Effects of Superoxide Anions on Endothelial Ca Signaling Pathways

Although the involvement of free radicals in the development of endothelial dysfunction under pathological conditions, like diabetes and hypercholesterolemia, has been proposed frequently, there is limited knowledge as to how superoxide anions (O2 ) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O2 -generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca release and capacitative Ca entry in response to bradykinin and ATP in a timeand concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4benzohydroquinone or thapsigargin was enhanced in O2 -exposed cells (138% and 132%, respectively). Increased Ca release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (1140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca store depletion with thapsigargin (1103% and 148%, respectively) and tyrosine kinase activity (197%). A comparison of bradykinin-initiated intracellular Ca release and thapsigargin-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca release and membrane viscosity. Because intracellular Ca is a main regulator of endothelial vascular function, our data suggest that O2 2 anions are involved in regulation of the vascular endothelium. (Arterioscler Thromb Vasc Biol. 1998;18:1470-1479.)