Mitochondrial ryanodine-sensitive Ca2+ channels of rat liver.

To examine ryanodine-sensitive Ca2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1µM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10µM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05µM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1µM or 1µM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05µM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1µM or 1µM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05µM, 0.1µM, or 1µM) causes differential effects on Ca2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca2+ concentration and affecting the energy state of mictochondria in hepatocytes.

[MITOCHONDRIA RESPIRATION AND OXIDATIVE PHOSPHORILATION OF RAT TISSUES AT TAURINE PER ORAL INJECTION].

Taurine--sulphur-containing amino acid is a necessary component of mitochondrial matrix, where it maintains pH and is included in mitochondrial transport RNA. But still it is unclear how taurine influences on ATP synthesis and mitochondrial respiration chain components activity. Thus, the aim of the work was to study the effect of long-term per oral taurine injection on mitochondrial respiration intensity in rat tissues: liver, brain, testes and thigh muscle. For this purpose male Wistar rats, that weighted 190-220 g, were divided in three groups, daily during 28 days they were injected drinking water (control group) or taurine solution 40 and 100 mg per kg of body weight (I and II research groups, correspondingly). Respiration intensity was measured polarogrifically with use of Clark electrode at endogenic substrates oxidation (V1), with exogenic α-ketoglutarate (5 mmol/l) or succinate (1 mmol/l;V4S) addition, at ADP addition to concentration 200 µmol/l (V3), and after ADP depletion (V4ATP). Phosphorylation time, oxidative phosphorilation efficacy (ADP/O), respiratory controls by Lardy (V3/V4S) and Chance (V3/V4ATP) were calculated. It was found that long term taurine injection increased V1 in animal brain and liver of both research groups, but it decreased in testes and muscles of I research group. In liver of I research group animals, when both α-ketoglutarate and succinate were oxidated, V4S, V3 and V4ATP were 4-7 times larger than in control. At the same time, Lardy respiratory control increased at succinate oxidation, this may indirectly point on increased coupling between respiration and oxidative phosphorylation. In liver of II research group animals V4S, V3 and V4/ATP when α-ketoglutarate was oxidated were significantly higher than in control. In muscles of I research group V4S, V3 and V4ATP when α-ketoglutarate and succinate was added were lower than in control. In thigh muscle of II research group animals at α-ketoglutarate oxidation V3 was higher than in control. When succinate was added V4S and V4ATP increased in testes mitochondria of both research groups and in brain of I research group. But in II research group animals mitochondria V4S brain was lower than in control. At the same time, coupling between respiration and oxidative phosphorytation in brain was on control level, in testes of I research group it was lower. In testes of II research group animals at α-ketoglutarate addition increased respiratory controls. Thus, the effect of long term per oral taurine injection on mitochondria respiration intensity is dose-dependent and tissue-specific and, obviously, has different significance and is implemented by different mechanisms.

ACTIVITY AND ISOZYME CONTENT OF LACTATE DEHYDROGENASE UNDER LONG-TERM ORAL TAURINE ADMINISTRATION TO RATS.

The effect of long-term oral taurine administration to rats on activity of lactate dehydrogenase (LDH), its isozyme content and activity in the whole blood, liver, thigh muscle, brain and testes tissues were studied in the present work. For this purpose male Wistar rats with body weight 190-220 g were randomly divided into three groups, they were orally administered drinking water (control group) or taurine solution 40 and 100 mg per kg of body weight ( groups I and II, respectively). The total lactate dehydrogenase activity was measured spectrophotometrically, the percentage content of isozymes was determined by electrophoresis in 7.5% poliacrylamide gel withfurther staining according to J. Garbus. It was found that the total lactate dehydrogenase activity increased in all studied tissues. In testes of animals of both groups and in brain of group I animals, the total percentage contents of isozymes that are responsible for lactate production (LDH4+LDH5) increased. In liver of animals of both groups and in whole blood of group II animals, the total percentage content of isozymes that produce pyruvate (LDH1+LDH2) increased. In thigh muscle of both groups and in brain of group II animals the balance between LDH1+LDH2 and LDH4+LDH5 content did not differ from control values, though total lactate dehydrogenase activity was significantly higher, than that in the control group. Thus, the increase in the lactate dehydrogenase activity under long-term oral taurine administration in different rat tissues was found to be tissue- and dose-dependent and was caused by the increase in the content of different isozymes. Such increase in group I animals might be explained by adaptive mechanisms to hypoxia caused by high doses of taurine. For group II animals high doses of taurine were toxic and directly affected metabolic processes in the animal bodies.

EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION.

To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and a-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 µM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 µg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml--by 50 µg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml--by 100 µg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 µg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate, on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 αg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

Mechanisms of respiration intensification of rat pancreatic acini upon carbachol-induced Ca(2+) release.

AIM: Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes, presumably due to increase in ATP hydrolysis or Ca(2+) transport into mitochondria. The aim of this work was to investigate the mechanisms of carbachol (CCh) action on respiration and oxidative phosphorylation of isolated pancreatic acini. METHODS: Respiration of intact or permeabilized rat pancreatic acini was studied at 37 °C using a Clark oxygen electrode. RESULTS: Respiration rate of isolated acini in rest was 0.27 ± 0.01 nmol O2 s(-1) 10(-6) cells. Addition of 10 µM CCh into respiration chamber evoked biphasic stimulation of respiration. Rapid increase of respiration by 20.1% lasted for approx. 1 min, followed by decrease to level by 11.5% higher than control. Addition of 1 µm CCh caused monophasic increase by 11.5%. Preincubation (5 min) with 1 or 10 µm CCh elevated respiration rate by 12.5 or 11.2% respectively. FCCP prevented the effect of CCh. Preincubation with 1 (but not 10) µm CCh increased FCCP-uncoupled respiration rate. Thapsigargin slightly elevated respiration, but ryanodine did not. Application of 2-aminoethoxydiphenyl borate or ruthenium red prevented the effects of CCh on respiration, while oligomycin abolished them. Preincubation with 1 µm CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation, but not at succinate oxidation. In contrast, preincubation with 10 µm CCh decreased pyruvate+malate oxidation. CONCLUSION: Medium CCh dose (1 µm) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca(2+) transport into mitochondria and activation of Ca(2+) /ADP-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 µm) might impair mitochondrial functions.

Measurement of the in-medium K0 inclusive cross section in pi(-) -induced reactions at 1.15 GeV/c.

The K0 meson production by pi(-) mesons of 1.15 GeV/c momentum on C, Al, Cu, Sn, and Pb nuclear targets was measured with the FOPI spectrometer at the Schwer-Ionen-Synchrotron accelerator of GSI. Inclusive production cross sections and the momentum distributions of K0 mesons are compared to scaled elementary production cross sections and to predictions of theoretical models describing the in-medium production of kaons. The data represent a new reference for those models, which are widely used for interpretation of the strangeness production in heavy-ion collisions. The presented results demonstrate the sensitivity of the kaon production to the reaction amplitudes inside nuclei and point to the existence of a repulsive KN potential of 20+/-5 MeV at normal nuclear matter density.

B Lymphocyte Regulation of Proliferation and Differentiation of Hematopoietic Stem Cells.

The fraction of bone marrow hematopoietic stem cells was isolated. To induce in vivo proliferation and differentiation of the hematopoietic fraction the helper action of B cells or T cell precursors was necessary. In presence of bone marrow T cell precursors, B cells suppressed spleen colony formation. The intensity of B cell helper or suppressive action depended on their organ localization. Bone marrow B cells were characterized with maximum helper and minimum suppressive activity. On the contrary, spleen B cells exhibited maximum suppressive action on hematopoietic stem cells without helper one. Lymph node B cells possessed intermediate activity compared to those from bone marrow and spleen. The induction of the colony formation was observed in the presence of either naive or activated B lymphocytes. In case of activated B lymphocytes the regulation of hematopoietic stem cells depended on the admixtured cells as Thy-1(+) and SC-1(+) cells, on the presence of precursors of T cells in transplant and on the organ localization of B cells, as well as on the type of the agent for B cell activation. The activation of B cells might resulted in their helper action changed into suppressive or vice-versa. The B cell regulation of hematopoietic stem cell was mediated by their soluble products of non-immunoglobulin nature. It was found, that helper and suppressive factors from B cell hybridoma supernatants regulated hematopoietic stem cells similar to B cells, of which the hybridomas were created. Thus, we attempted to work out the biotechnological basis for obtaining and researching the regulatory products secreted by B cells.

Is equilibrium of aligned Kerr black holes possible?

We show that equilibrium of two Kerr black holes can be achieved by placing between them a relativistic disk or a third Kerr black hole, the latter case demonstrating the existence of equilibrium configurations in the purely black hole systems with the number of constituents more than two.