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PubMed1993

Cyclosporin and carnitine prevent the anoxic death of cultured hepatocytes by inhibiting the mitochondrial permeability transition.

Cyclosporin A (CyA) and L-carnitine (LC) prevented the killing of cultured hepatocytes by anoxia and rotenone but not by cyanide. Neither CyA nor LC affected the rate or extent of the loss of the mitochondrial membrane potential or the rate or extent of the depletion of ATP. Atractyloside blocked the ability of both CyA and LC to protect, and D-carnitine antagonized the effect of LC but not that of CyA. Cell killing by cyanide was prevented when the phospholipase A2 inhibitor butacaine was added together with CyA. Butacaine by itself had no effect on cell killing. In a swelling assay with isolated rat liver mitochondria having a low calcium content, phenylarsine oxide or palmitoyl-CoA induced the inner membrane permeability transition when electron transport was inhibited by rotenone or cyanide. CyA prevented the permeability transition with rotenone but not with cyanide, and atractyloside reversed the effect of CyA. LC prevented the permeability transition occurring with palmitoyl-CoA plus rotenone but not with palmitoyl-CoA plus cyanide. Atractyloside and D-carnitine antagonized the protective effect of LC. Inhibition of the cyanide-dependent permeability transition in isolated liver mitochondria required the presence of both CyA and butacaine. These data document the close correlation between the effect of CyA and LC on the response of cultured hepatocytes to inhibition of mitochondrial electron transport and their ability to prevent the permeability transition in isolated mitochondria. It is concluded that the ability of CyA and LC to protect cultured hepatocytes is a consequence of their ability to prevent the mitochondrial permeability transition, indicating that this event is likely to be causally linked to the genesis of irreversible injury. Thus, cell death with anoxia or inhibitors of electron transport is related to a mitochondrial alteration by a mechanism that is independent of the maintenance of a membrane potential or cellular stores of ATP.

4-Aminobenzoic Acid↗
PubMed1976

Inhibition of mitochondrial electron transport by hydrophilic metal chelators. Determination of dehydrogenase topography.

The topography of the inner mitochondrial membrane was investigated using inhibitors of electron transport on preparations of beef heart mitochondria and electron transport particles of opposite orientation. Reductions of juglone, ferricyanide, indophenol, coenzyme Q, duroquinone, and cytochrome c by NADH are inhibited to different extents on both sides of the membrane by the impermeant hydrophilic chelators bathophenanthroline sulfonate and orthophenanthroline. The extent of inhibition for each acceptor increased in the order given. At least two chelator-sensitive sites are present on each membrane face between the flavoprotein and coenzyme Q and a chelator-sensitive site is present on the matrix face between the sites of coenzyme Q and duroquinone interaction. Duroquinol oxidation in mitochondria only is stimulated by bathophenanthroline sulfonate. Juglone reduction is stimulated in electron transport particles (only) by p-hydroxymercuribenzenesulfonate, but after mercurial treatment, juglone reduction in both particles and mitochondria is more sensitive to bathophenanthroline sulfonate. Succinate dehydrogenase components are inhibited by hydrophilic orthophenanthroline or bathophenanthroline sulfonate in mitochondria only. Electron flow between the dehydrogenases of succinate and NADH occurs via a chelator-sensitive site located on the matrix face of the membrane. Inter-complex electron flow is prevented by rotenone or thenoyltrifluoroacetone. The lack of succinate-indophenol reductase inhibition by bathophenanthroline sulfonate in the presence of rotenone or thenoyltrifluoroacetone indicates that the rotenone-sensitive site may be located on the matrix face and demonstrates that electrons flow between the NADH and succinate dehydrogenases via a hydrophilic chelator and rotenone-thenoyltrifluoroacetone-sensitive site on the matrix face of the membrane. Inhibiton by hydrophilic chelators only in mitochondria indicates that succinate dehydrogenase as well as NADH dehydrogenase has a transmembranous orientation.

Animals↗
PubMed1982

ATP synthesis during exogenous NADH oxidation. A reappraisal.

This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c.

Adenosine Triphosphatases↗
PubMed1977

Mitochondria from human term placenta. III. The role of respiration and energy generation in progesterone biosynthesis.

