Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

An investigation on the effect of oligomycin on state-4 respiration in isolated rat-liver mitochondria

The inhibitory action of oligomycin on State-4 respiration in rat-liver mitochondria has been investigated in detail with regard to the extent, mode and characteristics of the inhibition. The possibility that this effect may be due either to some damage of the mitochondrial preparation used or to the presence of heavy contaminations by microsomes has been excluded. It has been found that the concentration of specific binding sites is the same in State 4 as in State 3. The extent of the inhibition appears to be related to the ADP concentration, rather than to $\text{ATP}\text{ADP}$ ratios. The inhibition of this antibiotic on State-4 respiration does not depend on the experimental conditions used (i.e., choice of substrates or composition of the reaction medium). In agreement with these observations, it has been found that the membrane potential of State 4 is significantly increased when oligomycin is added. All these results provide further evidence to the conclusion that a large portion of State-4 respiration is linked to phosphorylation.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

An oligomycin-resistant Mg2+-dependent ATPase in rabbit bone marrow mitochondria

Summary Rabbit bone marrow mitochondria isolated by differential centrifugation showed typical oxypolarographic tracings with glutamate oxidation with ADP:O ratio of 2.9. Similar results were obtained with liver mitochondria of the same animal. When marrow mitochondria were oxydizing a substrate such as glutamate, added MgCl₂ markedly stimulated state-4 respiration giving a respiratory rate identical to that of state-3. In contrast, no Mg²⁺-stimulation was observed with liver mitochondria. Oligomycin completely blocked the stimulation by Mg²⁺ but further addition of 2,4-dinitrophenol reactivated the oxygen consumption by uncoupling. Further purification of marrow mitochondria by density gradient centrifugation in Percoll provided identical oxypolarographic results. Moreover, when marrow mitochondria were incubated without Mg²⁺, they showed a low ATPase activity that was stimulated by 2,4-dinitrophenol and blocked by oligomycin. The presence of Mg²⁺ in the incubation medium uncovered an additional ATPase activity which was resistant to oligomycin and apparently unaffected by 2,4-dinitrophenol. It is concluded that bone marrow mitochondria possess two types of ATPase activity distinguished on the basis of their reactivity with oligomycin, 2,4-dinitrophenol and Mg²⁺.Abbreviations EDTA ethylenediamine tetraacetate - DNP 2,4-dinitrophenol - BSA bovine serum albumin - BMM bone marrow mitochondria - LM liver mitochondria - Oligo. oligomycin - Anti A antimycin A Howard Hughes Investigator.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation

The effects of three tetrachlorobiphenylols [2′,3′,4′,5′-tetrachloro-2-biphenylol (1); 2′,3′,4′,5′-tetrachloro-4-biphenylol (2); and 2′,3′,4′,5′-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3′,5,5′-tetrachloro-4,4′-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase)] activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1–3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5–7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (≥0.4 μM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1–7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O₂ consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.     

Effect of scorpion Heterometrus fulvipes venom on metabolism of rat liver mitochondria

The scorpion venom Heterometrus fulvipes was able to uncouple the state-4 respiration of rat liver mitochondria. The venom caused mitochondrial swelling and was found to be inhibitory to mitochondrial succinate and glutamate dehydrogenases. Bovine serum albumin was able to protect against mitochondrial swelling.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na⁺ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na⁺ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.     

Effect of rubomycin, carminomycin and adriamycin on respiration in liver mitochondria in various metabolic states, respiratory control and ADP/O ratio

A comparative study on the effects of antitumour antibiotics of the anthracycline group (rubomycin, carminomycin and adriamycin) on respiration and oxidative phosphorylation in liver mitochondria in various metabolic states has been carried out for the first time. It was shown that the antibiotics under study cause partial inhibition of mitochondrial state 3 respiration, which is eliminated by an uncoupler. Treatment of liver mitochondria with the antibiotics decreases the ADP/O and respiratory control values and stimulates state 4 respiration. The latter is partly inhibited by oligomycin. The uncoupled respiration is decelerated in the presence of the antibiotics. Under these conditions the oxidation of succinate is inhibited by lower concentrations of the antibiotics than that of NAD+-dependent substrates. It was shown that the maximal activity is exerted by the most polar agent carminomycin, while the hydrophobic rubomycin is the least active. The experimental results are discussed in terms of the toxic effect of antitumour antibiotics.     

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.     

Respiration in Eggs of the Echiuroid, Urechis unicinctus, Before and After Fertilization

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization-induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p-phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization-induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.     

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

Stimulation of respiration by mitogens in rat thymocytes is independent of mitochondrial calcium.

The role of calcium in the control of respiration by the mitogen concanavalin A (ConA) was investigated in rat thymocytes. ConA induced an increase in both mitochondrial respiration and the mitochondrial calcium pool. The stimulation of respiration was shown to be independent of the increase in mitochondrial calcium: the calcium pool declined after 3 min, whereas the respiration increase was persistent, and was not affected by depletion of the calcium pool or by buffering intracellular Ca2+ transients with quin2. The mitogen phytohaemagglutinin stimulated respiration to the same extent as ConA, but did not increase the mitochondrial calcium pool. In addition, respiration was unaffected by changes in the mitochondrial calcium pool induced by increasing or decreasing extracellular calcium. These results indicate that control of respiration is not located in the Ca2+-sensitive mitochondrial dehydrogenases. The ConA-induced increase in respiration could be blocked by oligomycin, suggesting control by cytoplasmic ATP turnover, and was not associated with detectable changes in NAD(P)H fluorescence, indicating a balance between increased electron transfer and increased supply of reduced substrates.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

On the mechanism of oligomycin inhibition of Ca-induced mitochondrial respiration

The addition of oligomycin in the presence of Ca²⁺ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca²⁺-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca²⁺-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca²⁺ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.