H. Löw

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.

Coenzyme Q10, plasma membrane oxidase and growth control

The plasma membrane of eukaryotic cells contains an NADH oxidase which can transfer electrons across the membrane. This oxidase is controlled by hormones, growth factors and other ligands which bind to receptors in the plasma membrane. Oncogenes also affect activity of the oxidase. Natural serum components such as diferric transferrin and ceruloplasmin which stimulate proliferation also stimulate membrane oxidase activity. Additional growth factors can be required to complement the proliferative effect. Electron transport across the plasma membrane can be measured by the reduction of impermeable electron acceptors, such as ferricyanide, which also stimulate cell growth. The oxidants activate growth-related signals such as cytosolic alkalinization and calcium mobilization. Antiproliferative agents such as adriamycin and retinoic acid inhibit the plasma membrane electron transport. Flavin, Coenzyme Q and an iron chelate on the cell surface are apparent electron carriers for the transmembrane electron transport. Coenzyme Q10 stimulates cell growth, and Coenzyme Q analogs such as capsaicin and chloroquine reversibly inhibit both growth and transmembrane electron transport. Addition of iron salts to the depleted cells restores activity and growth. The ligand-activated oxidase in the plasma membrane introduces a new basis for control of signal transduction in cells. The redox state of the quinone in the oxidase is proposed to control tyrosine kinase either by generation of H2O2 or redox-induced conformational change.

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 apparentKm 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.

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.

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.

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.

Transmembrane redox in control of cell growth Stimulation of HeLa cell growth by ferricyanide and insulin

The impermeable electron acceptor ferricyanide stimulates the growth of HeLa cells in the absence of serum and increases cell replication with limiting amounts of serum (0.75%). Maximum growth stimulation occurs at low ferricyanide concentration from 0.01 to 0.1 mM. Higher ferricyanide concentrations inhibit growth on serum. Addition of insulin enhances the stimulating effect of ferricyanide. Increase in the transplasmalemma electron transport activity in the presence of insulin is also observed by measuring the rate of ferricyanide reduction by cells. There is a close correlation between insulin stimulation of ferricyanide reduction and insulin induction of cell proliferation and attachment. In addition to ferricyanide, the growth response is observed with other impermeable oxidants, such as indigotetrasulfonate and hexaamine ruthenium III, which are reduced by the transplasma membrane electron transport system. Inactive oxidants such as cytochrome c do not stimulate cell growth. Ferrocyanide does not stimulate growth. We propose that electron flow through the transplasma membrane electron transport system stimulates growth and that insulin acts to increase that flow.

A transmembranous NADH-dehydrogenase in human erythrocyte membranes

Evidence is presented for a transmembranous NADH-dehydrogenase in human erythrocyte plasma membrane. We suggest that this enzyme is responsible for the ferricyanide reduction by intact cells. This NADH-dehydrogenase is distinctly different from the NADH-cytochromeb5 reductase on the cytoplasmic side of the membrane. Pretreatment of erythrocytes with the nonpenetrating inhibitor diazobenzene sulfonate (DABS) results in a 35% loss of NADH-ferricyanide reductase activity in the isolated plasma membrane. Since NADH and ferricyanide are both impermeable, the transmembrane enzyme can only be assayed in open membrane sheets with both surfaces exposed, and not in closed vesicles. The transmembrane dehydrogenase has affinity constants of 90 µM for NADH and 125 µM for ferricyanide. It is inhibited byp-chloromercuribenzoate, bathophenanthroline sulfonate, and chlorpromazine.

Transformed liver cells have modified transplasma membrane redox activity which is sensitive to adriamycin

Electron transport across the plasma membrane is found in all cells which have been tested. This activity has been implicated in control of cellular growth, transport and hormone response. In virus transformed cells and tumor cells we find the activity is decreased and becomes sensitive to the antitumor drug adriamycin. Inhibition of transmembrane redox by adriamycin parallels cytoxicity to transformed cells.

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.