Reactive oxygen species generation at the plasma membrane for antibody control.

Generation of reactive oxygen species (ROS) at the plasma membrane can be a vehicle for oxidative unmasking or masking of auto antibodies in a tissue selective and controlled way. There are seven related NADPH oxidases (NOX 1-5, DuoNOX 1,2) which can be activated in various ways to produce superoxide and hydrogen peroxide at the plasma membrane. There is also a plasma membrane NADH oxidase which is under different control. ROS can also be produced by mitochondria or cytosolic oxidases under special conditions. The ROS generation provides oxidant for thiol oxidation or peroxynitrite formation which can be a basis for antibody modification. The specific controls of the oxidases in different tissues allow a basis for localized autoantibody modification in response to stress or environment.

Effect of analogs with modified prenyl side chains on growth of serum deficient HL60 cells.

This study was organized by Professor Karl Folkers with the objective of finding derivatives of coenzyme Q which could be more effectively absorbed and would give better biomedical effects. In this series all the compounds are 2,3 dimethoxy, 5 methyl p benzoquinone with modified side chains in the 6 position. The modifications are primarily changes in chain length, unsaturation, methyl groups and addition of terminal phenyl groups. The test system evaluates the growth of serum deficient HL60, 3T3 and HeLa cells in the presence of coenzyme Q10 or coenzyme Q analogs. Short chain coenzyme Q homologues such as coenzyme Q2 give poor growth but compounds with saturated short aliphatic side chains from C10 to C18 produce good growth. Introduction of a single double bond at the 2' or 8' position in the aliphatic chain retains growth stimulation at low concentration but introduces inhibition at higher concentration. Introduction of a 3' methyl group in addition to the 2' enyl site in the side chain decreases the growth response and maintains inhibition. Addition of a terminal phenyl group to the side chain from C5 to C10 can produce analogs which give strong stimulation or strong inhibition of growth. The action of the analogs is in addition to the natural coenzyme Q in the cell and is not based on restoration of activity after depletion of normal coenzyme Q. The effects may be based on any of the sites in the cell where coenzyme Q functions. For example, coenzyme Q2 is known to decrease mitochondrial membrane potential whereas the analog with a 10C aliphatic side chain increases potential. Both of these compounds stimulate plasma membrane electron transport. Inhibition of apoptosis by coenzyme Q may also increase net cell proliferation and the 10C analog inhibits the permeability transition pore.

Biochemical functions of coenzyme Q10.

Coenzyme Q is well defined as a crucial component of the oxidative phosphorylation process in mitochondria which converts the energy in carbohydrates and fatty acids into ATP to drive cellular machinery and synthesis. New roles for coenzyme Q in other cellular functions are only becoming recognized. The new aspects have developed from the recognition that coenzyme Q can undergo oxidation/reduction reactions in other cell membranes such as lysosomes. Golgi or plasma membranes. In mitochondria and lysosomes, coenzyme Q undergoes reduction/oxidation cycles during which it transfers protons across the membrane to form a proton gradient. The presence of high concentrations of quinol in all membranes provides a basis for antioxidant action either by direct reaction with radicals or by regeneration of tocopherol and ascorbate. Evidence for a function in redox control of cell signaling and gene expression is developing from studies on coenzyme Q stimulation of cell growth, inhibition of apoptosis, control of thiol groups, formation of hydrogen peroxide and control of membrane channels. Deficiency of coenzyme Q has been described based on failure of biosynthesis caused by gene mutation, inhibition of biosynthesis by HMG coA reductase inhibitors (statins) or for unknown reasons in ageing and cancer. Correction of deficiency requires supplementation with higher levels of coenzyme Q than are available in the diet.

Interactions between ascorbyl free radical and coenzyme Q at the plasma membrane.

A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has been recently recognized. The aim of this work was to study the interactions between reduced ubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understand ubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbate and decreased the steady-state levels of the semiascorbyl radical. The ability of cells to reduce ascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine and stimulated by supplementing cells with coenzyme Q10. Purified plasma membranes also reduced ascorbyl free radical in the presence of NADH. Free-radical reduction was not observed in quinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q10. Addition of reduced coenzyme Q10 to depleted membranes allowed them to reduce the signal of the ascorbyl free radical without NADH incubation and the addition of an extra amount of purified plasma membrane quinone reductase further stimulated this activity. Reduction was abolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blocking surface glycoconjugates with the lectin wheat germ agglutinin, which supports the participation of transmembrane electron flow. The activity showed saturation kinetics by NADH and coenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our results support that reduction of ascorbyl free radicals at the cell surface involves coenzyme Q reduction by NADH and the membrane-mediated reduction of ascorbyl free radical.

Coenzyme Q6 and iron reduction are responsible for the extracellular ascorbate stabilization at the plasma membrane of Saccharomyces cerevisiae.

