Effects of Superoxide Anions on Endothelial Ca²⁺ Signaling Pathways

From the Department of Medical Biochemistry, University of Graz, Graz, Austria.

Correspondence to Dr Wolfgang F. Graier, Department of Medical Biochemistry, University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria. E-mail wolfgang.graier{at}kfunigraz.ac.at

Abstract—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 Ca²⁺ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca²⁺ release and capacitative Ca²⁺ entry in response to bradykinin and ATP in a time- and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca²⁺ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O₂^--exposed cells (+38% and +32%, respectively). Increased Ca²⁺ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca²⁺ signaling, 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-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease 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 O₂^- anions are involved in regulation of the vascular endothelium.

Key Words: cytochrome P450 epoxygenase • inositol-1,4,5-trisphosphate • membrane fluidity • membrane potential • tyrosine kinase

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