Plasma membrane redox and regulation of cell growth

Summary Gene expression can be activated by external oxidants which are reduced at the cell surface by plasma membrane electron transport. The signals generated in response to the plasma membrane electron transport include activation of proton release, internal calcium changes, and change in reductant/oxidant ratio in the cytosol. H₂O₂ generated in response to ligands which bind to plasma membrane receptors can also activate protein tyrosine kinases and gene expression. Inhibition of oxygen radical generation at the cell surface in response to the mitogen, phorbol myristate acetate by retinoic acid is consistent with a role for the plasma membrane electron transport as the source for H₂O₂ in Balb 3T3 cells. Agents which affect the binding of coenzyme Q to redox sites in the plasma membrane electron transport may increase formation of semiquinone radicals in the membrane which can be a source of oxygen radicals and H₂O₂. The generation of H₂O₂ by transformed cells indicates that oncogene product expression in the plasma membrane may also increase quinone-based oxygen radical generation.     

Plasma membrane coenzyme Q10 and growth control

Summary Coenzyme Q is distributed among cellular membranes and it has a significant concentration at the plasma membrane. The plasma membrane contains a trans-membrane electron transport system, which is centered on coenzyme Q. This molecule is maintained reduced by NAD(P)H-dependent enzymes and can reduce other antioxidants such as tocopheroxyl quinone and ascorbate free radical. Its antioxidant property and its ability to maintain in the reduced state the other antioxidants offers a system to protect membrane components against oxidations and prevents oxidative-stress-dependent cellular damage. Growth factor withdrawal induces cell growth arrest and apoptosis through an oxidative-stress-induced pathway. Coenzyme Q can stimulate growth of different cell lines under serum deficiency, mainly by preventing apoptosis. The protection caused by coenzyme Q is independent of the Bcl-2 protein. Plasma membrane coenzyme Q appears to be essential in the regulation of the redox equilibrium of the cell and redox-dependent pathways.     

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.     

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.     

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q₁₀ and exogenous Q₉ among cell membranes in human HL-60 cells. Endogenous Q₁₀ synthesized using the radiolabelled precursor [¹⁴C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [¹⁴C]-Q₁₀. Brefeldin A prevented Q₁₀ incorporation in plasma membrane. Exogenous Q₉ was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.     

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     

Increased oxidative stress and coenzyme Q10 deficiency in juvenile fibromyalgia: amelioration of hypercholesterolemia and fatigue by ubiquinol-10 supplementation

Abstract

Fibromyalgia (FM) is characterized by generalized pain and chronic fatigue of unknown etiology. To evaluate the role of oxidative stress in this disorder, we measured plasma levels of ubiquinone-10, ubiquinol-10, free cholesterol (FC), cholesterol esters (CE), and free fatty acids (FFA) in patients with juvenile FM (n = 10) and in healthy control subjects (n = 67). Levels of FC and CE were significantly increased in juvenile FM as compared with controls, suggesting the presence of hypercholesterolemia in this disease. However, plasma level of ubiquinol-10 was significantly decreased and the ratio of ubiquinone-10 to total coenzyme Q10 (%CoQ10) was significantly increased in juvenile FM relative to healthy controls, suggesting that FM is associated with coenzyme Q10 deficiency and increased oxidative stress. Moreover, plasma level of FFA was significantly higher and the content of polyunsaturated fatty acids (PUFA) in total FFA was significantly lower in FM than in controls, suggesting increased tissue oxidative damage in juvenile FM. Interestingly, the content of monoenoic acids, such as oleic and palmitoleic acids, was significantly increased in FM relative to controls, probably to compensate for the loss of PUFA. Next, we examined the effect of ubiquinol-10 supplementation (100 mg/day for 12 weeks) in FM patients. This resulted in an increase in coenzyme Q10 levels and a decrease in %CoQ10. No changes were observed in FFA levels or their composition. However, plasma levels of FC and CE significantly decreased and the ratio of FC to CE also significantly decreased, suggesting that ubiquinol-10 supplementation improved cholesterol metabolism. Ubiquinol-10 supplementation also improved chronic fatigue scores as measured by the Chalder Fatigue Scale.     

