Adolfas Toleikis

The role of adenine nucleotide translocators in regulation of oxidative phosphorylation in heart mitochondria

The regulative role of adenine nucleotide translocators (ANTs) in oxidative phosphorylation has been estimated by the titration of respiration of isolated rabbit heart mitochondria with carboxyatractyloside in the presence of a non‐rate limiting creatine phosphokinase ADP‐regenerating system. It has been established that the respiration rate is not controlled by ANTs in the two extreme states, state 3 and state 4. On the other hand, at an intermediate respiration rate (30–70% of the state 3 respiration, which roughly corresponds to that under physiological conditions) the ANT control coefficient had a value of 0.62–0.75. Thus, ANTs seem to play a key role in the regulation of oxidative phosphorylation.

In the eye of the storm: mitochondrial damage during heart and brain ischaemia.

We review research investigating mitochondrial damage during heart and brain ischaemia, focusing on the mechanisms and consequences of ischaemia‐induced and/or reperfusion‐induced: (a) inhibition of mitochondrial respiratory complex I; (b) release of cytochrome c from mitochondria; (c) changes to mitochondrial phospholipids; and (d) nitric oxide inhibition of mitochondria. Heart ischaemia causes inhibition of cytochrome oxidase and complex I, release of cytochrome c, and induction of permeability transition and hydrolysis and oxidation of mitochondrial phospholipids, but some of the mechanisms are unclear. Brain ischaemia causes inhibition of complexes I and IV, but other effects are less clear.

Adenine Nucleotide Translocase Mediates the KATP-Channel-Openers-Induced Proton and Potassium Flux to the Mitochondrial Matrix

KATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 μM) and BA (20 μM) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the KATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of KATP channel openers with adenine nucleotides. CAT also abolished K+-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of KATP-channel-openers-induced K+ flux through the inner mitochondrial membrane.

The effect of crataegus fruit extract and some of its flavonoids on mitochondrial oxidative phosphorylation in the heart.

Crataegus (Hawthorn) fruit extracts (CE) are widely used for the treatment of various cardiovascular diseases (arrhythmias, heart failure, myocardial weakness, etc). Despite the fact that many of these diseases are associated with disturbances of the mitochondria, no data have been found on the effect of CE on their function. The aim of this study was to perform an oxygraphic investigation of the effect of CE (in concentration range from 70 ng/mL to 13.9 µg/mL of Crataegus phenolic compounds (PC)) and its several pure flavonoids on isolated rat heart mitochondria respiring on pyruvate + malate, succinate and palmitoyl‐L‐carnitine + malate. CE at doses under 278 ng/mL of PC had no effect on mitochondrial functions. At concentrations from 278 ng/mL to 13.9 µg/mL of PC, CE stimulated State 2 respiration by 11%–34% with all used substrates, and decreased the mitochondrial membrane potential by 1.2–4.4 mV measured with a tetraphenylphosphonium‐selective electrode and H2O2 production measured fluorimetrically. Similar uncoupling effects on mitochondrial respiration were observed with several pure CE flavonoids. The highest CE concentration also slightly reduced the maximal ADP‐stimulated and uncoupled respiration, which might be due to inhibition of the mitochondrial respiratory chain between flavoprotein and cytochrome c. Whether or not the uncoupling and other effects of CE on mitochondria may be realized in vivo remains to be determined. Copyright

Diazoxide and Pinacidil Uncouple Pyruvate-Malate-Induced Mitochondrial Respiration

We investigated the effects of KATP channel openers diazoxide and pinacidil on the respiration rate and membrane potential (ΔΨ) of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8–1348.3 μM) increased the V2 (-ADP) respiration rate accordingly by 13–208% and 30–273% and decreased the ΔΨ by 2–17% and 6–55%. These effects were also similar in the respiration medium without K+. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.

Effects of Ginkgo biloba extract on heart and liver mitochondrial functions: mechanism(s) of action.

Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H2O2 generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.

Direct effects of K(ATP) channel openers pinacidil and diazoxide on oxidative phosphorylation of mitochondria in situ.

KATP channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of KATP channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37°C). Our results showed that both pinacidil and diazoxide (60-1250 µM) in a concentration-dependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 ± 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 ± 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 µM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 ± 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 ± 0.2, n=4) by 4-27%. Thus, common effect for both KATP channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by KATP channel openers.

What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent Km ADP of oxidative phosphorylation increased significantly, by 70-100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent Km ADP value (234+/-24 microM ADP) compared with other preparations (14-28 microM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.

The function of ATP/ADP translocator in the regulation of mitochondrial respiration during development of heart ischemic injury.

Inhibitor titration studies were carried out in order to quantify the amount of control exerted by ATP/ADP translocator on the rate of succinate, palmitoylcarnitine + malate and pyruvate + malate oxidation in ischemia-damaged heart mitochondria. It was shown that after 30 min of total ischemia in vitro the maximal value of the control coefficient of the translocator was as high as in the control: 0.5-0.72 (succinate), 0.8-0.87 (palmitoylcarnitine + malate), 0.83-0.95 (pyruvate+malate). However, the translocator-controlled range of respiratory rates of ischemic mitochondria was narrower than that of normal mitochondria. The control coefficient of the translocator close to State 3 and State 4 was equal to 0-0.15. After 45 min ischemia the maximal value of the translocator control coefficient decreased by 25-30% in comparison with normal mitochondria with all substrates investigated. This value was preserved within a wide range of mitochondrial respiratory rates including the maximal rate in State 3. It was found that the amount of ATP/ADP translocator in mitochondria decreased by 20% after only 45 min ischemia. Our data show that the ATP/ADP translocator is one of the most important steps in regulation of oxidative phosphorylation in isolated mitochondria during development of heart ischemic injury.

The Effect of Collagenase and Temperature on Mitochondrial Respiratory Parameters in Saponin-skinned Cardiac Fibers

The results of a comparative study of the respiration rates of mitochondria in saponin-skinned rat cardiac fibers (SF) and in fibers treated with saponin and collagenase (SCF) suggest that only about half of the whole population of mitochondria manifest their activity in SF, in contrast to SCF, in response to extracellular substrates of oxidative phosphorylation. The apparent Km value for ADP with succinate as substrate, which was as high as 330±32 μM in SF in SF at 20 °C, decreased about 2-fold in SCF at the same temperature and in SF at 37 °C, and decreased further to 67±8 μM in SCF at 37 °C. Thus, weakening or breaking of cellular contacts by collagenase and the temperature-dependence of diffusion of substrates such as ADP, seem to be important factors that determine the respiratory activity and regulatory parameters of mitochondria in saponin-permeabilized cardiomyocytes.