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At least 613 records · Page 34Linked to original sources
PubMed1982

A role for 2,4-enoyl-CoA reductase in mitochondrial beta-oxidation of polyunsaturated fatty acids. Effects of treatment with clofibrate on oxidation of polyunsaturated acylcarnitines by isolated rat liver mitochondria.

1. beta-Oxidation of gamma-linolenoylcarnitine, arachidonoylcarnitine and docosahexaenoylcarnitine by isolated rat liver mitochondria is inhibited by uncoupling conditions. Partial re-activation is obtained with added ATP. With mitochondria from clofibrate-treated rats ATP-stimulated rates of beta-oxidation of docosahexaenoylcarnitine are higher than ADP-stimulated rates. This is not observed with the beta-oxidation of oleoylcarnitine. 2. beta-Oxidation of docosahexaenoylcarnitine, in the presence of rotenone, is inhibited by added oxaloacetate, analogous to previous findings with pent-4-enoylcarnitine [see Osmundsen (1978) FEBS Lett. 88, 219-222]. In the absence of rotenone added oxaloacetate stimulates the beta-oxidation of docosahexaenoylcarnitine, but has the opposite effect on the beta-oxidation of palmitoylcarnitine. 3. beta-Oxidation of polyunsaturated acylcarnitines by isolated rat liver mitochondria is selectively increased after treatment of the animals with a low dietary dose (0.2%, w/w) of clofibrate. Treatment with a higher dose of clofibrate (0.5%, w/w) resulted in a general stimulation of beta-oxidation. 4. The results presented suggest that long-chain fatty acids possessing a delta 4-double bond are not readily beta-oxidized unless the 2,4-enoyl-CoA reductase (EC 1.3.1.-) is operating.

2,4-Dinitrophenol↗
PubMed1983

The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.

2,4-Dinitrophenol↗
PubMed1990

Environmentally induced differential amplification of mitochondrial populations.

Resistance to the drug rutamycin, an inhibitor of mitochondrial ATPase, has been shown to be cytoplasmically inherited in a mouse fibroblast line (TL) on fusion of the cytoplast (enTL) with a nucleated recipient A9 [Lichtor & Getz (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 324-328]. The cytoplasmic hybrid (cybrid) so formed may be readily grown in the presence [CY(+)] or absence [CY(-)] of rutamycin. The ATPase of TL mitochondria is similarly resistant to rutamycin whether grown in the presence or absence of antibiotic. The ATPase of CY(+) mitochondria is resistant to rutamycin, but CY(-) mitochondrial ATPase is sensitive to rutamycin. Nevertheless, CY(-) can be readily grown in rutamycin after a brief lag. The pH optima of mitochondrial ATPase are 8.0 for A9 and CY(-) cells and 7.5 for TL cells, whereas the pH optimum for CY(+) spans the optima of A9 and TL. The TL mitochondrial NADH-cytochrome c reductase is resistant to rotenone, whereas that of A9 mitochondria is sensitive to this agent. CY(-) and CY(+) mitochondria are sensitive and resistant respectively to rotenone. Growth of cybrids in rutamycin for 2 weeks results in a 2-3-fold increase in mitochondrial mass, measured on the basis of electron microscopic morphometry, mitochondrial membrane enzyme assays, mass of cardiolipin, and quantification of mitochondrial DNA. These data suggest that the cybrid harbours two populations of mitochondria and that the proportions of the two populations dramatically influence morphology, growth and mitochondrial phenotype in the cybrid. Selective pressure appears to induce these changes through the differential amplification of mitochondria.

Adenosine Triphosphatases↗
PubMed1996

The specificity of mitochondrial complex I for ubiquinones.

We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.

Animals↗
PubMed2001

Light-dependent gene expression for proteins in the respiratory chain of potato leaves.

Expression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L. cv. Desiree) leaves. The recently characterized nda1 and ndb1 genes, homologues to genes encoding the non-proton pumping respiratory chain NADH-dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone-insensitive NADH oxidation. The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged. A variation in transcript level, specific for nda1, is seen at different times of the day with highest expression in the morning. This variation also influences the apparent developmental induction. Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re-induction when plants are returned to light. Corresponding immunodetected NDA protein is specifically decreased in mitochondria isolated from dark-treated plants, accompanied by a lower capacity for internal rotenone-insensitive NADH oxidation. Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins. Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence. In addition to the specific change in nda1, a general, slower down-regulation in darkness was seen for the other NAD(P)H dehydrogenase genes. The nda1 expression during development, and in response to light, indicates a specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.

