Data on effects of rotenone on calcium retention capacity, respiration and activities of respiratory chain complexes I and II in isolated rat brain mitochondria.

The data presented in this article are related to the research article entitled "Rotenone decreases ischemia-induced injury by inhibiting mitochondrial permeability transition in mature brains" (Rekuviene et al., 2017) [1]. Data in this article present the direct effects of rotenone on calcium retention capacity (CRC) in isolated normal cortex and cerebellum mitochondria, effects of rotenone intravenous infusion on leak and phosphorylating respiration rates of isolated cortex and cerebellum mitochondria, on activities of respiratory chain complexes I and II in freezed-thawed/sonicated cortex and cerebellum mitochondria after brain ischemia. In addition, detailed experimental procedures of isolation of brain mitochondria, measurements of CRC, respiration, activities of respiratory chain complexes and H2O2 generation in cortex and cerebellum mitochondria are described.

Rotenone decreases ischemia-induced injury by inhibiting mitochondrial permeability transition in mature brains.

The mitochondrial permeability transition pore (mPTP) is thought to be implicated in brain ischemia-induced cell death. Here we sought to determine whether complex I (CI) of the mitochondrial electron transfer system may be involved in regulation of mPTP opening during ischemia and whether a specific inhibitor of this complex - rotenone can protect against ischemia-induced cell death in an experimental model of total ischemia in adult rat brains. Anesthetized Wistar rats were administered a single injection of rotenone (0.01mg/kg) to the tail vein and brains were removed and subjected to 120min ischemia. We found that intravenous injection of rotenone 20min before ischemia increased resistance to Ca2+-induced mPTP opening and decreased production of reactive oxygen species (ROS) in mitochondria isolated from ischemia-damaged cortex and cerebellum. Rotenone administration before ischemia decreased infarct size in both brain regions (cortex and cerebellum). Rotenone added directly to normal, non-ischemic cortical or cerebellar mitochondria increased their resistance to Ca2+-induced mPTP opening at concentration which fully inhibited NAD-dependent mitochondrial respiration. Our data demonstrate that rotenone used intravenously may be protective against acute brain ischemia-induced injuries by inhibition of mPTP opening and ROS production. These findings suggest that CI of mitochondrial electron transfer system plays a role in mPTP regulation during cerebral ischemia in mature brains and that agents acting on CI activity may be clinically useful for stroke therapy.

The effects of cadmium chloride and sodium selenite on protein synthesis in mouse liver.

The study aimed at evaluating the effects of cadmium and selenite ions on protein synthesis and metallothioneins content in mice liver after 2 h, 8 h, 24 h and 14 days of exposure. Our studies revealed that cadmium suppressed protein synthesis after 2 h and 24 h, but activated after 8h and 14 days. Also, the endogenous mRNA translation were reduced under any exposure to cadmium, meanwhile, metallothioneins content was decreased after 2 h, but then was progressively increasing up to 492% after 14 days. Meantime, selenite did not influence metallothioneins content, caused mild activation of protein synthesis, and slightly suppressed the endogenous mRNA translation. The combined treatments with cadmium and selenite favored toward resisting of protein synthesis to cadmium after 2 h and 24 h of intoxication. Besides, selenite also protected translation against cadmium in cell-free systems, but did not attenuate effects of cadmium on metallothioneins content.

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G.

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.

Subacute effects of cadmium and zinc ions on protein synthesis and cell death in mouse liver.

