Post-receptor formation of active forms of oxygen in nonphagocytic cells]

Generation of reactive oxygen species (ROS) in A431 cells, NIH3T3 fibroblasts expressing normal epidermal growth factor (EGF) receptor, L929 fibroblasts, and in mouse peritoneal macrophages (professionally phagocytic cells) upon the effect of different activators has been studied. It has been shown that ROS formation in A431 and NIH3T3 cells upon the effect of EGF is time- and dose-dependent process. A variety of stimuli were used to stimulate macrophage ROS production. However, the effect of only phorbol ester, opsonized zymozan, peptide fMLP, and platelet activating factor led to ROS generation, whereas tumor necrosis factor alpha, interferon gamma, and lipopolysaccharide did not stimulate macrophage oxidative burst. The literature data on ROS generation in a variety of cell types are presented. ROS formed in cells acted upon certain agents are considered as the molecules participating in intracellular signaling.     

Spatial patterning of metabolism by mitochondria, oxygen, and energy sinks in a model cytoplasm

Metabolite gradients might guide mitochondrial localization in cells and angiogenesis in tissues. It is unclear whether they can exist in single cells, because the length scale of most cells is small compared to the expected diffusion times of metabolites. For investigation of metabolic gradients, we need experimental systems in which spatial patterns of metabolism can be systematically measured and manipulated. We used concentrated cytoplasmic extracts from Xenopus eggs as a model cytoplasm, and visualized metabolic gradients formed in response to spatial stimuli. Restriction of oxygen supply to the edge of a drop mimicked distance to the surface of a single cell, or distance from a blood vessel in tissue. We imaged a step-like increase of Nicotinamide adenine dinucleotide (NAD) reduction approximately 600 microm distant from the oxygen source. This oxic-anoxic switch was preceded on the oxic side by a gradual rise of mitochondrial transmembrane potential (Deltapsi) and reactive oxygen species (ROS) production, extending over approximately 600 microm and approximately 300 microm, respectively. Addition of Adenosine triphosphate (ATP)-consuming beads mimicked local energy sinks in the cell. We imaged Deltapsi gradients with a decay length of approximately 50-300 microm around these beads, in the first visualization of an energy demand signaling gradient. Our study demonstrates that mitochondria can pattern the cytoplasm over length scales that are suited to convey morphogenetic information in large cells and tissues and provides a versatile model system for probing of the formation and function of metabolic gradients.     

Brain hypoxia and the role of active forms of oxygen and of energy deficit in the neuron degeneration

The concepts about physiological mechanisms of oxygen transport to the brain have recently changed substantially. Precise data on the capillary blood flow rate, on a substantial dispersion of corresponding values, on the influence of the capillary blood flow rate on pO2 in the capillaries and tissues have evolved. Krog's paradigm about an exclusive role of capillaries in the gas exchange between the blood and tissues amounting to almost 100 years was abandoned. All these data also changed the concepts about the development of various types of hypoxia in the brain tissues. The study of pO2 in the brain at normoxia showed that pO2 exhibits the fluctuations from 1-2 to 80-85 mm Hg. This means, in particular, that hypoxic phenomena take place in the normal healthy brain. During hypoxia the mass adhesion of leukocytes to the walls of microvessels was shown to hamper the capillary blood flow and can become one of the reasons for the death of the brain during hypoxia. The brain hypoxia is not an occasional pathologic process. It exists in an intact brain owing to physiological fluctuations of pO2 in various microregions of the brain. It occurs during various physiological states in the norm and also during various illnesses associated with the changes and disruptions in the oxygen transport. The final stage of hypoxia is the destruction of the cells. The development of this process and its particular reasons are nowadays the subject of multiple physiological and biochemical studies. Certain changes are introduced into modern ideas about the reasons for the degradation of the nervous cells upon hypoxia. The degradation of the neurons during hypoxia or anemia is postulated to be associated not only with the cell generation of active forms of oxygen (AFO), but also with the energy deficiency. This means a deficient synthesis or a complete absence of ATP in a cell during hypoxia, anemia, and ishemia.     