Effect of mitochondrial dysfunction and oxidative stress on endogenous levels of coenzyme Q(10) in human cells

Little is known about the regulation of endogenous CoQ(10) levels in response to mitochondrial dysfunction or oxidative stress although exogenous CoQ(10) has been extensively used in humans. In this study, we first demonstrated that acute treatment of antimycin A, an inhibitor of mitochondrial complex III, and the absence of mitochondrial DNA suppressed CoQ(10) levels in human 143B cells. Because these two conditions also enhanced formation of reactive oxygen species (ROS), we further investigated whether oxidative stress or mitochondrial dysfunction primarily contributed to the decrease of CoQ(10) levels. Results showed that H(2)O(2) augmented CoQ(10) levels, but carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a chemical uncoupler, decreased CoQ(10) levels in 143B cells. However, H(2)O(2) and FCCP both increased mRNA levels of multiple COQ genes for biosynthesis of CoQ(10) . Our findings suggest that ROS induced CoQ(10) biosynthesis, whereas mitochondrial energy deficiency caused secondary suppression of CoQ(10) levels possibly due to impaired import of COQ proteins into mitochondria.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

Equivalent uptake of organic and inorganic zinc by monkey kidney fibroblasts, human intestinal epithelial cells, or perfused mouse intestine

Zinc (Zn) is recognized as an essential nutrient, and is added as a supplement to animal and human diets. There are claims that zinc methionine (ZnMet) forms a stable complex that is preferentially transported into tissues, and this has contributed to uncertainty about conflicting reports on the bioavailability of various Zn compounds. This study evaluated the cellular and intestinal uptake of inorganic and organic forms of Zn. Steady-state uptake of⁶⁵Zn by human intestine epithelial cells, and monkey kidney fibroblasts was not significantly different with zinc chloride (ZnCl₂), ZnMet, or zinc propionate (ZnProp) (P > 0.05). Uptake of⁶⁵Zn from zinc chelated with EDTA was significantly lower (P < 0.01). In live mice,⁶⁵Zn uptake by perfused intestine and deposition in intestine and liver showed no significant difference between ZnCl₂ and ZnMet. Equimolar [⁶⁵Zn]methionine and zinc[³⁵S]methionine were prepared according to a patented method that yields “ complexed” Zn. Cellular uptake of the radiolabeled methionine was <0.1% of the radiolabeled Zn from these complexes, indicating separate uptake of the Zn and methionine. Gel filtration did not distinguish between⁶⁵Zn in ZnCl₂, ZnProp, or reagent ZnMet, though feed-grade ZnMet containing >10% protein did give a higher-mol-wt form of⁶⁵Zn. Results of this study show equivalent uptake of Zn from inorganic and organic compounds, and support recent feed trials on Zn bioavailability.     

Phenazine methosulfate stimulation of ouabain-sensitive Rb+ uptake by HeLa cells: effects of respiratory inhibitors, anaerobiosis, and ascorbate

phenazine methosulfate (PMS) stimulates ouabain-sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 microM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS-stimulated ouabain-sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na+K-pump activity is closely related to the cellular redox state.     

Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines

We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product.     

Coregulation of electron transport through PS I by Cyt b 6 f,excitation capture by P700 and acceptor side reduction. Time kinetics and electron transport requirement

Regulation of electron transport rate through Photosystem I (PS I) was investigated in intact sunflower leaves. The rate constant of electron donation via the cytochrome b ₆ f complex (k_q, s^–1) was obtained from the postillumination P700⁺ reduction rate, measured as the exponential decay of the light-dark difference (D830) of the 830 nm transmission signal. D830 corresponding to maximum oxidisable P700 (D830_m) was obtained by applying white light flashes of different intensity and extrapolating the plot of the quantum yield Y vs. D830 to the axis of abscissae (Y->0). Maximum quantum yield of PS I at completely reduced P700 (Y_m) was obtained by extrapolating the same plot to the axis of ordinates (D830->0). Regulation of k_q, D830_m and Y_m under rate-limiting CO₂ and O₂ concentrations applied after air (21% O₂, 310 ppm CO₂) was investigated. The amplitude of the downregulation of k_q (photosynthetic control) was maximal when electron transport rate (ETR) was limited to about 3 nmol cm^–2 s^–1 and decreased when ETR was higher or lower. Downregulation did not occur in the absence of CO₂ and O₂. These gases acted only as substrates of ribulosebisphosphate carboxylase-oxygenase, no high-affinity reaction of O₂ leading to enhanced photosynthetic control (e.g. Mehler reaction) was detected. After the transition, D830_m at first decreased and then increased again, showing that the reduction of the PS I acceptor side disappeared as a result of the downregulation of k_q. The variation of Y_m had two reasons, PS I acceptor side reduction and variable excitation capture efficiency by P700. It is concluded that electron transport through PS I is coregulated by the rate of plastoquinol oxidation at Cyt b ₆ f, excitation capture efficiency by P700, and by acceptor side reduction.Abbreviations Cyt b ₆ f cytochrome b ₆ f complex - D830 difference of the 830 nm signal from the dark level - ETR electron transport rate - PAD photon absorption density nmol cm^–2 s^–1 - PFD incident photon flux density, nmol cm^–2 s^–1 - PS I Photosystem I - PS II Photosystem II - PQH₂ plastoquinol - P700 Photosystem I donor pigment - Y quantum yield of PS I electron transport, rel. un.     