OBJECTIVE: The aim of this study was to evaluate in vivo the effects of cadmium and zinc ions on translational machinery and death of mouse liver cells. MATERIAL AND METHODS: Outbred mice received intraperitoneal injections of cadmium chloride solution (1.4 micromoles cadmium per 1 kg of body weight) and/or zinc sulfate solution (4.8 micromoles zinc per kg of body weight) three times per week for six weeks. Analogical volume of saline solution was injected to the control mice. Protein synthesis was evaluated by incorporation of [(14)C]-labeled leucine into peptides and proteins. Total tRNAs were isolated using deproteinized extract of liver tissue. Postmitochondrial supernatant was as a source of leucyl-tRNA synthetase. Activities of tRNA(Leu) and leucyl-tRNA synthetase were measured by an aminoacylation reaction using [(14)C]-labeled leucine. Liver cell apoptosis was detected by TUNEL assay using in situ cell death detection kit. RESULTS: A decrease in incorporation of [(14)C]-labeled leucine into proteins was detected in liver, kidney, and heart as well as diminution of tRNA(Leu) acceptor activity in cadmium-exposed liver. Cadmium caused activation of the leucyl-tRNA synthetase and induced liver cell apoptosis. Pretreatment of mice with zinc sulfate solution favored to protection of protein synthesis and acceptor activity of tRNA(Leu) against cadmium-induced inhibition. Under co-exposure of mouse liver to cadmium and zinc, activity of the leucyl-tRNA synthetase was at the level of control. Zinc did not influence TUNEL-positive cell number in cadmium-exposed mouse liver. CONCLUSIONS: Under subacute intoxication of mice by cadmium, zinc ions protect the translation machinery against inhibition, but do not decrease the number of apoptotic cells in the liver.

Effects of cadmium ions on the initial stage of translation and the cell death in mouse liver.

OBJECTIVE: To evaluate in vivo and in vitro effects of cadmium ions on the activities of mice liver tRNA(Leu)and leucyl-tRNA synthetase and on the type of liver cells death. MATERIAL AND METHODS: White laboratory mice were intoxicated by intraperitoneal injection of cadmium chloride solution (1.6 mg cadmium ions/1 kg of body weigh). Total tRNAs were isolated by adding ethanol and isopropanol into the phenol-deproteinized supernatant of mouse liver homogenate. Post-mitochondrial fraction of the liver cells was used as a source of leucyl-tRNA synthetase. Acceptor activity of tRNA(Leu)and activity of leucyl-tRNA synthetase were measured in tRNA aminoacylation reaction with [14C]-labeled leucine as a substrate. An apoptotic cell death was assessed by the TUNEL assay using in situ cell death detection kit. DNA degradation was verified by electrophoresis. RESULTS: It was determined that 2-24 hours after intoxication with sublethal dose of cadmium ions the acceptor activity of mice liver tRNA(Leu)was decreased by 43-73% as compared to control. At the same time intervals, the activity of leucyl-tRNA synthetase was reduced about 20-30%. Experiments in vitro revealed that 10-20 microM concentrations of cadmium ions suppressed the activities of mice liver tRNA(Leu)and leucyl-tRNA synthetase by 40-98%. No significant difference was observed between the number of TUNEL positive apoptotic liver cells in the control mice and 24 hours after intoxication with cadmium chloride. Electrophoresis revealed extensive degradation of nuclear DNA. CONCLUSIONS: Cadmium ions significantly reduce activities of tRNA(Leu)and leucyl-tRNA synthetase in vivo and in vitro. There is no significant difference between the number of apoptotic cells in the control liver specimens and in those after 24 hours of intoxication with cadmium chloride. In latter specimens DNA electrophoresis revealed as extensive degradation of DNA, which is characteristic to the cell necrosis.

Apoptosis of cardiomyocytes in explanted and transplanted hearts. Comparison of results from in situ TUNEL, ISEL, and ISOL reactions.

We assessed the efficiency of detecting myocyte apoptosis within human hearts using in situ enzymatic reactions in paraffin-embedded tissue samples: in situ end labeling (ISEL), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and in situ oligoligation (ISOL). The reactions were carried out in explanted hearts (idiopathic dilatative cardiomyopathy, n = 6; ischemic heart disease, n = 3) and in endomyocardial biopsy specimens (EMBs; n = 32) obtained from transplanted human hearts. The results were verified by DNA laddering. The ISOL reaction led to a significantly (P = .027) smaller number of false-positive results (2/41 [5%]) compared with assessment by ISEL (9/41 [22%]) or TUNEL (9/41 [22%]). Only 1 ISEL+ apoptotic cardiomyocyte was found in specimens from explanted hearts. Among the EMBs, 1 specimens had TUNEL+ apoptotic cardiomyocytes and 1 specimen had ISEL+ apoptotic cardiomyocytes. This implies that verifying results by independent methods must be used for TUNEL and ISEL techniques. A smaller number of false-positive results makes interpretation of ISOL results easier, although the sensitivity of this reaction remains to be established.

Nitric oxide and calcium together inactivate mitochondrial complex I and induce cytochrome c release.

