Differential effects of superoxide, hydrogen peroxide, and hydroxyl radical on intracellular calcium in human endothelial cells

Changes in intracellular Ca2+ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine-xanthine oxidase system (X-XO) mobilizes Ca2+ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O2 species involved in the disruption of calcium homeostasis by X-XO, we studied the effect of X-XO on [Ca2+]i by spectrofluorimetry with fura-2 in human umbilical vein endothelial cells (HUVEC). The [Ca2+]i response to X-XO was essentially diminished by superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (200 U/ml), which scavenge the superoxide anion, O2-, or H2O2, respectively. The [Ca2+]i increase stimulated by 10 nmol H2O2/ml/min, generated from the glucose-glucose oxidase system, or 10 microM H2O2, given as bolus, was about a third of that induced by X-XO (10 nmol O2-/ml/min) but was comparable to that induced by X-XO in the presence of SOD. The X-XO-stimulated [Ca2+]i increase was significantly reduced by 100 microM o-phenanthroline, which inhibits the iron-catalysed formation of the hydroxyl radical. On the other hand, the [Ca2+]i response to low dose X-XO (1 nmol O2-/ml/min) was markedly enhanced in the presence of 1 microM H2O2, which itself had no effect on [Ca2+]i. More than 50% of this synergistic effect was prevented by o-phenanthroline. These results indicate that the effect of X-XO on calcium homeostasis appears to result from an interaction of O2- and H2O2, which could be explained by the formation of the hydroxyl radical.