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The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia.

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H2O2 formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H2O2 generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca(2+) (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5mM). We have shown that the H2O2 formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H2O2 formation through mitochondrial respiratory chain complex II. The H2O2 formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Glycine stabilizes energetics of brain mitochondria under conditions of brain hypoxia in vivo modeled by ligation of the common carotid artery in rats. Hypoxia reduced respiratory control in brain cortex mitochondria from 7.7 ± 0.5 to 4.5 ± 0.3. Preliminary oral administration of glycine almost completely prevented this decrease. In both in vitro models of hypoxia, similar phosphorylation disturbances were detected in both cortical slices and isolated brain mitochondria; they were effectively prevented by glycine. Hypoxia activates H(2)O(2) generation in mitochondrial suspension. The process is significantly reduced in the presence of 5 mM glycine. It is concluded that both in the model of hypoxia in vivo and during in vitro modeling of hypoxia in cortical slices and mitochondria, glycine acts as a protector inhibiting generation of reactive oxygen species in mitochondria and preventing energetics disturbances in brain mitochondria.

The effect of Ca2+ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca2+ at concentrations of 1-100 microM increased and at a concentration of 1 mM prevented the CN(-)-induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca2+ at concentrations of 0.1-1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN(-) was suppressed by 2 mM Ca2+ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 microM concentration prevented GC death induced by CN(-) or CN(-) + 0.1 mM Ca2+ but had no influence on respiration and photosynthetic O2 evolution in pea leaf slices. The generation of reactive oxygen species determined from 2',7'-dichlorofluorescein fluorescence was promoted by Ca2+ in epidermal peels from pea leaves.

Chitosan, CN(-), or H(2)O(2) caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H(2)O(2), or chitosan + H(2)O(2) on EC. H(2)O(2) formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN(-) and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN(-)-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN(-)-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H(2)O(2) on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H(2)O(2) as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5mM) and normal (10mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5mM), inhibitor of NMDA receptor MK-801 (10microM) and PTP inhibitor cyclosporine A (10microM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II-III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca(2+) and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.

Slow destructive processes in brain cortex were studied under deep hypoxia (anoxia). Study of the character and dynamics of DNA destruction showed that apoptosis and necrosis run in parallel under the experimental conditions. These processes typically develop in tens of hours. A similar conclusion was reached from electron microscopic study of the tissue ultrastructure. More detailed study revealed that a relatively rare type of apoptosis not involving cytochrome c release from the intermembrane space of mitochondria and not associated with opening of the mitochondrial nonspecific pore occurs under the experimental conditions. As this is occurring, the process can be slowed by high concentrations of glycine, an inhibitory neurotransmitter. The study of DNA destruction demonstrated that high concentrations of glycine selectively slow apoptosis but have almost no effect on necrosis. Glycine also drastically decreases changes in the tissue ultrastructure, particularly of mitochondria, arising under anoxia. Glycine does not notably influence the mitochondrial oxidative phosphorylation system. Study of impairment of mitochondrial function demonstrated that the oxidative phosphorylation system is not disturbed for 1 h, which is several times longer than the inhibition time of brain function under deep hypoxia. The mitochondrial respiratory system is preserved for a relatively long time (24 h). Malate oxidase activity is deactivated after 48 h. The succinate oxidase fragment of the mitochondrial respiratory chain proved especially resistant; it retains activity under anoxia for more than 72 h. A possible mechanism of the effect of high glycine concentrations is discussed.

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.