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PubMed1976

[Mechanism of opiate of oxidative phosphorylation in mitochondria].

Effect of morphine, codeine, dionine and nalorphine on the oxidative phosphorylation in rat liver mitochondria was studied. Morphine is found to inhibit both ATP-synthetase and ATP-ase activities in mitochondria, but not in submitochondrial particles. Morphine-suppressed oxidative phosphorylation was competitively reversed with high concentrations of ADP, but not of inorganic phosphate. The effect of other opiates (i.e. codeine, dionine, nalorphine) was similar. It is suggested, that opiates inhibit the transport of adenine nucleotides through inner mitochondrial membrane, as it does atractyloside. A significance of the hydrophobic interaction between the inhibitor and adenine nucleotide translocase is outlined, since the degree of the inhibition of oxidative phosphorylation is increased with the increase in the number of non-ionized opiate molecules (at alkaline pH values) and in the length of the carbon chain of narcotic molecule as follows: morphine--codeine--dionine--nalorphine.

Adenosine Diphosphate↗

PubMed2000

Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro.

The effect of inhibiting ATP production via oxidative phosphorylation during pericompaction of in vitro produced bovine embryos was investigated. This was achieved by: (i) varying the atmospheric O2 concentration (0, 1, 2, 4 and 7%); (ii) addition of oxidative phosphorylation inhibitors, NaN3 and antimycin A; and (iii) addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation from electron transport. The development of embryos under various O2 concentrations from day 5 to day 7 of development indicated that an optimal concentration occurred at about 2%. Addition of NaN3 revealed that doses above 100 mumol l-1 were toxic to embryo development, but that concentrations of 5-10 mumol l-1 stimulated embryo development by 10-25%. A similar result was observed after addition of 2,4-dinitrophenol, whereas antimycin A was inhibitory at doses as low as 1 mumol l-1. At concentrations of NaN3 or 2,4-dinitrophenol that stimulated embryo development, the number of cells of the resulting blastocysts was also significantly increased. Addition of NaN3 from day 1 of development inhibited subsequent development. Metabolic data of NaN3-treated embryos revealed that O2 uptake was significantly lower at inhibitory doses (100 mumol l-1). A significant (P < 0.05) log linear increase in glucose uptake was measured between the three concentrations of NaN3 (0, 10 and 100 mumol l-1). These results demonstrate that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro. However, transient (subacute) inhibition appears to be beneficial to embryo development and the number of cells, perhaps by creating a more favourable intracellular environment.

2,4-Dinitrophenol↗

PubMed2000

Genetic control of oxidative phosphorylation and experimental models of defects.

Energy in the form of ATP is continually produced by all cells for normal growth and function. Anaerobic glycolysis can provide enough ATP for some cells, but energetic cells such as cardiomyocytes and neurons require a more efficient ATP supply, which can only be provided by mitochondrial oxidative phosphorylation. Invented by bacteria that became symbiotically associated with other bacteria to form eukaryotic cells billions of years ago, oxidative phosphorylation carries with it a genetic legacy that is unique. The mitochondrial oxidative phosphorylation complexes are assembled from protein subunits encoded by both the mitochondrial genome (mtDNA) and the nuclear genome (nDNA, located in the chromosomes). The mtDNA is a remnant genome of the bacterial progenitor of mitochondria, and (unlike the biparental diploidy that characterizes the nuclear genome) is present in thousands of copies per cell, is replicated through life, and is inherited (cytoplasmically) only from the female parent. Oxidative phosphorylation comprises five multimeric enzyme complexes that act as a redox pathway, passing electrons from oxidizable intermediates produced by the metabolism of food to molecular oxygen in the mitochondrial matrix, while producing an electrochemical gradient by pumping protons into the intermembranal space. The proton (hydrogen ion) gradient across the inner mitochondrial membrane is used by the H+-transporting ATP synthase to produce ATP from ADP and inorganic phosphate, with the protons released into the mitochondrial matrix then combining with electronated oxygen to form water. Many of the details regarding the control of the synthesis of oxidative phosphorylation enzyme complexes remain to be elucidated. Transmitochondrial cell culture systems have been developed so that defective oxidative phosphorylation can be studied in a controlled nuclear background. Such systems may soon enable the development of mtDNA 'knockout' mice in order to better model mtDNA transmission and mitochondrial disease.