The interrelationships among the processes of progesterone biosynthesis, respiration and energy production in human term placental mitochondria were examined. All substrates (citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, glutamate, glycerol 3-phosphate, ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) previously found to stimulate oxygen uptake and ATP synthesis in placental mitochondria supported progesterone synthesis from endogenous and exogenous cholesterol. Citrate support of progesterone synthesis was stimulated by NADP+ but not NAD+. It was inhibited by fluorocitrate and trans-aconitate but not by arsenite, rotenone, antimycin, cyanide or 2,4-dinitrophenol. Ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine support of progesterone synthesis was stimulated by NAD+ and NADP+ and was inhibited by ADP, rotenone, antimycin, cyanide and 2,4-dinitrophenol. Cyanide inhibition was relieved by an exogenous ATP regenerating system and ADP inhibition was reversed by oligomycin. Progesterone synthesis supported by alpha-ketoglutarate + malonate was stimulated by NAD+ and NADP+, and was completely inhibited by arsenite. 2,4-Dinitrophenol was strongly inhibitory both in the absence and presence of rotenon or antimycin. Stimulation by ATP was enhanced by rotenon, antimycin and oligomycin and inhibited by 2,4-dinitrophenol. Thus, metabolic control of progesterone synthesis by the energy status of the mitochondrial system was demonstrated when reducing equivalents were supplied via NADH or the respiratory electron transport chain.

Cholesterol↗
PubMed1993

Inhibition of mitochondrial and Paracoccus denitrificans NADH-ubiquinone reductase by oxacarbocyanine dyes. A structure-activity study.

In this study, we determined that three structurally related oxacarbocyanine dyes, 3,3'-diethyloxacarbocyanine (DiOC2(3)), 3,3'-dipentyloxacarbocyanine (DiOC5(3)), and 3,3'-dihexyloxacarbocyanine (DiOC6(3)), and one oxadicarbocyanine, 3,3'-diethyloxadicarbocyanine (DiOC2(4)), inhibit bovine heart mitochondrial NADH oxidase activity and one of them, DiOC6(3), inhibits Paracoccus denitrificans NADH oxidase activity. The mitochondrial I50 values were 9 microM (DiOC2(3)), approximately 1 microM (DiOC5(3)) and DiOC6(3)), and approximately 3 microM (DiOC2(4)), whereas the I50 value for P. denitrificans was approximately 2 microM (DiOC6(3)). Neither succinate nor cytochrome oxidase (EC 1.9.3.1) activity was inhibited significantly by any of the compounds in either electron transport chain, localizing the inhibitory site of the oxacarbocyanine dyes to the respiratory chain segment between NADH and ubiquinone. With submitochondrial particles (SMP), NADH-dependent reduction of duroquinone and coenzyme Q1 was inhibited markedly by all four compounds with DiOC6(3) being the most potent inhibitor, and the reduction of menadione was inhibited substantially by DiOC6(3). When purified complex I was used, NADH-dependent reduction of ferricyanide was inhibited by DiOC5(3) and coenzyme Q1 reduction was inhibited by all oxacarbocyanines. With P. denitrificans membrane vesicles, DiOC6(3) substantially inhibited NADH-dependent reduction of coenzyme Q1. All the oxacarbocyanines were more effective inhibitors with membrane preparations than with complex I, suggesting that membrane interactions play a role in inhibition. The mechanism of inhibition of the oxacarbocyanines appears to be similar to that of rotenone since (a) essentially only electron acceptors affected by rotenone were affected by the compounds, (b) inhibition of menadione reduction was diminished drastically with rotenone-saturated SMP, and (c) inhibition of coenzyme Q1 was largely eliminated with rotenone-insensitive complex I, and P. denitrificans membrane vesicles.

Animals↗
PubMed1995

The antianginal agent ranolazine is a weak inhibitor of the respiratory complex I, but with greater potency in broken or uncoupled than in coupled mitochondria.