The generation of active microbicidal forms of oxygen by leukocytes transiting the vascular bed of the lungs

In experiments on dogs using the chemiluminescent method and nitroblue-tetrazole reaction the authors found out that leucocytes while passing through the pulmonary vessels bed, in contrast to the spleen increase the generation of active microbicidal forms of oxygen. Due to this fact we suppose that the lungs may take part in the formation of free radical status and phagocytic antiinfectious defence of the organism.     

The effect of dissolved oxygen tension on the energy metabolism of yeasts]

The specific ATP generation rate in yeasts was examined on the glycolytic pathway and on the respiratory chain as a function of the dissolved oxygen tension of the culture medium. Two different strains were used: Saccharomyces cerevisiae sensitive to the glucose effect and Kluyveromyces fragilis insensitive to the catabolite respression when growing on lactose. The oxidative ATP generation rate followed by these two strains a Michaelis Menten kinetics against the dissolved oxygen concentration. Dissolved oxygen tension only influenced the glycolytic ATP generation rate in Kluyveromyces fragilis. Thus glucose and Pasteur effects are two mutually exclusive regulatory mechanisms of the energy yielding metabolism of the yeasts.     

Food restriction affects energy metabolism in rat liver mitochondria

To examine the effect of 50% food restriction over a period of 3 days on mitochondrial energy metabolism, liver mitochondria were isolated from ad libitum and food-restricted rats. Mitochondrial enzyme activities and oxygen consumption were assessed spectrophotometrically and polarographically. With regard to body weight loss (-5%), food restriction decreased the liver to body mass ratio by 7%. Moreover, in food-restricted rats, liver mitochondria displayed diminished state 3 (-30%), state 4-oligomycin (-26%) and uncoupled state (-24%) respiration rates in the presence of succinate. Furthermore, "top-down" elasticity showed that these decreases were due to an inactivation of reactions involved in substrate oxidation. Therefore, it appears that rats not only adapt to food restriction through simple passive mechanisms, such as liver mass loss, but also through decreased mitochondrial energetic metabolism.     

The role of active forms of oxygen in platelet aggregation and deaggregation

The effect of hydrogen peroxide on ADP-induced platelet aggregation in the presence of active oxygen species scavengers was studied. It was shown that the superoxide radical and singlet oxygen, alongside with hydrogen peroxide, may play a role in platelet interactions.     

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria

We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extra-mitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extra-mitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.     

Luminol chemiluminescence and active oxygen generation by activated neutrophils.

Upon stimulation by various ligands and membrane perturbers, neutrophils produce various active oxygen species. Since luminol chemiluminescence (LCL) in neutrophils can be blocked by azide, an inhibitor of myeloperoxidase, LCL has been believed to reflect mainly the myeloperoxidase-catalyzed reaction. When cells were stimulated by formyl-methionyl-leucyl-phenylalanine, LCL was strongly inhibited by superoxide dismutase (SOD) and uric acid, a scavenger for hydroxy radical (.OH) and singlet oxygen, whereas it was stimulated by azide. LCL was also inhibited by .OH scavengers, such as mannitol, ethanol, and dimethylsulfoxide. However, when stimulated by phorbol myristate acetate or opsonized zymosan, LCL was strongly inhibited by azide but not by uric acid, and the inhibitory action of SOD was low. Thus, the qualitative and quantitative aspects of reactive oxygen generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the metabolic fate of active oxygens in neutrophils and, hence, their effect on microorganisms and the surrounding tissues might differ depending on the stimulus.     

