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

Changes in intracellular Ca 2+ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine‐xanthine oxidase system (X‐XO) mobilizes Ca 2+ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O 2 species involved in the disruption of calcium homeostasis by X‐XO, we studied the effect of X‐XO on [Ca 2+ ] i by spectrofluorimetry with fura‐2 in human umbilical vein endothelial cells (HUVEC). The [Ca 2+ ] 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, O 2 − , or H 2 O 2 , respectively. The [Ca 2+ ] i increase stimulated by 10 nmol H 2 O 2 /ml/min, generated from the glucose‐glucose oxidase system, or 10 μM H 2 O 2 , given as bolus, was about a third of that induced by X‐XO (10 nmol O 2 − /ml/min) but was comparable to that induced by X‐XO in the presence of SOD. The X‐XO—stimulated [Ca 2+ ] i increase was significantly reduced by 100 μM o‐phenanthroline, which inhibits the iron‐catalysed formation of the hydroxyl radical. On the other hand, the [Ca 2+ ] i response to low dose X‐XO (1 nmol O 2 − /ml/min) was markedly enhanced in the presence of 1 μM H 2 O 2 , which itself had no effect on [Ca 2+ ] 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 O 2 − and H 2 O 2 , which could be explained by the formation of the hydroxyl radical. © 1995 Wiley‐Liss, Inc.