Aging↗
PubMed2000

Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures.

Previous research has implicated mitochondrial physiology and, by extension, respiratory capacity in the initiation and progress of apoptosis of cells in culture and tissue environments. This hypothesis was tested by separating a hybridoma cell population into subpopulations of varying mitochondrial membrane potential (MMP) using Rhodamine 123 stain and fluorescence-activated cell sorter analysis and subjecting them to two apoptosis inducers, rotenone and staurosporin. Apoptotic death was characterized morphologically through the determination of apoptosis-related chromatin condensation and biochemically through the measurement of caspase-3 enzymatic activity. We found dramatic differences in the apoptotic death kinetics for the subpopulations, with the high MMP cells showing higher resistance to apoptotic death. After incubation with 30 microM rotenone, the low MMP cells exhibited one-third of the viability of the high MMP cells and a three-fold increase in the capsase-3 enzymatic activity. No changes were observed in the DNA content or the cell cycle distributions of the two cell subpopulations, which maintained their mean MMP difference after 20 generations. These results suggest that heterogeneity exists in mammalian cell populations with respect to mitochondrial physiology, which correlates with resistance to apoptotic death.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Endogenous and exogenous nitric oxide enhance the DNA strand scission induced by tert-butylhydroperoxide in PC12 cells via peroxynitrite-dependent and independent mechanisms, respectively.

A short-term exposure to tert-butylhydroperoxide (tB-OOH) promoted a concentration-dependent formation of DNA single-strand breaks in PC12 cells. These events were paralleled by an increase in the cytosolic concentration of Ca2+ that was in part cleared by the mitochondria. Unlike the extent of Ca2+ mobilization and/or mitochondrial Ca2+ clearance, the DNA strand scission evoked by the hydroperoxide was markedly reduced by the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO) or by the NO synthase inhibitor N-nitro-L-arginine methylester (L-NAME). Inhibitors of electron transport (rotenone and myxothiazol), ruthenium red (RR, a polycation which inhibits the calcium uniporter of mitochondria), or peroxynitrite scavengers (Trolox and L-methionine) were as effective as PTIO or L-NAME in inhibiting the DNA-damaging response mediated by tB-OOH. Rotenone, RR or peroxynitrite scavengers did not further reduce the residual DNA cleavage observed following treatment with tB-OOH in L-NAME-supplemented cells. Exogenous NO also increased the DNA damage caused by tB-OOH in L-NAME-supplemented cells and this response was blunted by RR or by inhibitors of electron transport but was insensitive to peroxynitrite scavengers. We conclude that both endogenous and exogenous NO enhance the DNA cleavage generated by tB-OOH in PC12 cells. However, only endogenous NO set the bases for an involvement of peroxynitrite in this DNA-damaging response.

Animals↗
PubMed1995

The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes.

The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK-21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [P(i)] ([ATP]i, [ADP]i, and [P(i)]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [P(i)], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] and lower [glucose-6-phosphate] and [fructose-6-phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [P(i)] rose under all pHe conditions. Simultaneously [glucose-6-phosphate] and [fructose-6-phosphate] decreased whereas [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1999

Effect of ATP depletion on the palmitoylation of myelin proteolipid protein in young and adult rats.

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.

Acylation↗
PubMed2001

Mitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: relevance to Parkinson's disease.

Deficiency of complex I in the respiratory chain and oxidative stress induced by hydrogen peroxide occur simultaneously in dopaminergic neurones in Parkinson's disease. Here we demonstrate that the membrane potential of in situ mitochondria (Delta Psi m), as measured by the fluorescence change of JC-l (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbezimidazolyl-carbocyani ne iodide), collapses when isolated nerve terminals are exposed to hydrogen peroxide (H(2)O(2), 100 and 500 microM) in combination with the inhibition of complex I by rotenone (5 nM-1 microM). H(2)O(2) reduced the activity of complex I by 17%, and the effect of H(2)O(2) and rotenone on the enzyme was found to be additive. A decrease in Delta Psi m induced by H(2)O(2) was significant when the activity of complex I was reduced to a similar extent as found in Parkinson's disease (26%). The loss of Delta Psi m observed in the combined presence of complex I deficiency and H(2)O(2) indicates that when complex I is partially inhibited, mitochondria in nerve terminals become more vulnerable to H(2)O(2)-induced oxidative stress. This mechanism could be crucial in the development of bioenergetic failure in Parkinson's disease.