Yeast plasma membrane contains an electron transport system that maintains ascorbate in its reduced form in the apoplast. Reduction of ascorbate free radical by this system is comprised of two activities, one of them dependent on coenzyme Q6 (CoQ6). Strains with defects in CoQ6 synthesis exhibit decreased capacity for ascorbate stabilization compared with wild type or with atp2 or cor1 respiratory-deficient mutant strains. Both CoQ6 content in plasma membranes and ascorbate stabilization were increased during log phase growth. The addition of exogenous CoQ6 to whole cells resulted in its incorporation in the plasma membrane, produced levels of CoQ6 in the coq3 mutant strain that were 2-fold higher than in the wild type, and increased ascorbate stabilization activity in both strains, although it was higher in the coq3 mutant than in wild type. Other antioxidants, such as benzoquinone or alpha-tocopherol, did not change ascorbate stabilization. The CoQ6-independent reduction of ascorbate free radical was not due to copper uptake, pH changes or to the presence of CoQ6 biosynthetic intermediates, but decreased to undetectable levels when coq3 mutant strains were cultured in media supplemented with ferric iron. Plasma membrane CoQ6 levels were unchanged by either the presence or absence of iron in wild type, atp2, or cor1 strains. Ascorbate stabilization appears to be a function of the yeast plasma membrane, which is partially based on an electron transfer chain in which CoQ6 is the central electron carrier, whereas the remainder is independent of CoQ6 and other antioxidants but is dependent on the iron-regulated ferric reductase complex.

Genetic evidence for coenzyme Q requirement in plasma membrane electron transport.

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3delta, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH-ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH-ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3delta strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3delta strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3-14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3delta strain. Ascorbate free radical reductase was not altered significantly in mutants atp2delta and cor1delta which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3delta mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH-ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

Ascorbate and alpha-tocopherol prevent apoptosis induced by serum removal independent of Bcl-2.

Cells require serum to maintain growth in vitro. Serum provides growth and survival factors and its removal causes an oxidative stress that induces peroxidations in membrane lipids and development of programmed cell death (apoptosis) in some cells. Cells containing Bcl-2 are partially protected against both lipid peroxidation and apoptosis and some cell lines, such as Daudi, which lack this protein, are very sensitive to serum removal. Thus, cells are grown for 48 h in the absence of fetal calf serum and apoptotic cells are scored. HL-60 cells containing a moderate amount of Bcl-2 show 30% apoptosis, while 55% cells are apoptotic of the Bcl-2-negative Daudi cell population. Apoptosis is reduced to 15% in the transiently transfected Daudi/Bcl-2 cells. Ascorbate (Asc) and alpha-tocopherol (alphaTOH) can prevent lipid peroxidation and apoptosis caused by serum withdrawal, when added to culture media, even in the absence of Bcl-2. Also, these two antioxidants increase survival of cells grown in the absence of serum independent of their Bcl-2 content. Immunostaining and quantification of Bcl-2 show that HL-60 cell line is a heterogeneous population relative to the expression of Bcl-2. When these cells are grown in the presence of serum, cells lacking Bcl-2 survive, but no Bcl-2-negative cells survive without serum. Part of this population of Bcl-2-negative cells is rescued by Asc and alphaTOH. Antioxidants effective at the plasma membrane such as Asc and alphaTOH can protect cells from oxidative damage and prevent apoptosis independent of Bcl-2 content.

Antioxidant ascorbate is stabilized by NADH-coenzyme Q10 reductase in the plasma membrane.

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ10 and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ10 reductase activity and its internal sequence being identical to cytochrome b5 reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ10 content in isolated plasma membranes. We show here the role of CoQ10 and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b5-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ10 and its NADH-dependent reductase.

Ascorbate stabilization is stimulated in rho(0)HL-60 cells by CoQ10 increase at the plasma membrane.

Long-term treatment with ethidium bromide of HL-60 cells induced a mitochondria-deficient rho degree cell line, where mitochondrial DNA can not be identified by PCR and cytochrome c oxidase activity was 80% decreased. These cells showed a progressive increase of ascorbate stabilization which was 52% higher in the established rho degree HL-60 cells. Both CoQ10 and NADH-ascorbate free radical reductase of the plasma membrane were increased in rho(0)HL-60 cells compared to parental cells, while NADH-cytochrome c reductase was unchanged. CoQ10 is a component of the ascorbate stabilization activity in the plasma membrane that would provide both a mechanism to deplete the excess of NADH produced in rho(0)HL-60 cells and for resistance to oxidative stress.

Inhibition of DNA synthesis in CCL 39 cells by impermeable iron chelators.

The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.

The diversity of coenzyme Q function.

