Effects of standardized extract of Ginkgo biloba leaves EGb761 on mitochondrial functions: mechanism(s) of action and dependence on the source of mitochondria and respiratory substrate.

In this work, the effects of standardized dry extract of Ginkgo biloba leaves, EGb761, on the respiration of rat heart and liver mitochondria were investigated. We revealed uncoupling of oxidative phosphorylation in rat heart mitochondria by EGb761 which was not observed in liver mitochondria respiring on pyruvate + malate; oxidation of succinate in heart mitochondria was inhibited by EGb761, concentration-dependently, almost completely at 1.00 mg/mL. Uncoupling effect of EGb761 was found to be due to increase in H(+) and K(+) permeability of inner membrane of mitochondria which is most likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. EGb761 depressed State 3 respiration with pyruvate + malate (similarly in heart and liver mitochondria) and succinate (stronger than with pyruvate + malate) but not respiratory chain Complex IV; inhibition of respiration was not restored by uncoupler indicating the inhibitory action of EGb761 on the respiratory complexes preceding to Complex IV and/or on the substrate transport. Moreover, EGb761 rapidly reduced pure cytochrome c. This property of EGb761 together with the observed uncoupling of oxidative phosphorylation and reduction of H2O2 accumulation may be beneficial for the cell in the prevention of apoptosis and protection of cellular functions in pathological situations.

The effect of Leonurus cardiaca herb extract and some of its flavonoids on mitochondrial oxidative phosphorylation in the heart.

Motherwort (Leonurus cardiaca) possesses antibacterial, antioxidant, anti-inflammatory, and analgesic activities, and is used as a complementary remedy to improve heart function and blood circulation. Since cardiovascular diseases are often associated with an alteration of mitochondria, the main producers of ATP in cardiac muscle cells, the aim of our work was to determine bioactive constituents present in motherwort aerial parts extract in ethanol and investigate their effects on the functions of cardiac mitochondria. Quantitative determination of polyphenols in L. cardiaca herb extract was performed by HPLC. Mitochondrial respiration rates were evaluated using a Clark-type oxygen electrode. Mitochondrial ROS generation was determined fluorimetrically with Amplex Red and horseradish peroxidase. The results showed that constituents (chlorogenic acid, orientin, quercetin, hyperoside, and rutin) of L. cardiaca herb extract uncouple (by 20-90 %) mitochondrial oxidation from phosphorylation, partially inhibit (by ~ 40 %) the mitochondrial respiratory chain in cases of pyruvate and malate as well as succinate oxidation, and effectively attenuate the generation of free radicals in mitochondria. Since partial uncoupling of mitochondria, respiratory inhibition, and decreased ROS production are proposed as possible mechanisms of cardioprotection, our results imply that L. cardiaca herb extract could be a useful remedy to protect cardiac muscles from the effects of pathogenic processes.

Influence of ethanol extract of Ginkgo biloba leaves on the isolated rat heart work and mitochondria functions.

In this study, we attempted to elucidate whether the effects of ethanol extract of Ginkgo biloba leaves (GBE) observed previously on isolated rat heart mitochondria may be realized in situ (in case of isolated heart perfused under normal conditions and under ischemia-reperfusion). We found that GBE at low concentrations (0.01, 0.05, and 0.1 µL/mL) does not affect the heart rate and parameters of electrocardiogram (ECG) but produces a small increase in the coronary flow. Higher concentration of GBE (0.2 and 0.3 µL/mL) diminished the heart rate, decreased the coronary flow, and tended to enhance the parameters of ECG. The contractility of isolated rat heart and mitochondrial nicotinamide adenine dinucleotide reduced form fluorescence decreased in a GBE concentration-dependent manner. Mitochondria isolated from hearts pre-perfused with GBE (0.05 µL/mL) for 20 minutes before nonflow global ischemia-reperfusion (45 min/15 min) showed higher respiratory rates with pyruvate + malate in state 2 and state 3, higher respiratory control index, and diminished H2O2 generation compared with untreated group. Higher GBE concentration, 0.4 µL/mL, had no effect on H2O2 generation and did not prevent the ischemia-reperfusion-induced decrease of pyruvate + malate oxidation in state 3 but even enhanced it. However, in the case of nonischemic perfusions, this GBE concentration had no significant effect on these parameters of respiratory functions of isolated heart mitochondria.

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

K(ATP) 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 K(ATP) 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 degrees C). Our results showed that both pinacidil and diazoxide (60-1250 muM) 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 muM) 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 K(ATP) 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 K(ATP) channel openers.

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. H(2)O(2) 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.

Fatty Acid Oxidation and Mitochondrial Morphology Changes as Key Modulators of the Affinity for ADP in Rat Heart Mitochondria.

