EFFECTS OF SUPEROXIDE ANIONS ON ENDOTHELIAL CA2+ SIGNALING PATHWAYS

Although the involvement of free radicals in the development of endoth elial dysfunction under pathological conditions, like diabetes and hyp ercholesterolemia, has been proposed frequently, there is limited know ledge as to how superoxide anions (O-2(-)) might affect endothelial si gnal transduction. In this study, we investigated the effects of prein cubation with the O-2(-)-generating system xanthine oxidase/hypoxanthi ne (XO/HX) on mechanisms for Ca2+ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased i ntracellular Ca2+ release and capacitative Ca2+ entry in response to b radykinin and ATP in a time- and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine k inase inhibitor tyrphostin A48. In addition, capacitative Ca2+ entry i nduced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-be nzohydroquinone or thapsigargin was enhanced in O-2(-)-exposed cells ( +38% and +32%, respectively). Increased Ca2+ release in response to br adykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affect ed other signal transduction mechanisms involved in endothelial Ca2+ s ignaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca2+ store depletion with thapsigargin (+103% and +48%, respectively) and tyrosine kinase activity (+97%). A comparison of bradykinin-initiated intracellular Ca2+ release and thapsigargin-i nduced hyperpolarization with membrane viscosity modulated by XO/HX (d ecrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca2+ release and membrane viscosity. Because intracellular Ca2+ is a main regulator of endothelial vascular function, our data suggest that O-2(-) anions are involved in regulation of the vascular endothelium.