Ranolazine (RS-43285) has shown antianginal effects in clinical trials and cardiac anti-ischaemic activity in several in vivo and in vitro animal models, but without affecting haemodynamics. Its mechanism is thought to mainly involve a switch in substrate utilisation from fatty acids to glucose to, thus, improve efficiency of O2 use; however, its precise molecular target(s) are unknown. In studies to investigate its action further, using isolated rat heart mitochondria, ranolazine was found to weakly inhibit (pIC50 values > 300 microM) respiration by coupled mitochondria provided with NAD(+)-linked substrates but not with succinate. With broken mitochondrial membranes or submitochondrial particles, ranolazine inhibited NADH but not succinate oxidation and with pIC50 values in the lower range of 3-50 microM. Studies with different electron acceptors and respiratory inhibitors indicated that it inhibits respiratory Complex I at a site between ferricyanide and menadione and ubiquinone-1 reduction (i.e. at a similar locus to rotenone). However, unlike rotenone, ranolazine was an uncompetitive inhibitor with respect to ubiquinone-1. Ranolazine inhibition of Complex I was reversible and occurred also with mitochondria from pig, guinea pig, and human heart, and rat liver. Further studies using rat heart mitochondria in different energisation states (i.e. broken, uncoupled, or coupled) showed a 50-100-fold shift to greater potency of ranolazine in the broken compared to the coupled; with the uncoupled it was about 2-fold less potent than the broken. These shifts in potency were not found with rotenone or amytal. Studies with radiolabelled ranolazine showed that it bound to mitochondrial membranes with greater affinity in the broken compared to the coupled or uncoupled conditions. Rotenone displaced radiolabelled ranolazine from its binding site. This property of ranolazine may play some role in its anti-ischaemic activity.

Acetanilides↗
PubMed1979

The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta.

1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. ADP and ATP stimulated the malate, succinate and alpha-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or alpha-ketoglutarate. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. alpha-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of alpha-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.

Adenosine Diphosphate↗
PubMed1997

Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon.

Basal (substrate alone) and maximum rates of H2O2 production, oxygen consumption and free radical leak in the respiratory chain were higher in heart mitochondria of the short-lived rat (4 years) than in the long-lived pigeon (35 years). This suggests that the low free radical production of pigeon heart mitochondria is due in part to both a low electron flow and a low percent leak of electrons out of sequence in the respiratory chain. Thenoyltrifluoroacetone did not increase H2O2 production with succinate either in rats or pigeons. Mitochondrial H2O2 production was higher with pyruvate/malate than with succinate in both animal species. Rotenone and antimycin A increased H2O2 production with pyruvate/malate to the maximum levels observed in each species. Addition of myxothiazol to antimycin A-treated mitochondria supplemented with pyruvate/malate decreased H2O2 production in both species. All the combinations of inhibitors added with pyruvate/malate resulted in higher rates of H2O2 production in rats than in pigeons. When succinate instead of pyruvate/malate was used as substrate, rotenone and thenoyltrifluoroacetone decreased mitochondrial H2O2 production in the rat and did not change it in the pigeon. The results indicate that Complexes I and III are the main H2O2 generators of heart mitochondria in rats and pigeons and that both Complexes are responsible for the low H2O2 production of the bird. p-Chloromercuribenzoate and ethoxyformic anhydride strongly inhibited the H2O2 production induced by rotenone with pyruvate/malate in both species. This suggests that the free radical generator of Complex I is located after the ferricyanide reduction site, between the ethoxyformic and the rotenone-sensitive sites.

Aging↗
PubMed2002

EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ.

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron-sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNS (slow-relaxing), and SQNx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQNf, because of its high sensitivities to DeltamicroH+ and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQNf signal at a much lower concentration than that required to quench the slower relaxing components (SQNs and SQNx). A close correlation was shown between the line shape alteration of the g// = 2.05 signal of the cluster N2 and the quenching of the SQNf signal, using two different experimental approaches: (1) changing the DeltamicroH+ poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQNf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex .

Animals↗
PubMed1986

Subcellular distribution of OM cytochrome b-mediated NADH-semidehydroascorbate reductase activity in rat liver.

Tissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.

Adrenal Glands↗
PubMed1979

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes.

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.

