Inhibition of the state 3 respiration of isolated mitochondria and its implications in comparative studies

The initial state 3 rates of respiration of mitochondria from plant tissue and from rat liver are usually less than the state 3 rates obtained after the mitochondria experience several state 3-state 4 cycles.Reduction of the absolute rate of respiration by decreasing the temperature or increasing the osmolarity of the reaction medium increased both the magnitude of the inhibition and its persistence in subsequent cycles. Under particularly adverse reaction conditions the initial state 3 was depressed to the extent that no distinction was observed in the state 3 and state 4 rates and the mitochondria appeared to have lost respiratory control. Thus, for comparative studies, invalid conclusions regarding the effect of these factors on respiration could be drawn unless the inhibition were removed.The inhibition was observed with substrates other than succinate and was therefore not attributed to oxaloacetate inhibition of succinate dehydrogenase.Increasing the amount of ADP added at each cycle did not appreciably reduce the inhibition.The inhibition of initial state 3 rates was satisfactorily relieved by incubating the mitochondria with substrate, and several cycles of ADP under conditions which increased the absolute rate of respiration. Once relieved of the inhibition, mitochondria could be stored at 0° for several hours without the inhibition being apparent again.This work was supported by a CSIRO, Australia, study grant awarded to J. M. Lyons while on sabbatical leave from the Department of Vegetable Crops, University of California, Riverside, California 92502.     

Acrolein inhibits respiration in isolated brain mitochondria

Lipid peroxidation is elevated in diseased regions of brain in several neurodegenerative diseases. Acrolein (2-propenal) is a major cytotoxic product of lipid peroxidation and its adduction to neuronal proteins has been demonstrated in diseased brain regions from patients with Alzheimer's disease. Mitochondrial abnormalities are implicated in several neurodegenerative disorders, and mitochondria are targets of alkenal adduction in vivo. We examined the effects of acrolein upon multiple endpoints associated with the mitochondrial involvement in neurodegenerative disease. Acrolein inhibited state 3 respiration with an IC(50) of approx. 0.4 micromol/mg protein; however, there was no reduction in activity of complexes I-V. This inhibition was prevented by glutathione and N-acetylcysteine. Acrolein did not alter mitochondrial calcium transporter activity or induce cytochrome c release. These studies indicate that acrolein is a potent inhibitor of brain mitochondrial respiration.     

Simulation of state 4 --> state 3 transition in isolated mitochondria

The mathematical dynamic model of oxidative phosphorylation developed previously and in the accompanying paper was modified to involve isolated mitochondria conditions; it was also used to simulate state 4 --> state 3 transition in rat liver mitochondria incubated with succinate as respiratory substrate and glucose-hexokinase as an ADP-regenerating system. Changes in the respiration rate, protonmotive force and reduction level of ubiquinone and cytochrome c as well as the internal (i) and external (e) ATP/ADP ratio between state 4 and state 3 were calculated and compared with the experimental data. Flux control coefficients with respect to oxygen consumption flux for different reactions and processes of oxidative phosphorylation were simulated for different values of the respiration rate (state 4, state 3 and intermediate states). Flux control coefficients for the oxidation, phosphorylation and proton leak subsystems with respect to the oxidation, phosphorylation and proton leak fluxes for different values of the respiration rate were also calculated. These theoretical data were compared with the experimental results obtained in the frame of metabolic control analysis and the 'top-down' approach to this analysis. A good agreement was obtained. Simulated time courses of the respiration rate, the protonmotive force (Deltap) and other parameters after addition of a small amount of ADP to mitochondria in state 4 mimicked at least semiquantitatively the experimentally measured time courses of these parameters. It was concluded, therefore, that in the present stage, the model is able to reflect different properties of the oxidative phosphorylation system in a broad range of conditions fairly well, allows deeper insight into the mechanisms responsible for control and regulation of this process, and can be used for simulation of new experiments, thus inspiring experimental verification of the theoretical predictions.     