Evidence for coordinate, selective regulation of eicosanoid synthesis in platelet-derived growth factor-stimulated 3T3 fibroblasts and in HL-60 cells induced to differentiate into macrophages or neutrophils

We used Swiss 3T3 fibroblasts stimulated with platelet-derived growth factor and HL-60 cells induced to differentiate into macrophages or neutrophils to study the regulation of prostaglandin and leukotriene synthesis. Addition of platelet-derived growth factor to quiescent 3T3 fibroblasts led within 4 h to a dramatic and preferential increase in prostacyclin synthesis from endoperoxide prostaglandin H2, and microsomal assays showed a strong platelet-derived growth factor-dependent increase in the maximal velocities (Vmax) of both prostaglandin H synthase and prostacyclin synthase. In contrast, addition of phorbol ester to HL-60 cells to induce differentiation into macrophages led within 4 h to a strong and preferential increase in thromboxane synthesis from prostaglandin H2, and microsomal assays disclosed a major rise in Vmax for both prostaglandin H synthase and thromboxane synthase. No comparable changes occurred in HL-60 cells that were differentiating into neutrophils, though upregulation of 5-lipoxygenase pathway enzymes occurred in both differentiation systems. Actinomycin D and cycloheximide prevented the appearance of all of these enzymes of eicosanoid synthesis in all three model systems. Thus, the distinctive patterns of eicosanoid synthesis that are seen in replicating fibroblasts and in differentiating macrophages and neutrophils appear to depend on a coordinate, selective upregulation of several enzymes of eicosanoid biosynthesis that is specific for each cell system.     

Ca2+-dependent stimulation of hexose transport by A23187, 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor in mouse fibroblasts

Ca2+ ionophore A23187 stimulated 2-deoxy-D-glucose (2DG) uptake in Swiss 3T3 mouse fibroblasts. Chelation of extracellular Ca2+ with ethylene-glycol-bis-(beta-aminoethylether) N,N'-tetraacetic acid (EGTA) inhibited the effect of A23187. Similarly, the stimulation of 2DG uptake by a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was prevented by EGTA, whereas the epidermal growth factor (EGF)-stimulated 2DG uptake was not affected by EGTA alone, but in the presence of both EGTA and A23187 which effectively depleted cellular Ca2+ content, EGF could no longer stimulate 2DG uptake. These results suggest that Ca2+ regulates hexose transport system in Swiss 3T3 mouse fibroblasts, the activation of which by TPA and EGF differently depends on Ca2+.     

Mild thermotolerance induced at 40 °C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 °C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 °C, 3 h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H₂O₂. Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and γ-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H₂O₂. This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H₂O₂-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.     

Mechanism of cell death induced by an antioxidant extract of Cratoxylum cochinchinense (YCT) in Jurkat T cells: the role of reactive oxygen species and calcium

YCT is a semipurified extract from Cratoxylum cochinchinense that has antioxidant properties and contains mostly mangiferin. We show here that YCT is selectively toxic to certain cell types and investigate the mechanisms of this toxicity in Jurkat T cells. By flow cytometric analyses, we show that YCT causes intense oxidative stress and a rise in cytosolic Ca²⁺. This is followed by a rise in mitochondrial Ca²⁺, release of cytochrome c, collapse of Δψ_m, a fall in ATP levels, and eventually cell death. The mechanism(s) of intense oxidative stress may involve a plasma membrane redox system, as cell death is inhibited by potassium ferricyanide. Cell death has some features of apoptosis (propidium iodide staining, externalization of phosphatidylserine, limited caspase-3 and -9 activities), but there was no internucleosomal DNA fragmentation.