3T3 Cells↗

PubMed1994

Effects of cadmium on the control and internal regulation of oxidative phosphorylation in potato tuber mitochondria.

The effect of cadmium on the distribution of control over oxidative phosphorylation in potato tuber mitochondria was quantified by measuring control coefficients using top-down metabolic control analysis. Oxidative phosphorylation was divided into three subsystems, namely substrate oxidation, the phosphorylation reactions and the proton leak. The control exerted by each of these subsystems over the system fluxes, the value of the protonmotive force and the effective P/O ratio was quantified in the presence of different concentrations of free cadmium (up to 21 microM). Cadmium is known to stimulate the proton leak and inhibit the substrate oxidation reactions, but it had little effect on the distribution of control over the system variables except to shift the pattern to lower rates. Control exerted by particular subsystems appeared to change or to stay the same as cadmium was varied, depending on whether the control coefficients were presented as a function of respiration rate or protonmotive force. The regulatory strength of protonmotive force on the system variables was also calculated, as partial internal response coefficients. These coefficients changed with ATP turnover rate and with cadmium concentration, showing how the internal regulation of oxidative phosphorylation shifts under different conditions. The values of control coefficients and partial internal response coefficients show where control lies and how intermediates regulate the system variables under different conditions of ATP demand and external effector (i.e. cadmium) concentration. However, they are not useful for identifying the sites of action of external effectors, for which elasticity and regulation analysis must be used.

Cadmium↗

PubMed1997

Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria.

The effect of myocardial ischemia on mitochondrial oxidative phosphorylation was investigated using isolated, buffer-perfused rabbit hearts. After 45 min of global ischemia, oxidative phosphorylation was decreased only in the subsarcolemmal population of mitochondria with all substrates tested. The oxidation of N,N,N',N' tetramethyl p-phenylenediamine-ascorbate, an electron donor to cytochrome oxidase via cytochrome c, was decreased in subsarcolemmal mitochondria [ischemia (n = 6): 76 +/- 3 vs. control (n = 5): 105 +/- 6 nanoatoms O.min-1.mg-1, P < 0.01] but not in interfibrillar mitochondria. Only minor morphological changes were observed by electron microscopy in the isolated mitochondria after ischemia. Neither cytochrome oxidase activity measured under conditions for maximal activity nor the apparent Michaelis constant and maximum velocity values of the two cytochrome c binding sites were different in subsarcolemmal mitochondria isolated from ischemic and control hearts. The cytochrome c content was decreased in subsarcolemmal mitochondria after ischemia (ischemia: 0.111 +/- 0.013 vs. control: 0.156 +/- 0.007 nmol/mg protein, P < 0.05). Thus ischemia decreased the rate of oxidative phosphorylation through cytochrome oxidase selectively in intact subsarcolemmal mitochondria. Ischemic damage to the terminal segment of the electron transport chain involves a decrease in the content of cytochrome c, whereas the expressible catalytic activity of cytochrome oxidase remains unchanged.

Adenosine Diphosphate↗

PubMedDate not supplied

[Respiration and oxidative phosphorylation in isolated liver cells under freezing conditions].

Respiration and oxidative phosphorylation of hepatocytes were studied as affected by low temperatures and cryoprotectors. Fast freezing of these cells down to -196 degrees C with the presence of mentioned substances causes considerable destruction of their structures. The intensity of the oxygen endogenic uptake lowers significantly, respiration and oxidative phosphorylation become uncoupled. Two-stage freezing down to -196 degrees C also causes a complete uncoupling of the oxidation and phosphorylation processes in the hepatocyte mitochondria. When freezing hepatocytes down to -196 degrees C by the multistage programme with the presence of cryoprotectors in a 10% concentration, the mitochondria retain 20, 30 and 40% of their functional properties, respectively.

Animals↗

PubMed2002

Effect of enzyme deficiencies on oxidative phosphorylation: from isolated mitochondria to intact tissues. Theoretical studies.