Cellular nitric oxide (NO) and calcium levels have been reported to increase during various pathologies, including particularly ischaemia. In this study, we investigated whether elevated NO and calcium levels can synergistically damage isolated rat heart mitochondria. We found that NO and calcium together inhibited the oxygen consumption of mitochondria respiring on pyruvate + malate, but not mitochondria respiring on succinate. In the same conditions, complex I activity was synergistically inhibited by NO and calcium, and this inhibition was completely prevented by superoxide dismutase or urate, suggesting that the inhibition was mediated by peroxynitrite. Indeed, we found NO and calcium-stimulated mitochondrial production of peroxynitrite. The inhibition of complex I activity by NO and calcium was reversed by reduced thiols or light (as was complex I inhibition by S-nitrosothiols or peroxynitrite) suggesting that the inhibition may involve S-nitrosation or Fe-nitrosylation of complex I. However, NO and calcium also caused loss of mitochondrial cytochrome c, and the induced inhibition of respiration was partially reversed by addition of exogenous cytochrome c. Thus, NO and calcium appear to synergistically inhibit mitochondrial respiration, partly by inactivation of complex I and partly by inducing cytochrome c release.

[Programmed cellular death and atherogenesis: from molecular mechanisms to clinical aspects]. / Programuota lasteliu mirtis ir aterogeneze: nuo molekuliniu mechanizmu iki klinikos.

Numerous recent investigations on the development and morphology of atherosclerotic lesions have shown programmed cell death or apoptosis to be an important factor in atherogenesis. Enzymes known as caspases are essential for completion of the apoptotic program. With regard to the origin of signals inducing apoptosis, there are two ways of initiating caspase activation: (a) cellular death receptor-mediated activation; and (b) activation mediated by mitochondrial permeability and expression of the p53 oncogene. Both of these pathways are involved in atherogenesis. Oxidative stress, angiotensin II and cholesterol overload are the primary factors that induce apoptosis in vascular cells. Considering apoptosis in endothelial cells, exposed phosphatidylserine on the cell membrane activates thrombin increasing the probability of arterial thromboses. Further progression of atherosclerosis is promoted by the formation of apoptotic bodies with oxidized phospholipids exposed on the membrane; these also activate adhesion of monocytes. Apoptosis of smooth muscle cells is usually observed in the fibrous portion of an atherosclerotic plaque in which the cells produce collagen important for plaque stability. As apoptosis occurs in smooth muscle cells, the fibrous cap grows thinner. This can result in both plaque rupture, formation of thrombi as well as calcification of the plaque from apoptotic smooth muscle cells remnants. Smooth muscle cells apoptosis is beneficial in that it offers protection to the walls of arteries against proliferative restenosis induced by invasive procedures. Apoptosis of macrophages contributes to the formation and progression of the lipidic core and promotes thrombosis of atherosclerosis in damaged arteries. By contrast, apoptosis of macrophages diminishes the production of matrix methaloproteinases that decompose collagen fibers. New facts concerning the effects of antioxidants (selenium, vitamin C and vitamin E), inhibitors of angiotensin converting enzyme, beta-blockers, calcium chanel blockers, and statins are also considered in this review.

[Programmed cell death: molecular mechanisms and detection]. / Programuota lasteliu mirtis: molekuliniai mechanizmai ir ju nustatymo metodai.

Apoptosis or programmed cell death is genetically determined process to destroy cells for the maintaining of cellular homeostasis in the tissue. This paper reviews the current knowledge on the molecular mechanisms of apoptosis. Activation of cysteine proteases called caspases plays a major role in the execution of apoptosis. These activated caspases selectively cleave cellular proteins, which result in apoptotic morphology (internucleosomal fragmentation of DNA into 180-200 base pair pieces, shrinkage of the cell and the nucleus as well and fragmentation of the cell into apoptotic bodies) and death of the cell. Now two pathways of caspase activation are reported. The first through triggering of cellular death-receptor superfamily. The second is mitochondrial pathway induced by the changes of the expression of pro- and anti-apoptotic genes in the cell. It leads to release of cytochrome c and apoptosis inducing factor from mitochondria. The paper reviews also currently used methods of detection of apoptotic cells in tissue samples, causes of false-positive or false-negative results of ISEL and TUNEL in situ reactions.