Redox environment in stem and differentiated cells: A quantitative approach.

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

[CELLS FORM AND THEIR SENSITIVITY TO LYTIC ACTIVITY OF NATURAL KILLER CELLS UNDER THE ANTIOXIDANT ACTION].

The present paper is an attempt to estimate the influence of cell surface morphology changes to functional activity under the effect of antioxidant, N-acetylcysteine (NAC), and alpha-lipoic asid (ALA). Two experimental parameters were used to characterize transformed fibroblasts 3T3-SV40 status. The functional one was the cell sensitivity to lysis by natural killer (NK) mouse splenocytes, and morphology index (cell form index) was a cell area. We showed that addition of NAC or ALA to the cell medium caused fast decrease of cell area and changes of cell form. On the other hand, their sensitivity to lysis NK cells gradually and significantly decreased. Then we compared NAC or ALA effect with the effects of other substances, which were non-antioxidants but caused cell responses which concurred with of antioxidants, at least partly. They were: latrunculin B, desorganizing actin filaments (as both antioxidants), OTZ reducing ROS level in the cell (as NAC), BSO (inhibitor of glutathione synthesis), increasing ROS level in the cell (as ALA), antibodies to gelatinases, MMP-2 and MMP-9 inactivating their activities (as both antioxidants). The results obtained showed a correlation between changes of morphology index and functional activity, sensitivity to lysis by NK cells. We suppose that geometry of cell surface might be a functional indicator of cell reaction to the antioxidant.

Different metabolic effects of ganglioside GM1 in brain synaptosomes and phagocytic cells.

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe2+-H2O2 system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of 45Ca2+ influx and oxidative inactivation of Na+,K+-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10(-11)-10(-8) M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10(-11)-10(-10) M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.

The role of reactive oxygen species in membrane potential changes in macrophages and astrocytes.

Involvement of reactive oxygen species (ROS) in changes of the plasma membrane potential of mouse peritoneal macrophages and astrocytes (U118 cell line) under the action of different agents has been studied. Membrane potential was measured using the voltage-dependent fluorescent oxonol dye DiBAC4(3). Agonists which stimulate macrophages to release ROS (the fMLP peptide and platelet activating factor) caused prolonged hyperpolarization. Experiments with the fluorescent probe 2',7'-dichlorofluorescein diacetate have shown that astrocytes release ROS upon the action of C5a complement anaphylatoxin (but not C3a). The effect of C5a was accompanied with hyperpolarization of the astrocyte plasma membrane. Treatment of the cells with agents which do not induce ROS generation (C3a, lipopolysaccharide, interferon-gamma) depolarized the plasma membrane. Hyperpolarization of both cell types was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2- -generating system caused a marked hyperpolarization of both cell types. The data obtained suggest that O2- is involved in the macrophage and astrocyte hyperpolarization response.

Quantitative studies on the respiratory burst generated in peritoneal macrophages.

The luminol-dependent chemiluminescence of macrophages during the zymosan-stimulated respiratory burst has been studied both in the absence and in the presence of the radical inhibitor 3,5 di-tert-butyl-4-hydroxyphenyl propionic acid. In addition, the consumption of luminol and of the inhibitor has been followed analytically. Based on the rates of the consumption of the inhibitor, an iteration procedure yields a value of 2.2 x 10(-7) M for the steady-state concentration of radicals generated by cells at the maximum of the chemiluminescence in the presence of inhibitor. Approximate calculations have indicated that under the experimental conditions applied, additional formation of superoxide anion radicals by the oxidation of luminol is negligible. By assuming that in an inhibitor-free system the disappearance of radicals takes place via their combination process as well as by their interaction with luminol and/or with luminol-derived species, numerical integration yields a calculated curve of radical concentration versus time in fair agreement with experimental data and a rate-constant value for the combination of radicals of approximately 10(6) M-1 s-1, supporting literature findings according to which primarily superoxide anion radicals are formed.

