Comparative Study of Functional Changes in Heart Mitochondria in Two Modes of Epinephrine Exposure Modeling Myocardial Injury in Rats.

The parameters of coupled respiration and transport of calcium ions in mitochondria isolated from the heart of rats were studied in two modes of exposure to epinephrine for modelling myocardial damage. In 24 h after injection of 1.5 mg/kg epinephrine to rats, we observed a decrease in the efficiency of oxidative phosphorylation in heart mitochondria in the presence of both NADH- and FADH-dependent respiratory substrates. Increasing the epinephrine dose and exposure (2 mg/kg, 72 h) led to a more pronounced decrease in the ADP/O coefficient when succinate was used as a substrate, which indicated a predominant decrease in the activity of complex II of the respiratory chain. The injection of epinephrine in the two modes resulted in a decrease in the rate of calcium entry in rat heart mitochondria, but had no effect on mitochondrial calcium retention capacity, which reflects the resistance of the organelles to the induction of the Са2+-dependent pore. These findings suggest that both cardiomyopathy models in rats can be used to study the effectiveness of pharmacological therapy using mitochondria-targeted agents.

[Effect of per os administration of dihydroquercetin aqueous form on energy exchange in blood lymphocytes of rats with experimental cardiomyopathy].

Cardiomyopathies are among the most severe myocardial pathologies, which are characterized by resistance to therapy and high mortality due to increasing heart failure and arrhythmia. Cardiomyocyte pathological changes upon cardiomyopathies are associated with mitochondrial dysfunction, leading to excessive formation of reactive oxygen species and the development of oxidative stress. In this regard, the study of the therapeutic potential of antioxidants in cardiomyopathies, as well as the mechanisms of their action on the functioning of mitochondria, is relevant and of high practical importance. The aim of this study was to determine the effect of oral 14-day administration of dihydroquercetin in a water-soluble form (DHQWF) on the activity of the key marker of mitochondrial respiration [succinate dehydrogenase (SDH)] and the cytoplasmic marker of glycolysis [lactate dehydrogenase (LDH)] in blood lymphocytes, as well as on the serum level of lipid peroxidation (LPO) in control rats and rats with experimental cardiomyopathy. Material and methods. Adult male Wistar rats (body weight 220-240 g) were used for the study. Isoprenaline hydrochloride was used to induce cardiomyopathy (IIC) in animals (twice subcutaneous injection at a dose of 150 mg/kg body weight, with a break of 24 hours). DHQ-WF was added to the drinking water for 14 days at the dose of 15 or 30 mg/kg body weight. SDH and LDH activity in lymphocytes was measured using a highly sensitive cytobiochemical method on a blood smear according to the reduction of nitrotetrazolium blue chloride to diformazan of dark blue color. The content of malone dialdehyde (MDA) in the blood serum, heart and liver mitochondria was determined spectrophotometrically using thiobarbituric acid. Mitochondria were isolated from rat tissues by the conventional method of differential centrifugation. Mitochondrial respiration was recorded using a polarographic method. Results. Experimental cardiomyopathy in rats was accompanied by a twofold increase in blood serum MDA level, as well as by a significant increase in SDH and LDH activity in blood lymphocytes. The oral administration of DHQ-WF in cardiomyopathy at a dose of 15 mg/kg body weight led to a significant decrease in serum MDA level, but did not reduce the activity of SDH and LDH in blood lymphocytes, compared with animals with cardiomyopathy that did not receive DHQ-WF. In the control group of animals, the use of DHQ-WF at a dose of 15 mg/kg body weight significantly increased blood lymphocyte LDH activity, but did not have a statistically significant effect on SDH activity and the parameters of mitochondrial respiration and oxidative phosphorylation, the level of MDA in heart and liver mitochondria. Increasing the dose of DHQ-WF administered to 30 mg/kg had less effect on changes in these parameters in control animals. Conclusion. The data obtained indicate that in experimental cardiomyopathy in rats, the course application of DHQ-WF at a dose of 15 mg/kg of body weight acts as an effective antioxidant that prevents the development of lipid peroxidation in blood serum, and can modulate energy metabolism towards the enhancement of glycolysis in blood lymphocytes in control animals.

Activities of Succinate Dehydrogenase and Lactate Dehydrogenase in Blood Lymphocytes in Yakut Ground Squirrels Spermophilus undulatus During Hibernation and in the Active State.

We studied energy metabolism in blood lymphocytes of Yakut ground squirrels Spermophilus undulatus in active state and during hibernation. Activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), marker enzymes of mitochondrial respiration and glycolysis, were measured by a cytobiochemical method based on quantitative assessment of a product of NBT reduction to diformazan in blood lymphocytes immobilized on glass. To measure SDH and LDH activities, cytobiochemical staining of immobilized cells was performed with succinate, lactate, and NAD. In the state of hibernation, SDH activity decreased by 3 times and LDH activity decreased by 10 times or more. These results suggest that the decrease in metabolic activity in lymphocytes of ground squirrels during hypothermia is associated with inhibition of glycolysis, rather than with mitochondrial energy supply.

Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

[Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

Hyperactivation of succinate dehydrogenase in lymphocytes of newborn rats.

We measured the activity of mitochondrial succinate dehydrogenase (SDH) within cells, in media with near-physiological composition, in lymphocytes immobilized in a blood smear on glass. SDH activity was studied in newborn rats characterized by natural hyperadrenergic status and also in adult animals injected with epinephrine. In most newborns very high activities were recorded, which exceeded the activities in adults at rest 7-8-fold or 3-fold according to the conventional calculation, or more than 30- and 6-fold according to our more precise calculation. The findings support our concept about a selective interaction between adrenergic stimulation and oxidation of succinic acid. According to this concept, epinephrine and norepinephrine specifically activate oxidation of succinic acid, whereas blood micromolar concentrations of the latter stimulate the release of catecholamines (the receptor-mediated signaling effect). This interaction is half of a substrate-hormonal regulatory system responsible for connection of vegetative nervous system with oxidation in mitochondria of the innervated organs. The increase in succinate oxidation by catecholamines includes activation of the faster pathways of succinate generation than the complete Krebs cycle, in particular, the glyoxylate cycle that is shown in the newborn rats in the present study.

Role of the impairment of oxidative metabolism in pathogenesis of lower urinary tract symptoms with benign prostatic hyperplasia and their treatment by alpha1-adrenoblocker alfuzosin.

The role of impairment of general oxidative and energy metabolism in pathogenesis of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH) and their correction by (1-adrenoblocker alfuzosin was studied. One group of patients (N = 126) was examined by standard methods for determination of the severity of LUTS by IPSS and mean effective volume of urinary bladder (MEVUB). In the second group (N = 29) in addition to functional examinations, metabolic indicators in blood were measured: antioxidant activity (AOA) and succinate dehydrogenase activity (SDA). Severity of LUTS depends greatly on the MEVUB. It was the first to show a practically complete correlation between LUTS, AOA and SDA. Severity of LUTS exactly correlates with indicators of oxidative and energy metabolism. In patients with more heavy LUTS, lowest AOA and SDA values were found. In the course of effective treatment, both phenomena developed an improvement of clinical symptoms and a rise of biochemical parameters. Close correlation between functional and metabolic phenomena is evidence of an essential role of metabolic mechanisms in the pathogenesis of LUTS with BPH. This opens perspectives to use antioxidants and energy metabolism activators for correction of UB dysfunction in patients with BPH.