The role of state 4 electron transport in the activation of state 3 respiration in potato mitochondria

The initial state 3 respiration rate of potato mitochondria is markedly depressed, or attenuated. With several consecutive state 3/state 4 cycles the state 3 rate rises to a maximum, while the state 4 rate remains essentially unchanged. The development of state 3 respiration has been termed conditioning. An analysis of the process has indicated that state 4 is a better conditioner than state3 per se. Conditioning is also attained by preincubation in state 2, or under conditions designated pseudostate 2, wherein ADP is present, with or without oligomycin, and inorganic phosphate is absent. ADP implements the conditioning process in the absence of oxidative phosphorylation. The action of ADP in its secondary or modulator role appears to be positively cooperative, the kinetics of ADP involvement being second-order. S_0.5 for ADP as a modulator of the conditioning process is approximately 62 M, a value in excess of the K _s for ADP in oxidative phosphorylation. Electron transport is indispensable for conditioning, and it is suggested that conditioning and ATP synthesis represent alternative uses of respiratory energy. It is further suggested that to some extent state 4 underlies state 3.     

Thyroid-hormone control of state-3 respiration in isolated rat liver mitochondria.

Oxidative phosphorylation can be treated as two groups of reactions; those that generate protonmotive force (dicarboxylate carrier, succinate dehydrogenase and the respiratory chain) and those that consume protonmotive force (adenine nucleotide and phosphate carriers. ATP synthase and proton leak). Mitochondria from hypothyroid rats have lower rates of respiration in the presence of ADP (state 3) than euthyroid controls. We show that the kinetics of the protonmotive-force generators are unchanged in mitochondria from hypothyroid animals, but the kinetics of the protonmotive-force consumers are altered, supporting proposals that the important effects of thyroid hormone on state 3 are on the ATP synthase or the adenine nucleotide translocator.     

Inhibition of oxidative phosphorylation and respiration by ozone in tobacco mitochondria

Ozone was found to inhibit oxidative phosphorylation and oxygen uptake in mitochondria of tobacco leaves (Nicotiana tabacum, L. var. White Gold). The inhibition appeared to occur at both substrate and electron-transport chain levels. The inhibition increased with the length of exposure to ozone, however, the phosphorylative system was more sensitive to ozone than the respiratory system. With mitochondria from detached leaves after being treated with ozone at 1 ppm for 1 hour, uncoupling of phosphorylation was demonstrated without any detectable change in the rate of respiration in the early stage of ozone effect. Inhibition of phosphorylation by ozone was also demonstrated in isolated mitochondria without apparent change in optical density of the mitochondrial suspension at 520 mμ. Therefore, mitochondrial swelling appears not to be a necessary first step for ozone-induced uncoupling of phosphorylation. The evidence suggests that inhibition of oxidative phosphorylation in mitochondria may be a primary effect of ozone in tobacco leaves.

Sucrose and glucose, when fed to the detached tobacco leaves before ozone treatment, tended to raise the phosphorylative activity of mitochondria. Mannitol and lactose were less effective.

     

Effect of bilirubin and its photodegradation products on the respiration of mitochondria isolated from rat liver

In 0.05--0.1 mmol.l-1 concentration, bilirubin inhibits ADP-activated respiration of isolated liver mitochondria; it has no effect on respiration in the absence of ADP. Bilirubin-induced inhibition of respiration is not abolished by serum albumin, but bilirubin bound to serum albumin and the photodegradation products of bilirubin have no inhibitory effect.     

Inhibition of cyanide-resistant respiration in pea cotyledon mitochondria by chloroquine

The action on mitochondrial respiration of a ubiquinone analog, chloroquine, has been studied using purified mitochondria from the cotyledons of germinating peas (Pisum sativum L. var. Homesteader). Chloroquine at 3 millimolar did not inhibit malate or succinate oxidation at pH 7.2, but it did inhibit malate (but not succinate) oxidation at pH 8.2. Cyanide-resistant respiration was also inhibited.     