Fatty acids are the main respiratory substrates important for cardiac function, and their oxidation is altered during various chronic disorders. We investigated the mechanism of fatty acid-oxidation-induced changes and their relations with mitochondrial morphology and ADP/ATP carrier conformation on the kinetics of the regulation of mitochondrial respiration in rat skinned cardiac fibers. Saturated and unsaturated, activated and not activated, long and medium chain, fatty acids similarly decreased the apparent KmADP. Addition of 5% dextran T-70 to mimic the oncotic pressure of the cellular cytoplasm markedly increased the low apparent KmADP value of mitochondria in cardiac fibers respiring on palmitoyl-l-carnitine or octanoyl-l-carnitine, but did not affect the high apparent KmADP of mitochondria respiring on pyruvate and malate. Electron microscopy revealed that palmitoyl-l-carnitine oxidation-induced changes in the mitochondrial ultrastructure (preventable by dextran) are similar to those induced by carboxyatractyloside. Our data suggest that a fatty acid oxidation-induced conformational change of the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier (M-state to C-state, condensed to orthodox mitochondria) may affect the oxidative phosphorylation affinity for ADP.

Effects of intrusive force on selected determinants of pulp vitality.

OBJECTIVE: To determine the activity of aspartate aminotransferase (AST) in the pulp of orthodontically intruded teeth and to test the sensitivity of these teeth by means of electrical pulp testing (EPT). MATERIALS AND METHODS: The study sample consisted of 21 healthy subjects who needed extraction of first premolars for orthodontic reasons. In every subject, one premolar included in a 0.016''- 0.022'' stainless steel spring from the first molar and loaded by the force was regarded as a test tooth. The magnitude of the intrusive tipping force for every tooth was calculated with the use of ANSYS 10.0 software. The contralateral premolar was used as a control tooth. After 7 days, the spring was removed, and EPT was applied to test and control teeth. The teeth were extracted, and the dental pulp was removed. AST activity in the pulp was determined spectrophotometrically at 20 degrees C. RESULTS: Estimated mean AST activity values ranged from 0.572 +/- 0.097 U/mg in the test teeth to 0.348 +/- 0.053 U/mg in the control teeth (P < .01). The EPT test showed significant differences between test and control teeth (P < .001). The mean estimated magnitude of the intrusive tipping force was 61 +/- 4.5 g. CONCLUSION: Seven days of orthodontic intrusion can cause metabolic changes in the pulp expressed by increased AST activity. The increased threshold in the pulp reaction to EPT indicates changes in the neural response of the pulp.

Effect of Ginkgo biloba extract on the rat heart mitochondrial function.

Ginkgo biloba L. (Ginkgoaceae) originated from China, first introduced to Europe in the 18th century, it is now distributed all over the world. The leaves of Ginkgo biloba include a rich complex of active compounds responsible for various pharmacological properties. Ginkgo biloba extract improves blood circulation, protects against oxidative cell damage, blocks platelet aggregation that could be important for prevention of cardiovascular diseases. Therefore the fluid extract from Ginkgo biloba leaves was prepared and tested for it is effect on rat mitochondrial function. Our data showed that 0.5 microl/ml of GE (containing 0.57 ng/ml of rutin, 0.23 ng/ml of quercitrin, 0.105 ng/ml of hyperosid and 0.02 ng/ml of quercetin) had no effect on the State 2 respiration rate of mitochondria with all used substrates: pyruvate+malate, succinate and palmitoyl-L-carnitine. Further increase in GE concentration (2 and 4 microl/ml), increased the State 2 respiration rate with all respiratory substrates in a dose-dependent manner (by 35-116%). The State 3 respiration rate was not affected by GE. In order to identify which compounds of GE could be responsible for the observed effects, we measured the effect of pure flavonoids: rutin, quercetin, hyperosid and quercitrin on mitochondrial respiration. All flavonoids (except of hyperosid) at maximal used concentration, comparable/identical to that in GE, stimulated the State 2 respiration rate only by 8-20%, i.e. less effectively as compared to GE. Therefore, for the explanation of the GE-induced uncoupling of oxidative phosphorylation, other biologically active compounds of GE have to be taken into account in future studies.

Influence of propolis water solution on heart mitochondrial function.

The effect of propolis water solution (PWS) on the respiration of rat heart mitochondria with NAD-linked (pyruvate + malate), FAD-linked (succinate) substrates and fatty acids (palmitoyl-L-carnitine) was investigated in this study. PWS at the lowest concentration of 4 microg mL(-1) of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 microg mL(-1) of PC caused a significant decrease of basal (24 and 54%) and maximal (58 and 70%) respiration rates with succinate as substrate. At these PWS concentrations the oxidation of pyruvate + malate and palmitoyl-L-carnitine was diminished to a lower degree: the basal respiration rate decreased by 13-18% and the maximal respiration rate by 15-28%. Succinate oxidation was affected, probably because of the inhibition of succinate dehydrogenase by the 1,2-benzenedicarboxylic acid esters found in PWS. The PWS-caused decrease in the mitochondrial respiration rate with pyruvate + malate and fatty acids could be due to diminished activities of respiratory chain complexes and/or ADP/ATP translocator.

A user-friendly PC-based data acquisition and analysis system for respirometric investigations.