Formation of active forms of oxygen by rat peritoneal macrophages under the effect of cytotoxic dust]

The velocity of superoxide radicals (O2) production by rat peritoneal macrophages, phagocyting the dust particles (quartz and crocidolite-asbestos was measured by using the method of cytochrome c reduction. Generation of hydroxyl radicals (HO) by cells and intensity of lipid peroxidation in the membranes of phagocytes were also investigated. It was found, that under the action of quartz the cells form mainly O2, and under the action of crocidolite--O2 and HO(.). The differences observed were caused by catalytic properties of the surface of asbestos fiber, where the reaction of HO. formation from O2 takes place. The quartz particles increased the concentration of malondialdehyde in macrophages by 53% as compared with control; and lipid peroxidation intensity in the presence of crocidolite-asbestos fibers increased fourfold. The role of hydroxyl radicals in initiating of lipid peroxidation, cytotoxicity and mutagenicity of asbestos is discussed.     

Role of active forms of oxygen in inducing luminol-dependent chemiluminescence in macrophages

The luminol-dependent chemiluminescence of mouse peritoneal macrophages during phagocytosis of opsonized zymosan was studied by using specific active oxygen scavengers and metabolic inhibitors. Extracellular hydrogen peroxide and superoxide anion were shown to contribute immensely to the induction of the chemiluminescence. The role of the hydroxyl radical was rather insignificant, whereas singlet oxygen was not involved in this process. The interaction between luminol and peroxide was shown to be peroxidase-dependent. An inhibitory analysis revealed that the interaction between luminol, peroxide and superoxide anion obeyed a hybrid enzyme-free radical mechanism.     

Effect of corticosterone treatment on energy metabolism in rat liver mitochondria.

1. Effect of in vivo treatment (40 mg/kg body wt) with corticosterone on energy metabolism in rat liver mitochondria was examined under acute and chronic conditions in 20-, 35- and 60-day-old rats. 2. Acute treatment did not affect body or liver weight. However, chronic treatment caused increased liver weight in the former two age groups; in the 60-day-old animals the liver weight decreased. 3. Acute treatment resulted in a generalized decrease in state 3 respiration rates and state 4 respiration rates without having any significant effect on ADP/O ratios with glutamate, succinate and ascorbate + TMPD as substrates. However, rates of ATP synthesis decreased significantly. The effect was age-dependent, older animals showed increased resistance. 4. Chronic treatment resulted in uncoupling of oxidative phosphorylation without having significant effects on respiration rates. Once again, the effects were age-dependent. Consequently, the ATP synthesis rates were significantly lowered. However, it was apparent that the underlying mechanisms were entirely different. 5. With succinate as the substrate the state 3 respiration rates increased with age to reach adult values by day 60. The coupling efficiency was also exhibited via maturational changes.     

Long-chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria

In the heart, a nutritional state (fed or fasted) is characterized by a unique energy metabolism pattern determined by the availability of substrates. Increased availability of acylcarnitines has been associated with decreased glucose utilization; however, the effects of long-chain acylcarnitines on glucose metabolism have not been previously studied. We tested how changes in long-chain acylcarnitine content regulate the metabolism of glucose and long-chain fatty acids in cardiac mitochondria in fed and fasted states. We examined the concentrations of metabolic intermediates in plasma and cardiac tissues under fed and fasted states. The effects of substrate availability and their competition for energy production at the mitochondrial level were studied in isolated rat cardiac mitochondria. The availability of long-chain acylcarnitines in plasma reflected their content in cardiac tissue in the fed and fasted states, and acylcarnitine content in the heart was fivefold higher in fasted state compared to the fed state. In substrate competition experiments, pyruvate and fatty acid metabolites effectively competed for the energy production pathway; however, only the physiological content of acylcarnitine significantly reduced pyruvate and lactate oxidation in mitochondria. The increased availability of long-chain acylcarnitine significantly reduced glucose utilization in isolated rat heart model and in vivo. Our results demonstrate that changes in long-chain acylcarnitine contents could orchestrate the interplay between the metabolism of pyruvate–lactate and long-chain fatty acids, and thus determine the pattern of energy metabolism in cardiac mitochondria.