Tumour necrosis factor-alpha uncouples respiration in isolated rat mitochondria

Recent studies have demonstrated the existence of an intracellular (associated with mitochondria) tumour necrosis factor-alpha (TNF) binding protein. In an attempt to elucidate if this receptor could be involved in TNF action, we have incubated liver isolated mitochondria in the presence of recombinant murine TNF. The results show that the addition of TNF at concentrations as low as 10(-6) U/microl resulted in a clear uncoupling respiration of liver isolated mitochondria, therefore suggesting that TNF can indeed exert intracellular effects, which are possibly linked with its cytotoxic mechanism.     

Inhibition of respiration in mitochondria and in digitonin-treated rat hepatocytes by podophyllotoxin

Summary The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain.     

Hyperglycemia does not alter state 3 respiration in cardiac mitochondria from type-I diabetic rats

The possible role of nitric oxide on the exercise-induced changes in bleomycin-detectable iron (BDI) in the liver, spleen, bone marrow cells and heart was investigated. Female Sprague-Dawley rats were randomly assigned to four groups: S1 (Sedentary), S2 (Sedentary + L-NAME [N-nitro-L-arginine methyl ester]), E1 (Exercise) and E2 (Exercise + L-NAME). Animals in the E1 and E2 swam for 2 h/day for 3 months. L-NAME in the drinking water (1 mg/ml) was administrated to rats in the S2 and E2 groups for the same period. At the end of the 3rd month, nitrite and nitrate (NOx), BDI and non-heme iron (NHI) contents in the liver, spleen, bone marrow cells and heart were measured. The ratio of BDI/NHI was calculated. The exercise induced a significant increase in NOx and BDI contents and/or BDI/NHI ratio in the spleen, bone morrow cells and heart. Treatment with L-NAME, an inhibitor of NOS, led to a significant decrease in NOx and an increase in BDI levels and BDI/NHI ratios in these tissues. The correlative analysis showed that there is significantly positive correlation between NOx levels and BDI contents and/or BDI/NHI ratios in the spleen, bone marrow cells and heart. These results suggest that the increased nitric oxide might be one of the reasons leading to the increased BDI levels in these tissues in the exercised rats. In contrast to the above tissues, in the liver, exercise led to a significant decrease rather than increase in BDI levels and BDI/NHI ratios with a significant increase in NOx contents. Treatment with L-NAME led to a significant increase in BDI levels and BDI/NHI ratios and a decrease in NOx contents in the tissue. These findings plus the results reported by others imply that nitric oxide might have an inhibitory effect on BDI in the liver.     

The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochondria

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A.castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.     

An analysis of the control of phosphorylation-coupled respiration in isolated plant mitochondria

The control of phosphorylation-coupled respiration in isolated turnip (Brassica rapa) mitochondria was investigated according to the principles of metabolic control analysis as developed by H. Kacser and J. A. Burns ([1973] Symp Soc Exp Biol 32: 65-104) and R. Heinrich and T. A. Rapoport ([1974] Eur J Biochem 42: 97-105). Inhibitor titration studies were used to determine quantitatively the amount of control exerted by four individual processes—cytochrome bc₁, cytochrome oxidase, H⁺-ATPase, and the adenine nucleotide carrier—on respiratory flux under ADP-excess (state 3) and ADP-limited (state 4) conditions with a range of respiratory substrates. Under state 3 conditions control strength was found to be distributed between cytochrome oxidase, cytochrome bc₁, and H⁺-ATPase in decreasing order of importance. The adenine nucleotide carrier exerted no control on respiratory flux under these conditions. Control strength at each step was found to vary with different substrates and with the respiratory flux as altered by ADP supply, i.e. virtually zero control strength at cytochrome oxidase and cytochrome bc₁ under state 4 conditions.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and alpha-ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1-50 microM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and -ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1–50 μM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

The effect of nucleotides and inhibitors on respiration in isolated wheat mitochondria

The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticum aestivum L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re-established coupled respiration in the presence of the dinucleotides.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.