NADH Oxidation In Submitochondrial Particles Research Articles

Metformin selectively targets redox control of complex I energy transduction

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

Total Synthesis and Biological Activity of Neopeltolide and Analogues

Combining the core structure of neopeltolide, lactone 16 a, with the oxazole-containing side chain 23 via a Mitsunobu reaction provided the cytotoxic natural product neopeltolide (2). The side chain 23 was prepared from oxazolone 24 via the corresponding triflate. Key steps in the preparation of 23 were a Sonogashira coupling, an enamine alkylation, and a Still-Gennari Horner-Emmons reaction. By changing the Leighton reagent in the allylation step, the 11-epimer of lactone 16 a, compound 50 was prepared. This led to 11-epi-neopeltolide 51. The 5-epimer of neopeltolide, compound 52, could be obtained from the minor isomer of the Prins cyclization. Furthermore, a range of analogues with modifications in the side chain were prepared. All derivatives were checked for toxicity effects on mammalian cell cultures and inhibitory effects on NADH oxidation in submitochondrial particles of bovine heart. Modifications in the lactone part are tolerated to some degree. On the other hand, shortening the distance between the oxazole and the lactone causes a significant drop in activity. Analogue 65 with an additional double bond is equally or even more active than neopeltolide itself.

Inhibition and uncoupling of oxidative phosphorylation by nonsteroidal anti-inflammatory drugs: Study in mitochondria, submitochondrial particles, cells, and whole heart

The effects of the anti-inflammatory drugs diclofenac, piroxicam, indomethacin, naproxen, nabumetone, nimesulide, and meloxicam on mitochondrial respiration, ATP synthesis, and membrane potential were determined. Except for nabumetone and naproxen, the other drugs stimulated basal and uncoupled respiration, inhibited ATP synthesis, and collapsed membrane potential in mitochondria incubated in the presence of either glutamate + malate or succinate. Plots of membrane potential versus ATP synthesis (or respiration) showed proportional variations in both parameters, induced by different concentrations of nimesulide, meloxicam, piroxicam, or indomethacin, but not by diclofenac. The activity of the adenine nucleotide translocase was blocked by diclofenac and nimesulide; diclofenac also slightly inhibited mitochondrial ATPase activity. Naproxen did not affect any of the mitochondrial parameters measured. Nabumetone inhibited respiration, ATP synthesis, and membrane potential in the presence of glutamate + malate, but not with succinate. NADH oxidation in submitochondrial particles also was inhibited by nabumetone. Nabumetone inhibited O 2 uptake in intact cells and in whole heart, whereas the other five drugs stimulated respiration. These observations revealed that in situ mitochondria are an accessible target. Except for diclofenac, a negative inotropic effect on cardiac contractility was induced by the drugs. The data indicated that nimesulide, meloxicam, piroxicam, and indomethacin behaved as mitochondrial uncouplers, whereas nabumetone exerted a specific inhibition of site 1 of the respiratory chain. Diclofenac was an uncoupler too, but it also affected the adenine nucleotide translocase and the H +-ATPase.

Evidence that the inhibition sites of the neurotoxic amine 1-methyl-4-phenylpyridinium (MPP+) and of the respiratory chain inhibitor piericidin A are the same

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. Proof that these classical respiratory chain inhibitors and MPP+ react at the same site in the complex NADH dehydrogenase molecule has been difficult to obtain because none of these compounds bind covalently to the target. The 4'-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself, but still dissociate on washing the membrane preparations, with consequent re-activation of the enzyme. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin.

マイコトキシンと生体膜の相互作用 キノン色素によるミトコンドリア電子伝達阻害作用を中心として

Variety of quinone and quinoid compounds have been isolated from natural sources. Knowledges on the physiological significances of quinone production in biological systems, are, however, quite limited except those of ubiquinones and plastoquinones in the biological oxidation systems. In the present review, our brief works on the interactions of several naturally occurring, toxic quinones with respriratory chain of mitochondria were described.Flavoglaucin, a benzohydroquinone derivative from Eurotium chevalieri, exerted an uncoupling effect on oxidative phosphorylation in mitochondria. The uncoupling effect of flavoglaucin, which does not release proton at neutral pH range, was considered to be closely correlated with its strong induction of mitochondrial swelling. Flavoglaucin depressed mitochondrial respiration by directly interacting with the respiratory chain at higher concentrations than those for uncoupling activity. The site of inhibition was identified to be localized mainly at complex I and inferiorly at complex III of the respiratory chain.Xanthomegnin, a naphthoquinone pigment originally from dermatophytes Trichophyton and Microsporum, was later isolated from several species of saprophytic fungi Aspergillus and Penicillium and was shown to uncouple oxidative phosphorylation. Xanthomegnin exhibited a redox response upon NADH oxidation in submitochondrial particles and in dissolved system, in Which rotenone- and antimycin-inhibitions were no longer observed. It was concluded that xanthomegnin made an electron transport-shunt between complex I and IV in the presence of cytochrome c.Averufin, an anthraquinone mycotoxin which is a biosynthetic precursor of an extremely potent hepato-carcinogen aflatoxin B1, was found to be a strong uncoupler of oxidative phosphorylation in mitochondria. At the higher concentrations than those for uncoupling activity, averufin inhibited mitochondrial respiration directly interacting with the respiratory chain at cytochrome bc1 segment, showing a similarity to the mode of inhibition by antimycin A which is a specific inhibitor to the electron transport between cytochromes b and c1.

