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At least 919 records · Page 51Linked to original sources

PubMed2000

Functional expression of NAD(P)H oxidase p47 in lung microvascular endothelial cells.

Vascular endothelial cell superoxide (O(*)(2)) has an important role in intracellular signaling, in interaction with other reactive species such as nitric oxide, and in vascular dysfunction. Little is known regarding the source and function of O(*)(2) from microvascular endothelial cells from specific tissues. Mouse lung microvascular endothelial cells stimulated with phorbol ester (PMA) or NADPH generated significant O(*)(2), which was inhibited by diphenyleneiodonium (DPI) but not by allopurinol, rotenone, indomethacin, or quinacrine. Optimal O(*)(2) generation required cytosolic as well as particulate cell fractions of cells. In parallel studies, PMA induced increased expression of the p47 component of the NAD(P)H oxidase in the particulate fraction, which was inhibited by staurosporine and calphostin. These data demonstrate that NAD(P)H oxidase is an important source of O(*)(2) generation in lung microvascular endothelial cells.

Allopurinol↗

PubMed1997

Stimulation of oxygen consumption promotes mitochondrial calcium accumulation, a process associated with, and causally linked to, enhanced formation of tert-butylhydroperoxide-induced DNA single-strand breaks.

The NADH-linked substrates pyruvate, L-glutamine, and beta-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause-effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate L-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca(2+)-dependent process.

3-Hydroxybutyric Acid↗

PubMed2001

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions.

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.

Adenosine Triphosphate↗

PubMed1999

Impaired glutamate clearance as a consequence of energy failure caused by MPP(+) in astrocytic cultures.

Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP(+)) metabolite. The mechanism by which MPP(+) is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP(+), glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP(+) and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP(+)-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent alpha-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP(+) on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP(+) impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.

1-Methyl-4-phenylpyridinium↗

PubMed2001

Differential cytotoxicity of Mn(II) and Mn(III): special reference to mitochondrial [Fe-S] containing enzymes.

Manganese (Mn)-induced neurodegenerative toxicity has been associated with a distorted iron (Fe) metabolism at both systemic and cellular levels. In the current study, we examined whether the oxidation states of Mn produced differential effects on certain mitochondrial [Fe-S] containing enzymes in vitro. When mitochondrial aconitase, which possesses a [4Fe-4S] cluster, was incubated with either Mn(II) or Mn(III), both Mn species inhibited the activities of aconitase. However, the IC(10) (concentration to cause a 10% enzyme inhibition) for Mn(III) was ninefold lower than that for Mn(II). Following exposure of mitochondrial fractions with Mn(II) or Mn(III), there was a significant inhibition by either Mn species in activities of Complex I whose active site contains five to eight [Fe-S] clusters. The dose-time response curves reveal that Mn(III) was more effective in blocking Complex I activity than Mn(II). Northern blotting was used to examine the expression of mRNAs encoding transferrin receptor (TfR), which is regulated by cytosolic aconitase. Treatment of cultured PC12 cells with Mn(II) and Mn(III) at 100 microM for 3 days resulted in 21 and 58% increases, respectively, in the expression of TfR mRNA. Further studies on cell growth dynamics after exposure to 25-50 microM Mn in culture media demonstrated that the cell numbers were much reduced in Mn(III)-treated groups compared to Mn(II)-treated groups, suggesting that Mn(III) is more effective than Mn(II) in cell killing. In cells exposed to Mn(II) and Mn(III), mitochondrial DNA (mtDNA) was significantly decreased by 24 and 16%, respectively. In contrast, rotenone and MPP+ did not seem to alter mtDNA levels. These in vitro results suggest that Mn(III) species appears to be more cytotoxic than Mn(II) species, possibly due to higher oxidative reactivity and closer radius resemblance to Fe.

Aconitate Hydratase↗

PubMed1983

Effect of diastereoisomeric epoxyrotenones on hepatic epoxide hydrase activity.

The effect on epoxide hydrase activity of the two diastereomeric (6'R) and (6'S)-6',7'-epoxyrotenones formed during the oxidative metabolism of rotenone on the isopropenyl side chain is reported. The activity of microsomal epoxide hydrase was determined using styrene oxide as substrate. The results indicate that the two diastereomeric 6',7'-epoxyrotenones are good substrates for epoxide hydrase and are noncompetitive inhibitors for the hydration of styrene oxide. The more polar of the two isomers is more active as inhibitor of this enzyme.

Animals↗

PubMed1980

Dynamics of O2 consumption in rat pancreatic islets.

