Redox regulation of calcium signaling in cancer cells by ascorbic Acid involving the mitochondrial electron transport chain.

Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca(2+) release by ascorbic acid, the effects of specific inhibitors of the electron transport chain components on mitochondrial reactive oxygen species (ROS) production and Ca(2+) mobilization in HEp-2 cells were investigated. It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca(2+) release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca(2+) release from intracellular stores in HEp-2 cells caused by ascorbic acid and antimycin A. In the presence of rotenone, antimycin A stimulates ROS production by mitochondria. Ascorbate-induced Ca(2+) release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca(2+) release in HEp-2 cells caused by ascorbic acid is associated with induced mitochondrial ROS production. The data obtained are in line with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets.

Redox regulation of morphology, cell stiffness, and lectin-induced aggregation of human platelets.

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 µM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.

Redox buffer capacity of the cell: theoretical and experimental approach.

Reactive oxygen species (ROS) are involved in a variety of biological phenomena, such as mutation, carcinogenesis, inflammation, aging, development, and signal transduction. Intracellular generation of ROS might lead to the activation of redox signaling or oxidative stress. Nonetheless, it is difficult to estimate whether ROS-induced intracellular events are beneficial or deleterious to the cell. The quantitative basis of changes in the intracellular redox state of cells is not well-defined, thus leading to the dilemma that redox changes induced by oxidants in distinct cell types cannot be predicted. To overcome this limitation this study undertakes to analyze on a theoretical as well as on an experimental basis the intracellular redox state changes occurring inside cells upon addition of oxidants or reductants. 2,7-Dichlorodihydrofluorescein (H(2)DCF) was used to characterize the redox buffer capacity in erythrocytes. It was shown that the redox buffer capacity of erythrocytes in the relation to peroxynitrite (ONOO(-)) is 2.1 times lower than the redox buffer capacity of erythrocytes in the relation to hydrogen peroxide (H(2)O(2)). The feasibility of redox buffer capacity assessment as an innovative tool for investigation and description of redox signaling events in cells is discussed.

Intracellular redox state: towards quantitative description.

Redox state is a widely used term for the description of redox phenomena in biological systems. The regulating mechanisms responsible for maintaining the redox state are not yet fully known. But it was shown that changes in the redox state might lead to a cascade of intracellular events, beneficial or deleterious to the cell. There are several methods for the description of the intracellular redox state. These methods are based on using measured intracellular concentrations of reduced and oxidized glutathione in the Nernst equation. However, glutathione is not always a basic redox component in biological fluids, organelles, cells or tissues. As a result, changes in the intracellular redox state are not always accompanied by considerable changes of glutathione concentration. In this work it was proposed to use the concept of effective reduction potential for the quantitative characteristic of the intracellular redox state. The effective reduction potential was substantiated on the basis of a thermodynamic description. A new equation for the calculation of the effective reduction potential was derived. This equation summarizes the contribution of different oxidizing and reducing agents in the formation of an effective redox potential. The theoretical estimation of the effective reduction potential values for the different biological fluids and cells was carried out with the use of a method developed.