We have developed an easy-to-use computer-based system for recording, displaying, storing and analyzing signals generated by Clark-type oxygen electrodes. A user-friendly interface of Windows-based program BioMed significantly increases the productivity of investigations. It allows to process, control, present and archive the experimental data in real time. A 12-bit analog-to-digital-converter, analog and digital filters, a possibility to zoom the obtained respiratory curves and calculation of the respiration rates by a linear regression method increase the resolution of the estimated oxygen consumption rates. The new system enables to register even small changes, such as 3-5 ngatoms O/min, in respiration rates of biological objects -- enzymes, mitochondria and permeabilized muscle fibers. The system has been developed and is regularly used for the respirometric investigations at the Laboratory of Biochemistry, Institute for Biomedical Research, Kaunas University of Medicine.

Cytochrome C effect on respiration of heart mitochondria: influence of various factors.

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.

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.

Change in dental pulp parameters in response to different modes of orthodontic force application.

OBJECTIVES: (1) To evaluate dental pulp sensitivity by electrical pulp testing and measure aspartate aminotransferase activity in the pulp after 14 days of orthodontic intrusion, and (2) to compare those measurements with measurements obtained in teeth after 7 days of intrusion and 7 days of rest. MATERIALS AND METHODS: The study sample included 13 subjects (mean age = 16.5 +/- 2.7 years). For every subject, before extraction, two contralateral premolars were included in a spring and loaded by a force. Two study groups were formed: Group A, teeth with 14 days of mechanical load, and Group B, teeth with 7 days of mechanical load plus 7 days of rest. Electrical pulp testing and aspartate aminotransferase activity measurements were performed after 14 days in all tested teeth. After extraction, aspartate aminotransferase activity in the pulp was determined spectrophotometrically at 20 degrees C. RESULTS: Mean aspartate aminotransferase activity values were 0.21 U/mg (SD = 0.15) in Group A and 0.27 U/mg (SD = 0.17) in Group B. Mean electrical pulp testing readings were 38.92 microA (SD = 24.61) in Group A and 36.77 microA (SD = 26.84) in Group B. Mean values of the intrusive force magnitude did not differ in both groups. CONCLUSIONS: Different durations of orthodontic intrusion, defined as 14 days of load and 7 days of load followed by 7 resting days, were not reflected by electrical pulp testing or by aspartate aminotransferase activity levels in the pulp of the affected teeth. However, the response threshold to electrical pulp stimulation was elevated in all tested teeth.

Myocardial protective effect of warm blood, tepid blood, and cold crystalloid cardioplegia in coronary artery bypass grafting surgery.

AIM: To compare the myocardial effects of cardioplegia by warm blood, tepid blood, and cold crystalloid during coronary artery bypass grafting (CABG). METHODS: Patients undergoing CABG surgery at Kaunas University Hospital between 2000 and 2004 were randomized into three groups (n=156), receiving a different method of cardioplegia. Intermittent antegrade warm blood cardioplegia was used in 51 patients, tepid blood cardioplegia in 50 patients, and cold crystalloid cardioplegia in 55 patients. Mitochondrial function, myocardial ultrastructure, troponin T, and hemodynamic and clinical data were analyzed after surgery. RESULTS: All cardioplegic methods similarly affected structural and functional properties of mitochondria and coupling of oxidative phosphorylation, and all lowered the capacity of mitochondria to synthesize ATP. Ultrastructure of myocytes showed slight to moderate injury in the cold crystalloid cardioplegia group. The concentration of troponin T was significantly lower in the warm blood cardioplegia group than in the tepid blood cardioplegia and cold crystalloid cardioplegia groups at 12 hours (0.8+/-0.1 ng/mL, 1.9+/-0.2 ng/mL, and 2.8+/-0.3 ng/mL, respectively; P<0.001) and 24 hours after surgery (1.0+/-0.1 ng/mL, 2.2+/-0.3 ng/mL, and 2.5+/-0.3 ng/mL, respectively; P<0.001). Echocardiographic examination after surgery revealed that the changes in the left ventricle diastolic function were similar in all groups, and that systolic function did not change. The warm blood cardioplegia group showed shorter duration of intubation and hospitalization. There were no differences in the need of catecholamine administration, incidence of complications, and duration of stay in the intensive care unit. CONCLUSIONS: Intermittent antegrade warm blood cardioplegia provides better myocardial protection during CABG surgery, as assessed by the lower release of troponin T, lower fluid balance, shorter duration of tracheal intubation and hospital stay.

Diazoxide and pinacidil uncouple pyruvate-malate-induced mitochondrial respiration.

We investigated the effects of K(ATP) channel openers diazoxide and pinacidil on the respiration rate and membrane potential (deltapsi) of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8-1348.3 microM) increased the V2 (-ADP) respiration rate accordingly by 13-208% and 30-273% and decreased the deltapsi 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.

Adenine nucleotide translocase mediates the K(ATP)-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 microM) and BA (20 microM) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K(ATP) 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 K(ATP) channel openers with adenine nucleotides. CAT also abolished K+-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K(ATP)-channel-openers-induced K+ flux through the inner mitochondrial membrane.