The present article briefly summarizes the theoretical studies made by the authors and co-workers on the effect of inborn enzyme deficiencies on oxidative phosphorylation in intact tissues and on the genesis of mitochondrial diseases. The dynamic computer model of oxidative phosphorylation developed previously allowed to extrapolate experimental data (especially: threshold curves describing the dependence of oxygen consumption and ATP turnover on activities/concentrations of particular oxidative phosphorylation enzymes) obtained for isolated muscle mitochondria in state 3 at saturating oxygen concentrations to more physiological conditions prevailing in intact tissues. In particular, theoretical studies demonstrated that the threshold value of the relative activity/concentration of a given mitochondrial complex, below which a significant decrease in the respiration rate takes place, increases with an increase in energy demand. This fact was proposed as a possible explanation of the tissue specificity of mitochondrial diseases. Additionally, a decreased oxygen concentration was shown to increase the threshold value (and flux control coefficient) for cytochrome oxidase. We subsequently developed a model called 'binary mitochondria heteroplasmy', in which there are only two subpopulations of mitochondria: one 'wild-type' and one containing only defected molecules of a given enzyme. In this model we show that a defect has a pronounced effect on oxidative phosphorylation, significantly increasing the threshold value. It was also proposed that a parallel activation in the ATP supply-demand system during an increased energy demand significantly lessens the effect of enzyme deficiencies on oxidative phosphorylation (decreases the threshold value). Finally, the necessity of substrate activation may lead to an instability in the system and to appearance of a second threshold, below which respiration suddenly drops to zero, which is equivalent to the energetic death of a cell.

Adenosine Triphosphate↗

PubMed1995

[The effect of p-chloromercuribenzoate on regulation of oxidative phosphorylation by ADP and ATP and stimulation of liver mitochondrial respiration by palmitate].

The effect of p-chloromercuribenzoate (pCMB) on oxidative phosphorylation and palmitate-stimulated respiration of liver mitochondria has been studied. pCMB (1 microM) does not affect oxidative phosphorylation but reduces the inhibiting effect of ATP on this process. Used at the same concentration, pCMB eliminates the inhibiting effect of ADP and ATP on mitochondrial respiration in the presence of 10 and 20 microM palmitate. pCMB has no effect on inhibition of oxidative phosphorylation by carboxyatractyloside or on palmitate-stimulated respiration of mitochondria. It is concluded that the SH-groups of mitochondria localized in the hydrophilic region outside the inner mitochondrial membrane participate in regulation of oxidative phosphorylation by ATP and in regulation of palmitate-stimulated respiration by ADP + ATP.

Adenosine Diphosphate↗

PubMed1997

Effect of 2',3'-dideoxycytidine on oxidative phosphorylation in the PC12 cell, a neuronal model.

Peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) could result from the previously shown inhibition of mtDNA replication by the action of ddC on the mitochondrial enzyme DNA polymerase gamma. Such inhibition would be expected to impair oxidative phosphorylation, and this was demonstrated in the present study for the PC12 cell, a model of a peripheral neuron. The dramatic rise in lactate formation upon exposure of the cell to ddC indicated that increased glycolysis was needed to produce ATP. A concomitant rise in O2 uptake indicated that oxidative phosphorylation had become uncoupled. When tested in a standard respiratory control system (isolated rat liver mitochondria), however, we found ddC not to be an uncoupler. Rather, the uncoupling most likely resulted from the failure of synthesis of one or more mitochondrial gene products necessary for oxidative phosphorylation. We also observed an important distinction between the manner in which ddC and 3'-azido-3'-deoxythymidine (AZT) act. ddC-exerted inhibition of oxidative phosphorylation was delayed for several days. This is consistent with the inhibition occurring indirectly, most likely as a result of the prior destruction of the mitochondrial genome, which encodes many of the components of the oxidative phosphorylation system. In contrast, we have shown previously that although AZT also impairs replication of the mitochondrial genome (in the Friend murine erythroleukemic cell), it also attacks directly an additional primary target leading to impairment of oxidative phosphorylation; its initial inhibition of this process is immediate, not occurring via inhibition of mitochondrial DNA replication.

Animals↗

PubMedDate not supplied

Uncoupling of oxidative phosphorylation does not induce thermotolerance in cultured Chinese hamster cells.