The O2 consumption of rat pancreatic islets was determined by monitoring pO2 in the perifusate from groups of 200-300 islets. Basal respiration was maintained for up to 2 h. The insulin secretagogues, glucose and 4-methyl-2-oxopentanoate, provoked an immediate (< 5 s) increase in islet respiration which attained a new steady-state within 10-40 min. The respiratory changes were immediately reversible upon removal of the substrate and were paralleled by changes in insulin release and substrate oxidation. The concentration dependence of glucose-induced respiratory changes was sigmoidal with a threshold at 3 mmol/l. The concentration dependence with 4-methyl-2-oxopentanoate was characterised by a hyperbolic relationship. The weak insuln secretagogues 3-methyl-2-oxobutyrate and d,l-3-methyl-2-oxopentanoate, although stimulating islet respiration were not more effective than 4- methyl-2-oxopentanoate at non-insulinotropic concentrations. Rotenone, antimycin and oligomycin inhibited both basal O2 consumption and the ability of glucose and 4-methyl-2-oxopentanoate to increase islet respiration. 2,4-Dinitrophenol increased islet O2 consumption. The omission of Ca2+ and Mg2+ from the perifusing media, or the addition of the ionophore A23187, had little effect on respiration. The omission of K+ inhibited glucose-induced changes but had a lesser effect in the absence of substrate or in the presence of 4-methyl-2-oxopentanoate. The omission of HCO3-reduced both basal and secretagogue-induced changes in islet respiration. It is concluded that mitochondrial O2 consumption linked to oxidative phosphorylation is a major component in the respiratory response, and that some energy consuming process in the islets depends on the availability of HCO3-. Mitochondrial reactions may generate a signal initiating the secretory process.

Acetoacetates↗

PubMed1994

Chemical induction of quadruple and octuple chromosomes in Chinese hamster CHO-K1 cells and relationship between their three-dimensional structure and spatial distribution of BrdU-labeled chromatids.

Double endoreduplication of Chinese hamster CHO-K1 cells that exhibited quadruple chromosomes at metaphase was induced by a combination of rotenone and ammonium vanadate treatments. Analysis of sister chromatid differential staining patterns (using 5-bromo-2'-deoxyuridine) revealed that approximately 50% of the quadruple chromosomes did not keep the scheme of "outside replication" of DNA. Based on the ratio of the staining patterns observed, we suggest that the two diplochromosomes forming a quadruple chromosome are held together by a physical link connecting the two original chromatids. Metaphases with octuple chromosomes were also produced by the same treatment. Each chromosome constituting an octuple chromosome was longer and thinner than ordinary metaphase chromosomes. This suggests incomplete chromosome condensation at metaphase. The majority of octuple chromosomes showed the eight constituent chromosomes to be so enmeshed that a planar alignment could not be observed in air-dried preparations.

Aneuploidy↗

PubMed1975

The electron transport system of the anaerobic Propionibacterium shermanii: cytochrome and inhibitor studies.

1. Electron transport particles obtained from cell-free extracts of Propionibacterium shermanii by centrifugation at 105000 times g for 3 hrs oxidized NADH, D,L-lactate, L-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too. 2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome alpha1 and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80--90%. 3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or "site I region", in the electron transport system of P. shermanii. 4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor and reached 80--100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase. 5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions. 6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.

Aerobiosis↗

PubMed1982

Mitochondrial chemiluminescence elicited by acetaldehyde.

Acetaldehyde-dependent chemiluminescence has been found to be a sensitive technique for the study of superoxide and hydrogen peroxide formation in beef heart mitochondria. The system responds to ATP and antimycin A with increased emission intensities and to ADP and rotenone with decreased intensities, indicating that the chemiluminescence reflects the energy status of the mitochondrion. These effects are based on the ability of acetaldehyde to react with superoxide and hydrogen peroxide to form metastable intermediates which decay spontaneously with the emission of light. Additionally, these intermediates can react with cyanide to give alternative products which can also decay with the emission of light, the cyanide-evokable chemiluminescence. The interaction of acetaldehyde with mitochondria is complex because acetaldehyde can serve as a hydrogen source for NADH and as an inhibitor (at high concentration) of electron transport, and appears to be a reducing agent for a heat-stable site that autoxidatively generates HOOH from O2-.. Inasmuch as acetaldehyde is a metabolite of ethanol, this broad spectrum of reactivity may play a role in the hepatic and cardiac toxicity that is associated with alcoholism. The heat-stable site that generates HOOH from O2-. has been studied further and appears to contain vicinal dithiol which is primarily responsible for the cyanide-evokable chemiluminescence.

Acetaldehyde↗

PubMed1994

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia.

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4 and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Non-synaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4-7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they resulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/reperfusion damage, showing changes earlier than the hippocampal ones.

Animals↗

PubMed1985

DNA-mediated transfer of complex I genes into three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of electron transport chain.

We have used genomic DNA from human or mouse cells as a calcium phosphate precipitate to transfect three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of the electron transport chain. Transformants were selected in DMEM containing galactose, a medium in which respiration-deficient cells do not grow. Evidence for the DNA-mediated transformation of these respiration-deficient cells with a putative complex I gene includes: the clones are respiration-positive and respire at rates comparable to those of wild-type human, hamster, or mouse cells; the clones have rotenone-sensitive NADH oxidase activities, indicating a functional complex I of the electron transport chain; and the clones appear to be true transformants, as demonstrated by hybridization and Southern blot analyses. These experiments provide the basis for the isolation and subsequent characterization of several of the genes involved with complex I of the mammalian electron transport chain.

