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 O-2 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, O-2(-), or H2O2, respectively. The [Ca2+](i) increase stimulated by 10 nmol H2O2/ml/min, generated from the glucose-glucose oxidase system, or 10 mu M H2O2, 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 [Ca2+](i) increase was significantly reduced by 100 mu M 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 O-2(-)/ml/min) was markedly enhanced in the presence of 1 mu M 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 O-2(-) and H2O2, which could be explained by the formation of the hydroxyl radical. (C) 1995 Wiley-Liss, Inc.