Animals↗
PubMed2003

1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3.

1-Methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces apoptosis in cerebellar granule neurons (CGNs). We have tested the hypothesis that organic cation transporter (OCT) 3 mediates the accumulation and, hence, the toxicity of MPP+ in CGNs. CGNs in primary culture express OCT3 but do not express mRNA for OCT1, OCT2 or the dopamine transporter. Cerebellar astrocytes are negative for OCT3 protein by immunocytochemistry. [3H]MPP+ accumulation by CGNs exhibits first-order kinetics, and a Kt value of 5.3 +/- 1.2 micro m and a Tmax of 0.32 +/- 0.02 pmol per min per 106 cells. [3H]MPP+ accumulation is inhibited by corticosterone, beta-estradiol and decynium 22 with Ki values of 0.25 micro m, 0.17 micro m and 4.0 nm respectively. [3H]MPP+ accumulation is also inhibited by desipramine, dopamine, serotonin and norepinephrine, but is not affected by carnitine (10 mm), mazindol (9 micro m) or GBR 12909 (1 micro m). MPP+-induced caspase-3-like activation and cell death are prevented by pretreatment with 5 micro mbeta-estradiol. In contrast, the neurotoxic effects of rotenone are unaffected by beta-estradiol. Interestingly, GBR 12909 protects CGNs from both MPP+ and rotenone toxicity. In summary, CGNs accumulate MPP+ in manner that is consistent with uptake via OCT3 and the presence of this protein in CGNs explains their sensitivity to MPP+ toxicity.

1-Methyl-4-phenylpyridinium↗
PubMed1999

Sodium arsenate induces overproduction of interleukin-1alpha in murine keratinocytes: role of mitochondria.

It has recently been demonstrated that arsenic induces overexpression of keratinocyte-derived growth factors, which are likely to have a significant role in arsenic-induced skin hyperkeratoses and cancer. The mechanism(s) involved in this induction are, however, still elusive. The purpose of this study was to investigate the early intracellular events that follow in vitro treatment with sodium arsenate in a murine keratinocyte cell line (HEL30), which leads to cytokine overproduction. First, we observed that sodium arsenate induced a concentration-dependent production of interleukin-1alpha and a significant increase in cell proliferation, that could be suppressed by the addition of a neutralizing antibody against murine interleukin-1alpha, confirming the ability of arsenic to induce keratinocyte growth-promoting cytokines. Electron microscopic analysis revealed that arsenate induced a dramatic alteration in keratinocyte mitochondria. This effect could be prevented by rotenone pretreatment, which suggests the possible involvement of mitochondria-derived reactive oxygen species. Arsenic induced a concentration- and time-dependent increase in cellular oxidative activity, which was followed by activation of redox-sensitive transcription factors such as nuclear factor-kappaB and activator protein-1, that are essential for interleukin-1alpha synthesis. Prior treatment with rotenone or prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, to deplete cells of functional mitochondria, completely prevented sodium arsenate-induced interleukin-1alpha production, this indicates the pivotal role of these organelles in sodium arsenate-induced keratinocyte growth factors.

Animals↗
PubMed1977

Unexpected additional mode of energization of amino-acid transport into Ehrlich cells.