Animals↗
PubMed2004

A transcriptomic and proteomic characterization of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction.

Mitochondria import hundreds of cytosolically synthesized proteins via the mitochondrial protein import apparatus. Expression analysis in various organs of 19 components of the Arabidopsis mitochondrial protein import apparatus encoded by 31 genes showed that although many were present in small multigene families, often only one member was prominently expressed. This was supported by comparison of real-time reverse transcriptase-polymerase chain reaction and microarray experimental data with expressed sequence tag numbers and massive parallel signature sequence data. Mass spectrometric analysis of purified mitochondria identified 17 import components, their mitochondrial sub-compartment, and verified the presence of TIM8, TIM13, TIM17, TIM23, TIM44, TIM50, and METAXIN proteins for the first time, to our knowledge. Mass spectrometry-detected isoforms correlated with the most abundant gene transcript measured by expression data. Treatment of Arabidopsis cell culture with mitochondrial electron transport chain inhibitors rotenone and antimycin A resulted in a significant increase in transcript levels of import components, with a greater increase observed for the minor isoforms. The increase was observed 12 h after treatment, indicating that it was likely a secondary response. Microarray analysis of rotenone-treated cells indicated the up-regulation of gene sets involved in mitochondrial chaperone activity, protein degradation, respiratory chain assembly, and division. The rate of protein import into isolated mitochondria from rotenone-treated cells was halved, even though rotenone had no direct effect on protein import when added to mitochondria isolated from untreated cells. These findings suggest that transcription of import component genes is induced when mitochondrial function is limited and that minor gene isoforms display a greater response than the predominant isoforms.

Arabidopsis↗
PubMed1967

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors.

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.Malonate inhibition of succinate oxidation follows a classical type of competitive inhibition with an inhibitor dissociation constant of 0.13 mm. There is no inhibition detectable when malate is used as substrate. In contrast to animal mitochondria, amytal is capable of inhibiting 20 to 40% of succinate oxidation and 90 to 100% of malate oxidation, but inhibition due to rotenone amounts to only 0 to 20% of succinate oxidation and 40 to 50% of malate oxidation. The half-maximal inhibition caused by amytal occurs at 2 to 2.5 mm and that by rotenone at 3 mmumoles/mg protein.The maximal inhibition caused by either antimycin A or NOQNO is 70 to 80% of the state 3 respiration. Very little inhibition was observed on the state 4 respiration, and both inhibitors were capable of titrating stoichiometrically with mitochondrial protein with identical titers, 0.22 mmumoles/mg protein for half-maximal inhibition. They differ, however, in that NOQNO does uncouple oxidative phosphorylation in mung bean mitochondria, but antimycin A does not do so. Both cyanide and azide inhibit the state 3 rate 65 to 80%. Inhibition of state 4 respiration can be up to 50% by cyanide, while almost none by azide. Uncoupling action was noted with cyanide, but very little with azide.It is concluded that the second state 3 rate of succinate oxidation includes 80% succinoxidase, the remaining 20% being contributed by the NADH pathway. Malate oxidation apparently does not involve succinoxidase. Malate oxidation is completely sensitive to amytal, but only 50% inhibited by rotenone. A difference between animal and plant mitochondria appears to be in the flavoproteins associated with NADH oxidation.From the observations that antimycin A, NOQNO, cyanide, and azide do not cause complete inhibition, it is suggested that a leakage of electrons to oxygen exists before the site of inhibition of antimycin A or NOQNO.

Journal Article↗
PubMed1971

The effect of calcium and inhibitors on corn mitochondrial respiration.

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations.

Journal Article↗
PubMed1980

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway.

MALATE OXIDATION IN PLANT MITOCHONDRIA PROCEEDS THROUGH THE ACTIVITIES OF TWO ENZYMES: a malate dehydrogenase and a NAD(+)-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD(+) stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD(+) are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD(+)/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD(+)/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.

Journal Article↗
PubMed1991

Effects of hypoxia and metabolic inhibitors on production of prostacyclin and endothelium-derived relaxing factor by pig aortic endothelial cells.

