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.

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Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca2+ release by ascorbic acid, the effects of specific inhibitors of the electron transport chain components on mitochondrial reactive oxygen species (ROS) production and Ca2+ mobilization in HEp-2 cells were investigated. It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca2+ release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca2+ 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 Ca2+ release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca2+ 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.

Effects of ascorbic acid on calcium homeostasis of human laryngeal carcinoma cells were studied. Intracellular concentration of free calcium and intracellular pH were measured by fluorescent analysis. Ascorbic acid in concentrations of 3-10 mM caused pH drop and sharply increased concentrations of free Ca ions in HEp-2 cells. Intracellular concentration of free Ca ions resulted from Ca ion release from the thapsigargin-sensitive Ca depots.

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