Ehrlich cells treated with dinitrophenol and iodoacetate rapidly recover their 30-sec uptake of 2-(methyl-amino)-isobutyrate on treatment with 0.1 mM phenazine methosulfate + 20 mM sodium ascorbate before they begin to recover from the severely depressed ATP levels and alkali-ion gradients. Addition of 10 mM pyruvate also restores uptake of methylaminoisobutyrate before the alkali-ion gradients rise. This restoration is prevented by rotenone, but rotenone does not handicap restoration by phenazine methosulfate/ascorbate. Na+-independent uptake of 2-aminonorbornane-2-carboxylate by Ehrlich cells is affected the same way. Quinacrine almost completely suppresses uptake of methylaminoisobutyrate within the 30-sec uptake test, even when ATP levels are sustained by pyruvate and alkali-ion gradients are not depressed. Ouabain prevents restoration of both Na+-dependent and Na+-independent amino-acid transport by phenazine methosulfate/ascorbate or pyruvate. We interpret these results to indicate that amino-acid transport can be energized not only by known means, but also by reducing equivalents, which presumably reach the plasma membrane in the form of NADH from the mitochondria when the source of energy is pyruvate. In support of this hypothesis, the distribution of methylaminoisobutyrate between plasma membrane vesicles and their supporting media was influenced in the predictable way by NADH, quinacrine, and an uncoupling agent, proceeding on the assumption that more of the vesicles had the everted rather than the natural orientation.

Adenosine Triphosphate↗
PubMed1997

The content of intracellular mitochondrial DNA is decreased by 1-methyl-4-phenylpyridinium ion (MPP+).

1-Methyl-4-phenylpyridinium ion (MPP+), an oxidative metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is considered to be directly responsible for MPTP-induced Parkinson's disease-like symptoms by inhibiting NADH-ubiquinone oxidoreductase (complex I) in the mitochondrial respiratory chain. Here we demonstrate that 25 microM MPP+ decreases the content of mitochondrial DNA to about one-third in HeLa S3 cells. On the contrary, 0.1 microM rotenone, which inhibits complex I to the same extent as 25 microM MPP+ in the cells, increases the content of mitochondrial DNA about 2-fold. Hence, the effect of MPP+ on mitochondrial DNA is not mediated by the inhibition of complex I. To examine the replication state of mitochondrial DNA, we measured the amount of nascent strands of mitochondrial DNA. The amount is decreased by MPP+ but increased by rotenone, suggesting that the replication of mitochondrial DNA is inhibited by MPP+. Because the proper amount of mitochondrial DNA is essential to maintain components of the respiratory chain, the decrease of mitochondrial DNA may play a role in the progression of MPTP-induced Parkinson's disease-like symptoms caused by the mitochondrial respiratory failure.

1-Methyl-4-phenylpyridinium↗
PubMed1998

Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency.

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (rho degrees), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characterization of oxidative phosphorylation function in these cells. Mitochondrial complexes II, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as limiting substrates showed that the Vmax was decreased in HXC by approximately 40%, and the Km for the NADH was significantly increased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced a mild effect in control cells (0-10% inhibition) but produced a marked inhibition of HXC respiration (50-75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their relative amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.

Animals↗
PubMed2001

Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form.

Alpha-synuclein exists as at least two structural isoforms: a helix-rich, membrane-bound form and a disordered, cytosolic form. Here, we investigated the role of membrane-bound alpha-synuclein in the aggregation process. In a cell-free system consisting of isolated brain fractions, spontaneous and progressive aggregation of alpha-synuclein was observed in membranes starting at day 1, whereas no aggregation was observed in the cytosolic fraction in a 3-day period. The addition of antioxidants reduced the aggregation in the membrane fraction, implicating the role of oxidative modifications. When excess cytosolic alpha-synuclein was added to brain membranes, the rate of aggregation was increased, while the lag time was unaffected. Incorporation of cytosolic alpha-synuclein into membrane-associated aggregates was demonstrated by fractionation and co-immunoprecipitation experiments. In our recent study, we showed that mitochondrial inhibitors such as rotenone, induced alpha-synuclein aggregation in cells. In the present study using rotenone-treated cells, the earliest appearance of alpha-synuclein oligomeric species was observed in membranous compartments. Furthermore, alpha-synuclein-positive inclusions were co-stained with DiI, a membrane-partitioning fluorescent dye, confirming the presence of lipid components in alpha-synuclein aggregates. These results suggest that membrane-bound alpha-synuclein can generate nuclei that seed the aggregation of the more abundant cytosolic form.

Animals↗
PubMed2002

Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death.

Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.

Amino Acid Sequence↗
PubMed2003

NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons.

NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes ( approximately 10%) and neurons ( approximately 90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult.

1-Methyl-4-phenylpyridinium↗