanide reduction by coenzyme Q₁₀. The stimulatory effect on both ferricyanide reduction and cell growth by the short side-chain coenzyme Q₂ is much less than that of the long side-chain coenzyme Q₁₀. Ferricyanide reduction by HeLa cells is inhibited by coenzyme Q analogs such as 2,3-dimethoxy-5-chloro-6-naphthyl-mercapto-coenzyme Q and 2-methoxy-3-ethoxyl-5-methyl-6-hexadecyl-mercapto-coenzyme Q. However, these inhibitions are reversed by coenzyme Q₁₀. The growth inhibition of HL-60 cells by other coenzyme Q analogs, such as capsiacin can also be reversed by coenzyme Q₁₀. These data indicate that plasma membrane-based NADH oxidation or modification of the membrane quinone redox balance may be a basis for the growth stimulation.     

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.     

Effect of growth factors and antitumor drugs on normal and Vall2Ha-ras transformed C3H 10T1/2 cell transplasma membrane electron transport and growth

Summary Bothras and the transplasma membrane electron transport system have been implicated in the control of cell growth. Since the loss of growth regulation is at the heart of the cancerous phenotype, we have investigated the effect of factors like antitumor drugs, which inhibit cell growth, and growth factors which either cause or prepare the cells for growth. We have found that growth factors increase the plasma membrane electron transport of the normal cells but not that of the cells which have been transfected with oncogenicras (which have a 2–5-fold faster basal rate). In the presence of adriamycin the activity of the electron transport system is and cell numbers are decreased more in normal cells than in the cells which have been transfected with the oncogenic version of the Harveyras gene. Cisplatin inhibits cell growth but does not inhibit ferricyanide reduction. Ferricyanide alone or in conjunction with EGF or TPA does not stimulate cell proliferation with either cell line in the absence of fetal calf serum, so simple activation of plasma membrane electron transport is not sufficient to activate a complete growth response.Abbreviations EGF epidermal growth factor - TPA phorbol myristate acetate - PDGF platelet derived growth factor - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromides - SDS sodium dodecyl sulfate     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

Adriamycin effects of transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbal myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

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.     

Extracellular Calmodulin Stimulates the Transplasma Membrane Redox Reaction of Root Protoplasts in Zea mays

Using ferricyanide as the membrane impermeable electron acceptor, the effects of extracellular calmodulin on transplasma membrane redox reaction of the root protoplasts in Zea mays L. were studied. The calmodulin antagonists (calmidazolium, W7-agarose) and anti-calmodulin serum had inhibitory effect on the extracellular reduction of ferricyanide with their concentration that yielded 50% inhibition were 1.5 μmoL/L, 10 μmol/L and 10 mg/L respectively. Inhibition of calmidazolimn could be restored by calmodulin completely. And the reduction of ferricyanide could be specifically stimulated by the exogerous purified calmodulin. These results suggested that transplasma membrane redox system of root protoplasts in Zea mays L. could be modulated by calmodulin outside the plasma membrane.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

The role of plasma membrane redox activity in light effects in plants

Stimulations by light of electron transport at the plasma membrane make it possible that redox activity is involved in light-induced signal transduction chains. This is especially true in cases where component(s) of the chain are also located at the plasma membrane. Photosynthetic reactions stimulate transplasma membrane redox activity of mesophyll cells. Activity is measured as a reduction of the nonpermeating redox probe, ferricyanide. The stimulation is due to production of a cytosolic electron donor from a substance(s) transported from the chloroplast. It is unknown whether the stimulation of redox activity is a requirement for other photosynthetically stimulated processes at the plasma membrane, but a reduced intermediate may regulate proton excretion by guard cells. Blue light induces an absorbance change (LIAC) at the plasma membrane whose difference spectrum resembles certainb-type cytochromes. This transport of electrons may be due to absorption of light by a flavoprotein. The LIAC has been implicated as an early step in certain blue light-mediated morphogenic events. Unrelated to photosynthesis, blue light also stimulates electron transport at the plasma membrane to ferricyanide. The relationship between LIAC and transmembrane electron flow has not yet been determined, but blue light-regulated proton excretion and/or growth may depend on this electron flow. No conclusions can be drawn regarding any role for phytochrome because of a paucity of information concerning the effects of red light on redox activity at the plasma membrane.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

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.     

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentaammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E ₀) from 305 to –42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.     

Transplasmalemma electron transport is changed in simian virus 40 transformed liver cells

Transplasma membrane electron transport activity by fetal rat liver cells (RLA209-15) infected with a temperature-sensitive strain of SV40 has been measured with cells grown at the restrictive temperature (40°C) and permissive temperature (33°C). The transformed cells grown at 33°C had only one-half the rate of external ferricyanide reduction as the nontransformed cells held at 40°C. Both theK _m andV _max for ferricyanide reduction were changed in the transformed state. The change inV _max can be based on a decrease of NADH in the transformed cells. The change in rate with ferricyanide does not depend on change in surface charge. Reduction of external ferricyanide was accompanied by release of protons from the cells. The ratio of protons released to ferricyanide reduced was higher in the transformed cells than in the non-transformed cells. Since the transplasma membrane electron transport has been shown to stimulate cell growth under limiting serum, the changes in the plasma membrane electron transport and proton release in transformed cells may relate to modification of growth control.     

Properties of a transplasma membrane electron transport system in HeLa cells

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK _m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.     

Diferric transferrin reduction stimulates the Na+/H+ antiport of HeLa cells

Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.