Daiva Majiene

Uncoupling and antioxidant effects of ursolic acid in isolated rat heart mitochondria.

Ursolic acid (1), a pentacyclic triterpene acid, is one of the major components of certain traditional medicinal plants and possesses a wide range of biological effects, such as anti-inflammatory, antioxidative, and cytotoxic activities. Furthermore, 1, when present at 1.6-5 ng/mL concentrations in commercial herbal preparations used for patients with cardiac disorders, may also exert pro-cardiac activities. There are several indirect suggestions that the cardioprotective mechanism of ursolic acid could involve the mitochondria; however the mechanism of action is still not known. Therefore, the effects of 0.4-200 ng/mL ursolic acid (1) on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate, or palmitoyl-l-carnitine plus malate were investigated. It was found that 1 induced a statistically significant uncoupling of oxidative phosphorylation. A statistically significant decrease in H₂O₂ production in the mitochondria was observed after incubation with 5 ng/mL 1. This effect was comparable to the effectiveness of the classical uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Since mild mitochondrial uncoupling has been proposed as one of the mechanisms of cardioprotection, the present results indicate that ursolic acid (1) has potential use as a cardioprotective compound.

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

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.

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP

The present study on saponin‐treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl‐CoA+carnitine, palmitoyl‐L‐carnitine and octanoyl‐L‐carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K m of oxidative phosphorylation for ADP. Octanoyl‐D‐carnitine, as well as palmitate, palmitoyl‐CoA, and palmitoyl‐L‐carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.

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 μgmL−1 of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 μ gmL−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.

The Effect of Ginkgo biloba Extract on Mitochondrial Oxidative Phosphorylation in the Normal and Ischemic Rat Heart

Free radical‐induced myocardial damage and impairment of vascular endothelium‐dependent relaxation are amongst the most important mechanisms responsible for ischemic heart injury. Ginkgo biloba leaf extract (GE) has been reported to improve blood circulation in the brain and have a beneficial impact on the cardiovascular system but its cardioprotective effects have not been elucidated yet. Therefore, this study investigated the influence of GE in 70% ethanol (1:5) administered orally to rats on the functions of isolated heart mitochondria under normal and ischemic conditions. Wistar rats were given GE or ethanol (solvent control) at a dosage of 0.32 mL/kg in drinking water for 10 and 18 days, while the control animals received untreated drinking water. Mitochondrial respiration rates were determined oxygraphically. Pyruvate and malate, succinate or palmitoyl‐l‐carnitine and malate were used as substrates.

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5–1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase. (Mol Cell Biochem 174: 87–90, 1997)

Neuroprotective properties of anthocyanidin glycosides against H2O2-induced glial cell death are modulated by their different stability and antioxidant activity in vitro

The neuroprotective effect of several anthocyanins in combination with their stability and antioxidant/pro-oxidant activity has been investigated against H2O2-induced oxidative stress in C6 glial cells. First it was found that delphinidin (Dp) 3-O-glucoside and 3-O-rutinoside were degraded within an hour, and at the same time stimulated the production of H2O2 in the micromolar concentration range. The stability of peonidin, pelargonidin (Pg), malvidin (Mv) and cyanidin (Cy) 3-O-glucosides and Cy 3-O-rutinoside was significantly higher than that of Dp 3-O-glycosides, with Pg3G showing the highest percent recovery over time. Based on these findings and chemical difference (according to the set of functional groups on the B-ring) of tested anthocyanins Cy3G, Mv3G and Pg3G were selected as candidates for the protection of glial cells against H2O2-induced oxidative stress. It was revealed that Cy3G (5-20μM) and Mv3G (10-20μM) but not Pg3G protected glial cells against H2O2-induced necrotic cell death. Moreover, these anthocyanins sustained the glutathione antioxidant defence system. Finally, to the extent of our knowledge we were the first to demonstrate the protective effect of Cy3G on the resting mitochondrial respiration rate in H2O2-affected glial cells. The results suggest that Cy3G, as the most prominent antioxidant among tested anthocyanins, could be a potential adjuvant for the prevention or reduction of necrotic glial cell death during the oxidative stress conditions met in neurodegenerative diseases. However, further elucidation of other possible mechanisms for anthocyanins to protect the nervous system is encouraged.