Cell-cycle-dependent changes in membrane potential of L-929 cells caused by the effect of hydrogen peroxide.

The changes in membrane potential of L-929 fibroblasts caused by H2O2 (10-50 microM) at different phases of the cell cycle were investigated. The membrane potential of cells in G0, early G1, and G2/M responded to H2O2 by hyperpolarizing, while cells in late G1/S responded by depolarizing. Quinidine (50 microM), a blocker of Ca2+-dependent K+ conductance, inhibited the hyperpolarization response of L-929 cells to H2O2 in all cases. Measurements of reactive oxygen species (ROS) production by the cells at the same cell cycle phases indicated that the hyperpolarization response of the cells is linked with minimal production of ROS, and that the depolarization response is associated with maximal production of ROS inside the cell.

Roles of reactive oxygen species: signaling and regulation of cellular functions.

Reactive oxygen species (ROS) are the side products (H2O2, O2.-, and OH.) of general metabolism and are also produced specifically by the NADPH oxidase system in most cell types. Cells have a very efficient antioxidant defense to counteract the toxic effect of ROS. The physiological significance of ROS is that ROS at low concentrations are able to mediate cellular functions through the same steps of intracellular signaling, which are activated by natural stimuli. Moreover, a variety of natural stimuli act through the intracellular formation of ROS that change the intracellular redox state (oxidation-reduction). Thus, the redox state is a part of intracellular signaling. As such, ROS are now considered signal molecules at nontoxic concentrations. Progress has been achieved in studying the oxidative activation of gene transcription in animal cells and bacteria. Changes in the redox state of intracellular thiols are considered to be an important mechanism that regulates cell functions.

Superoxide release is involved in membrane potential changes in mouse peritoneal macrophages.

Participation of reactive oxygen species (ROS) in the changes in macrophage membrane potential resulted from effects of different agonists has been studied. Treatment of macrophages with chemotactic peptide fMLP or platelet-activating factor (PAF) caused a brief depolarization followed by a long-lasting hyperpolarization. Lipopolysaccharide and interferon-gamma only depolarized the plasma membrane. Chemiluminescence measurements indicated that only fMLP and PAF activated macrophages to release ROS. The hyperpolarization response of the cell was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2.- -generating system, xanthine plus xanthine oxidase, caused a marked hyperpolarization. In all the cases, the hyperpolarization induced by fMLP, PAF and O2.- -generating system was found to depend on the concentration of intracellular Ca2+ and extracellular K+. Furthermore, in the presence of quinidine, a blocker of Ca2+-dependent K+ conductance fMLP and PAF caused only prolonged depolarization while the effect of O2.- was reduced to a minimum. These data suggest that the macrophage hyperpolarization response to fMLP and PAF involves superoxide-mediated Ca2+-dependent alteration of the relative membrane permeability to K+.

Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils.

Directed locomotion of mouse peritoneal neutrophils under agarose was studied, and activity of hydrogen peroxide (H2O2) as a chemoattractant was tested in its concentration range of 10(-6) to 10(-3) M. It has been found that H2O2 at low concentrations (about 10 microM) induces chemotactic activity. This activity was not affected by the presence of serum in the agarose medium. Use of bovine serum albumin instead of the heat-inactivated bovine serum in the medium had no effect on cell locomotion. The H2O2-induced chemotaxis was significantly reduced by catalase. Involvement of [Ca2+]i transients in the H2O2-induced chemotactic response was shown. These data indicate that H2O2 itself in small quantities can act as a chemoattractant without interacting with a plasma precursor to form a chemotactic factor. It has been suggested that H2O2 may form an important link similar to the second messenger in communication between the cells.

Activation of murine macrophages by hydrogen peroxide.

Hydrogen peroxide at concentrations from 0.1 to 20 microM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralleled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H2O2 are compared with those for a natural activator, peptide N-formyl-methionyl-leucyl-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.