Animals↗

PubMed1976

Some properties of external NADH oxidation by human placental mitochondria.

Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.

Animals↗

PubMed1975

Respiratory inhibition and reversible fusion of frog blastomeres.

Rotenone and high doses of chloramphenicol, both of which specifically inhibit electron transport between NADH and flavoprotein in the respiratory chain, caused fully separated Rana pipiens blastomeres to refuse, as shown by syncytium counts on embryos reconstructed from serial sections. With chloramphenicol, the effect was completely reversible: re-cleavage and normal development followed drug removal. The blastomere fusion effect was not produced by the succinic dehydrogenase-specific respiratory inhibitor, thenoyltrifluoroacetone, nor by a non-mitochondrial protein synthesis inhibitor, cycloheximide, both of which instead produced simple arrest of cleavage.

Animals↗

PubMed1997

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria.

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.

Adenosine↗

PubMed2000

Nephritogenic ochratoxin A interferes with mitochondrial function and pH homeostasis in immortalized human kidney epithelial cells.

The ubiquitous nephritogenic and carcinogenic fungal metabolite ochratoxin A (OTA) has been shown to interact with renal cell function at low nanomolar concentrations. This is possibly brought about through changes in cellular pH (pHc) homeostasis and mitochondrial function. We assessed the effect of nanomolar concentrations of OTA on pHc homeostasis and the possible involvement of mitochondria using immortalized human kidney epithelial (IHKE1) cells. Within seconds OTA evoked a decrease of pHc with a threshold concentration of 0.1 nmol/l, followed by a sustained alkalinization. Acidification was the same in bicarbonate and non-bicarbonate Ringer solution. When Ca2+ entry across the plasma membrane was prevented, virtually no OTA-induced pH changes could be observed. Inhibition of Na+/H+-exchange (NHE, Na+-free solution) and H+-ATPase (bafilomycin A1) did not reduce the OTA-induced acidification. By contrast, determination of NHE activity as a function of pHc revealed that OTA stimulates NHE (maximal flux increases) in a Ca2+-dependent manner. OTA exposure did not increase lactic acid production, indicating that anaerobic glycolysis was not enhanced. Inhibiting complexes I, III and IV of the mitochondrial electron transport chain (ETC) with rotenone, antimycin A and CN- prevented the OTA-induced acidification almost completely. Completely inhibiting F1FO-ATPsynthase with oligomycin reduced the effect of OTA by approximately equal 50%. In addition, OTA induced a hyperpolarization of the mitochondrial membrane potential (psim) in a Ca2+-dependent manner. Furthermore, OTA exposure resulted in a mitochondria-dependent increase of the cellular ATP content. We conclude that OTA activates mitochondria and NHE by interfering with cellular Ca2+ homeostasis. Stimulation of mitochondrial metabolism leads to enhanced "proton production". Anaerobic glycolysis is not enhanced.

Antimycin A↗

PubMed2004

NADH-ubiquinone oxidoreductase activity in the kinetoplasts of the plant trypanosomatid Phytomonas serpens.

NADH-ubiquinone oxidoreductase activity is present in mitochondrial lysates of Phytomonas serpens. Rotenone at 2-10 microM inhibited the activity 50-75%, indicating that it belongs to respiratory complex I. The activity was also inhibited 50-60% in the presence of 10-30 nM atovaquone suggesting that inhibition of complex I represents a likely mechanism of the known antileishmanial activity of this drug. The complex was partially purified by chromatography on DEAE-Sepharose CL-6B and gel-filtration on Sepharose CL-2B. The NADH:ubiquinone oxidoreductase activity in this preparation was completely inactivated by 20 nM atovaquone. The partially purified complex was present in a low amount and its subunits could not be discerned by staining with Coomassie. However, one of its components, a homologue of the 39 kDa subunit of the bovine complex I, was identified immunochemically in the original lysate and in the partially purified material.

Animals↗

PubMed1999

Tumor necrosis factor-alpha plus actinomycin D-induced apoptosis of L929 cells is prevented by nitric oxide.

Treatment with the nitric oxide-(NO)-generating compound S-nitroso-N-acetylpenicillamine protected cul-tured L929 cells from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D, as determined by the detection of DNA fragmentation and morphological changes. NO also prevented an enhancement of the production of reactive oxygen intermediates by TNF-alpha plus actinomycin D, as assessed by the oxidation of dihydrorhodamine 123 and hydroethidine. Because the inhibition of mitochondrial respiration by rotenone or antimycin A suppressed the increased oxidation of both dihydrorhodamine 123 and hydroethidine, it was suggested that TNF-alpha accelerated the leakage of reactive oxygen intermediates from the mitochondrial electron transport system. Polarography showed that NO reversibly inhibited mitochondrial respiration at either complexes I-III, II-III, or IV, thus suggesting the inhibition of cytochrome oxidase. Taken together, these findings indicate that the decreased mitochondrial formation of reactive oxygen intermediates in the presence of NO might have a protective effect against TNF-alpha plus actinomycin D-induced apoptosis.

Animals↗