NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca2+, but whether NADPH oxidase activation directly affects Ca2+ signaling is unknown. NADPH stimulated the production of superoxide anion and H2O2 in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rad, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H2O2 each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca2+ and shifted the InsP(3)-Ca2+ release dose-response curve to the left, Concentrations of H2O2 as low as 3 mu M increased the sensitivity of intracellular Ca2+ stores to InsP(3) and decreased the InsP(3) EC50 from 423.2 +/- 54.9 to 276.9 +/- 14.4 nM. The effect of NADPH on InsP(3)-stimulated Ca2+ release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H2O2 increases the sensitivity of intracellular Ca2+ stores to InsP(3) in human endothelial cells. Since Ca2+-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.