1. The content of adenosine triphosphate (ATP) and basal and bradykinin-stimulated production of prostacyclin and endothelium-derived relaxing factor (EDRF) was measured in primary cultures of porcine aortic endothelial cells under normoxic (14.4% O2) and hypoxic (2.8% O2) conditions, and following treatment with rotenone and 2-deoxy glucose, which inhibit oxidative and glycolytic metabolism, respectively. 2. ATP content and basal and bradykinin-stimulated production of prostacyclin were similar under normoxic and hypoxic conditions. EDRF production, assessed as endothelial guanosine 3':5'-cyclic monophosphate (cyclic GMP) content, was also similar under both conditions. 3. Treatment with rotenone (0.3 microM) had no effect on ATP content or basal or bradykinin-stimulated production of prostacyclin or of EDRF, measured as endothelial cyclic GMP content. Elevation of cyclic GMP content by atriopeptin II was also unaffected. 4. Treatment with 2-deoxy glucose (20 mM) in glucose-free Krebs solution lowered ATP content, reduced bradykinin-stimulated production of prostacyclin and abolished the bradykinin-stimulated elevation of cyclic GMP content. Resting production of prostacyclin was unaffected but basal content of cyclic GMP was lowered in some experiments. Elevation of cyclic GMP content by atriopeptin II was abolished. 5. Combined treatment with rotenone (0.3 microM) and 2-deoxy glucose (20 mM) lowered ATP content more than with 2-deoxy glucose alone. Basal production of prostacyclin rose slightly and bradykinin-stimulated production was powerfully inhibited. Basal content of cyclic GMP was unaffected, but bradykinin-stimulated production was abolished. Elevation of cyclic GMP by atriopeptin II was also abolished. 6. Cascade bioassay experiments using endothelium-denuded rings of rabbit aorta as a detector system confirmed that bradykinin-stimulated production of EDRF was blocked by 2-deoxy glucose, but not by rotenone. 7. These data indicate that porcine aortic endothelial cells in culture operate under mainly glycolytic metabolism and this probably explains why production of prostacyclin and EDRF is unaffected under hypoxic conditions. They also indicate that glycolytic metabolism is required for agonist-stimulated production of prostacyclin and EDRF by these cells.

6-Ketoprostaglandin F1 alpha↗
PubMed2003

Inhibitory effects of antiparkinsonian drugs and caspase inhibitors in a parkinsonian flatworm model.

It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP(+), a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration- and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.

Animals↗
PubMedDate not supplied

In vitro search for synergy between flavonoids and epirubicin on multidrug-resistant cancer cells.

The drug accumulation of a human multidrug resistance 1 (mdr1) gene-transfected mouse lymphoma cell line and a multidrug resistance protein (MRP)-expressing human breast cancer cell line MDA-MB-231 was compared in the presence of sixteen flavonoids and five isoflavonoids. The expression of the 170-kDa P-glycoprotein (P-gp) (MDR1) and 190-kDa multidrug resistance protein (MRP) in both cell lines was confirmed by immunocytochemistry. The rhodamine 123 accumulation of the P-glycoprotein (P-gp)-expressing cells increased up to 46.4, while 2,7'-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM) accumulation of the MRP-expressing cells increased up to 1.6, in fluorescence activity ratio (FAR). Major P-gp-mediated efflux pump modifiers are formononetin, amorphigenin, rotenone and chrysin, while MRP-mediated efflux pump modifiers are formononetin, afrormosin, robinin, kaempferol and epigallocatechin. In antiproliferative assay, afrormosin, amorphigenin, chrysin and rotenone exhibited the strongest antiproliferative effects in L5178 (max. ID50: 19.70) and MDA-MB-231 cell lines (max. ID50: 55.47). In a checkerboard microplate method in vitro, furthermore, the most effective multidrug resistance (MDR) resistance modifiers, amorphigenin, formononetin, rotenone and chrysin, were assayed for their antiproliferative effects in combination with epirubicin. Rotenone and afrormosin showed additive effects. Chrysin and amorphigenin on the mouse lymphoma cell line and formononetin on the MDA-MB-231 cell line synergistically enhanced the effect of epirubicin.

ATP Binding Cassette Transporter, Subfamily B↗