Two uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), were tested for their ability to modify the survival of cultured Chinese hamster ovary (CHO) and Chinese hamster V79 cells treated with hyperthermia. The uncouplers were used under conditions that inhibit oxidative ATP synthesis, as judged from measurements of cellular ATP levels. Incubation of CHO cells in glucose-free Hanks' balanced salt solution (HBSS) containing 1 mM DNP for 1 h at 37 degrees C followed by reincubation at 37 degrees C in complete growth medium for 3 or 16 h, showed no substantial changes in the 45 degrees C heat survival curve as compared to heated cells not exposed to DNP. Thus, DNP treatment of CHO cells did not induce thermotolerance. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), tested under similar experimental conditions, did alter cellular heat resistance. The major change in the 45 degrees C survival curve of CHO cells pretreated with CCCP was an increase in the width of the shoulder: the Dq value increased from 14 min to 24 min, for the control and CCCP-treated cells respectively. The D0 value did not change appreciably. In contrast, heat-induced thermotolerance (10 min, 45 degrees C + 16 h, 37 degrees C) was characterized primarily by an increase in the D0 parameter from 4 min (unheated cells) to 17 min. Similar results were observed with CCCP-treated V79 cells. The data demonstrate that heat resistance induced by 1.2 microM CCCP was manifest as an increased cellular capacity to accumulate and/or repair hyperthermia damage, rather than an induction of thermotolerance, and that this effect probably was not related to the action of CCCP as an uncoupler of oxidative phosphorylation.

2,4-Dinitrophenol↗

PubMed1996

Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies.

1. The dynamic model of oxidative phosphorylation developed previously for rat liver mitochondria incubated with succinate was adapted for muscle mitochondria respiring on pyruvate. We introduced the following changes considering: (1) a higher external ATP/ADP ratio and an ATP/ADP carrier less displaced from equilibrium; (2) a substrate dehydrogenation more sensitive to the NADH/NAD+ ratio; and (3) the respiratory chain, ATP synthase and phosphate carrier being more displaced from equilibrium. The experimental flux control coefficients already determined in state 3 for respiratory rate and ATP synthesis were used to adjust some parameters. This new oxidative phosphorylation model enabled us to simulate the whole titration curves obtained experimentally in state 3. These curves, which mimic the effect of mitochondrial complex deficiencies on oxidative phosphorylation, show a threshold effect, which is reproduced by the model. 2. the model was also used to simulate other physiological conditions such as (i) state 3.5, conditions in-between state 4 and state 3; and (ii) hypoxic conditions. In both cases a profound change in the pattern of the control coefficients was shown. 3. This model was thus found useful in investigating a variety of new conditions, the most interesting of which can then be experimentally studied.

Adenosine Diphosphate↗

PubMed1997

Inhibition of oxidative phosphorylation, vascular tone, and [Ca2+]i in the perfused rat tail artery.

We investigated the possibility that the inhibition of oxidative phosphorylation in vascular smooth muscle attenuates norepinephrine- or KCl-evoked vasoconstriction with no change in mobilization of intracellular calcium concentration ([Ca2+]i). Experiments were performed in perfused segments of the rat tail artery loaded with the intracellular calcium dye fura 2, in the absence and presence of dinitrophenol or sodium cyanide; inhibition of oxidative phosphorylation was evaluated from the fall in intracellular ATP levels. The metabolic inhibitors reduced vasoconstriction with no change in [Ca2+]i handling, suggesting that 1) inhibition of oxidative phosphorylation attenuates vasoconstriction via a mechanism downstream of [Ca2+]i, and 2) [Ca2+]i homeostasis (both increases and decreases in [Ca2+]i) can be maintained in the presence of inhibitors of oxidative phosphorylation.

Adenosine Triphosphate↗

PubMed1965

Effect of salicylate on oxidative phosphorylation and respiration of mitochondrial fragments.

1. The effects of salicylate on oxidative phosphorylation and respiration were investigated in liver and brain mitochondria, and in sonically prepared mitochondrial fragments. 2. Salicylate was shown to uncouple oxidative phosphorylation in mitochondrial fragments, as well as in intact mitochondria. The effects of salicylate on mitochondria and oxidative phosphorylation resemble those produced by dinitrophenol. 3. The quantitative effects of salicylate on respiration in vitro were shown to be complex. The final effect may be the resultant of an interaction of a multiplicity of factors, such as the tissue, the substrate, mitochondrial and extramitochondrial influences, and the direct effect on the respiratory chain itself.