Effects of tricyclic antidepressant drugs on energy-linked reactions in mitochondria

The effects of impramine and chlorimipramine on energy-linked reactions in mitochondria were characterized. Both compounds exhibited some characteristics of classical uncouplers of oxidative phosphorylation, i.e. they released respiratory control, hindered ATP synthesis, and enhanced ATPase activity of isolated rat liver mitochondria. Unlike classical uncouplers, however, these compounds only weakly stimulated proton uptake in intact mitochondria. They also exhibited unusual effects on energy-linked reactions in beef heart submitochondrial particles (SMP). Both compounds inhibited NADH oxidation in SMP in an “oligomycin-like” manner, and inhibited ATPase activity of SMP and the soluble F 1-ATPase. In contrast, the drugs weakly inhibited ATPase activities of bovine adrenal gland chromaffin granules and resealed granule ghosts. The mechanisms responsible for the multiple effects on mitochondrial energy-linked processes are unclear. They may be related to the hydrophobicity of the drugs, as has been shown for other hydrophobic amines.

NADH oxidation in submitochondrial particles protects respiratory chain activity against damage by adriamycin-Fe3+.

Oxidative decomposition of polyunsaturated fatty acid moieties of membrane lipid in pig heart submitochondrial particles, as initiated by ferric ion complexes of the antineoplastic drug adriamycin and concomitant inactivation of oxidase activities, is counteracted by EDTA, low oxygen pressure, a phenolic antioxidant and NADH oxidation through the respiratory chain but not by scavengers of reactive oxygen species. Protection by NADH is strengthened by removal of cytochrome c from the submitochondrial particles and by antimycin A but abolished by rotenone. Inhibition of cytochrome c oxidase activity by the adriamycin-Fe3+ complex is reversible and activity is recovered upon cholate solubilization of the particles. ADP inhibits binding of the complex to the submitochondrial particles and protects both cytochrome c oxidase activity and membrane lipid. The results are discussed in relation to the possible role of mitochondrial function in protection against free-radical-mediated effects of adriamycin.

Effect of monovalent cations on the inhibition by NAD + of NADH oxidation in submitochondrial particles

Oxidation of NADH in submitochondrial particles, with O 2 or ferricyanide as electron acceptor, was inhibited by micromolar concentrations of NAD + when measured in 240 mM sucrose or, in a lesser extent, in 120 mM NaCl or LiCl. In 120 mM solutions of either KCl, RbCl, CsCl or NH 4Cl the inhibition by up to 100 μM concentrations of NAD + did not occur. The inhibition observed in the sucrose medium disappeared after solubilization of the particles with detergents and re-appeared when the membranes were reconstituted. The inhibitory effect was potentiated by palmitoyl-CoA. The possibility is discussed that the inhibition of NADH oxidation by low concentrations of NAD + and its release by K +, Rb +, Cs + and NH 4 + depend on the interaction between NAD + and the negatively charged mitochondrial membrane.

Effect of mercurials on NADH oxidation and energy-linked hydrogen ion gradient in submitochondrial particles

Earlier work has shown that the sensitivity of NADH oxidation in submitochondrial particles to inhibition by mercurial increases, if the particles are ‘preconditioned’ by allowing them to oxidize a small amount of NADH before exposure to the inhibitor. This inhibition is relieved by the addition of ethylenediamine tetraacetate (EDTA). The effect of EDTA and mersalyl on the decolorization of membrane-bound bromthymol blue, when it is energized by NADH oxidation, has been measured. Under conditions where EDTA overcame the inhibition of oxidation by mersalyl, it did not affect the bromthymol blue response. The involvement of an -SH in the energy-driven establishment of a proton gradient thus becomes apparent.