Coenzyme Q is uniquely designed as an electron and proton carrier within the lipid phase of membranes. It now appears that this unique chemistry has diverse application to important functions in all cellular membranes. The first function of coenzyme Q was defined in the energy transduction process in mitochondria. New studies show that the presence of coenzyme Q in other cellular membranes has dynamic rather than passive significance. Coenzyme Q functions in the plasma membrane electron transport involved in activation of signalling protein kinases related to gene activation for cellular proliferation. Furthermore, the antioxidant potential of the reduced coenzyme Q is now taken on a new significance in the evidence that the reduced quinone can act to maintain tocopherol in the reduced state in membranes and ascorbate reduced both inside and outside the cell.

Cytokine inhibition of transplasma membrane election transport.

Two cytokines, interferon gamma and tumor necrosis factor alpha, which can inhibit cell proliferation or induce cell death, have been found to inhibit transplasma membrane electron transport. The concentrations required for inhibition of election transport are similar to concentrations effective in inhibition of cell growth. Since inhibition of election transport has been related to apoptosis and modification of election transport can cause oxygen radical formation, the changes in electron transport induced by the cytokines can contribute to known mechanisms of cytokine cytotoxicity.

Extracellular ascorbate stabilization as a result of transplasma electron transfer in Saccharomyces cerevisiae.

The presence of yeast cells in the incubation medium prevents the oxidation of ascrobate catalyzed by copper ions. Ethanol increases ascorbate retention. Pyrazole, an alcohol dehydrogenase inhibitor, prevents ascorbate stabilization by cells. Chelation of copper ions does not account for stabilization, since oxidation rates with broken or boiled cells or conditioned media are similar to control rates in the absence of cells. Protoplast integrity is needed to reach optimal values of stabilization. Chloroquine, a known inhibitor of plasma membrane redox systems, inhibits the ascorbate stabilization, the inhibition being partially reversed by coenzyme Q6. Chloroquine does not inhibit ferricyanide reduction. Growth of yeast in iron-deficient media to increase ferric ion reductase activity also increases the stabilization. In conclusion, extracellular ascorbate stabilization by yeast cells can reflect a coenzyme Q dependent transplasmalemma electron transfer which uses NADH as electron donor. Iron deficiency increases the ascorbate stabilization but the transmembrane ferricyanide reduction system can act independently of ascorbate stabilization.

Determination of structure-activity relationships of Annonaceous acetogenins by inhibition of oxygen uptake in rat liver mitochondria.

A new group of natural compounds, the Annonaceous acetogenins, have recently been determined to inhibit ATP production at a similar site of action and higher levels of potency as rotenone, i.e., at NADH-ubiquinone oxido-reductase, complex I of the mitochondrial electron-transport chain. The acetogenins had earlier been determined to be pesticidal, antimalarial, antimicrobial, anti-parasitic, cytotoxic, and in vivo active as potentially new antitumor agents. In order to determine structural activity relationships (SARs) among these compounds, at the subcellular level, several available acetogenins have been tested. Data obtained, from the inhibition of oxygen consumption by rat liver mitochondria, demonstrated that all of the twenty acetogenins tested are active with IC50 values in the range of 15-800 nM/mg protein. The IC50 value of rotenone was 17 nM/mg protein. The bis-adjacent THF ring acetogenins and the bis-nonadjacent THF ring compounds are about ten times more active than the mono-THF ring acetogenins. Overall, 30-OH and 31-OH-bullatacinone were the most active and were slightly more active than rotenone. The least active were the 4-deoxy bis-adjacent THF ring compounds followed by the mono-THF ring group. There was some variation between the groups, e.g., within the bis-adjacent and mono-THF ring groups, the alpha, beta-unsaturated-gamma-lactones were less active than the keto-lactones, but this observation was reversed for one of the pairs of bis-nonadjacent THF ring acetogenins. Additional hydroxylations, to a maximum of three, seemed to increase activity within all of the groups. Before final decisions on SARs can be made, additional comparisons of the results of this subcellular assay (as an in vitro assay) with the results of in vivo assays should be made. Also, future investigations into the exact site of action within complex I and other possible sites of action (such as the NADH oxidase of plasma membranes) need to be conducted for a more. complete understanding of the utility and potential of this new group of very potent compounds.

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane.

A 34 kDa coenzyme Q reductase has been solubilized and purified from pig liver plasma membranes. The solubilized enzyme reduced coenzyme Q0 with NADH. Ubiquinones with longer isoprenoid side chain such as Q2 and Q10 were also reduced when the quinones and the enzyme were reconstituted into phospholipid liposomes. N-terminal sequencing of an internal peptide showed identity to bovine NADH-cytochrome b5 reductase. Biochemical characterization of the purified enzyme indicated that the coenzyme Q reductase corresponds to an unusual form of NADH-cytochrome b5 reductase.

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.

Iron chelators hydroxyurea and bathophenanthroline disulfonate inhibit DNA synthesis by different pathways.

We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.