Journal Article↗

PubMed2000

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat.

BACKGROUND: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation. AIMS: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat. METHODS: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control. RESULTS: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin. CONCLUSIONS: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.

Animals↗

PubMed2006

Control of cellular proliferation by modulation of oxidative phosphorylation in human and rodent fast-growing tumor cells.

The relationship between cell proliferation and the rates of glycolysis and oxidative phosphorylation in HeLa (human) and AS-30D (rodent) tumor cells was evaluated. In glutamine plus glucose medium, both tumor lines grew optimally. Mitochondria were the predominant source of ATP in both cell types (66-75%), despite an active glycolysis. In glucose-free medium with glutamine, proliferation of both lines diminished by 30% but oxidative phosphorylation and the cytosolic ATP level increased by 50%. In glutamine-free medium with glucose, proliferation, oxidative phosphorylation and ATP concentration diminished drastically, although the cells were viable. Oligomycin, in medium with glutamine plus glucose, abolished growth of both tumor lines, indicating an essential role of mitochondrial ATP for tumor progression. The presumed mitochondrial inhibitors rhodamines 123 and 6G, and casiopeina II-gly, inhibited tumor cell proliferation and oxidative phosphorylation, but also glycolysis. In contrast, gossypol, iodoacetate and arsenite strongly blocked glycolysis; however, they did not affect tumor proliferation or mitochondrial metabolism. Growth of both tumor lines was highly sensitive to rhodamines and casiopeina II-gly, with IC(50) values for HeLa cells lower than 0.5 microM, whereas viability and proliferation of human lymphocytes were not affected by these drugs (IC(50) > 30 microM). Moreover, rhodamine 6G and casiopeina II-gly, at micromolar doses, prolonged the survival of animals bearing i.p. implanted AS-30D hepatoma. It is concluded that fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target.

Adenosine Triphosphate↗

PubMed2001

Limits to sustainable muscle performance: interaction between glycolysis and oxidative phosphorylation.

This paper proposes a mechanism responsible for setting the sustainable level of muscle performance. Our contentions are that the sustainable work rate is determined (i) at the muscle level, (ii) by the ability to maintain ATP supply and (iii) by the products of glycolysis that may inhibit the signal for oxidative phosphorylation. We argue below that no single factor 'limits' sustainable performance, but rather that the flux through and the interaction between glycolysis and oxidative phosphorylation set the level of sustainable ATP supply. This argument is based on magnetic resonance spectroscopy measurements of the sources and sinks for energy in vivo in human muscle and rattlesnake tailshaker muscle during sustained contractions. These measurements show that glycolysis provides between 20% (human muscle) and 40% (tailshaker muscle) of the ATP supply during sustained contractions in these muscles. We cite evidence showing that this high glycolytic flux does not reflect an O(2) limitation or mitochondria operating at their capacity. Instead, this flux reflects a pathway independent of oxidative phosphorylation for ATP supply during aerobic exercise. The consequence of this high glycolytic flux is accumulation of H(+), which we argue inhibits the rise in the signal activating oxidative phosphorylation, thereby restricting oxidative ATP supply to below the oxidative capacity. Thus, both glycolysis and oxidative phosphorylation play important roles in setting the highest steady-state ATP synthesis flux and thereby determine the sustainable level of work by exercising muscle.

Adenosine Diphosphate↗

PubMed1993

A threshold membrane potential accounts for controversial effects of fatty acids on mitochondrial oxidative phosphorylation.

The uncoupling effect of free fatty acids on oxidative phosphorylation in mitochondria has been known for more than 35 years. The mechanism of action, however, remains controversial. In this report the physicochemical basis of uncoupling was elucidated by studying the effect of free fatty acids on the proton permeability and membrane potential of proteoliposomes containing reconstituted cytochrome c oxidase (COX). A threshold membrane potential of about 125 mV was identified for fatty acid-induced proton permeability. Only above this potential do free fatty acids translocate protons across the biological membrane. The data explain the controversial effects of long-chain fatty acids on oxidative phosphorylation as well as their role on non-shivering thermogenesis in larger mammals.

Adipose Tissue↗

PubMed1989

Mechanism of uncoupling of oxidative phosphorylation by gramicidin.

The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of delta mu H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and delta pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