Effects of Superoxide Anions on Endothelial Ca ²⁺ Signaling Pathways

Abstract

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 (O 2 Ϫ ) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O 2 Ϫ -generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca 2ϩ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca 2ϩ release and capacitative Ca 2ϩ 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 Ca 2ϩ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O 2 Ϫ -exposed cells (ϩ38% and ϩ32%, respectively). Increased Ca 2ϩ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (ϩ140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca 2ϩ signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca 2ϩ store depletion with thapsigargin (ϩ103% and ϩ48%, respectively) and tyrosine kinase activity (ϩ97%). A comparison of bradykinin-initiated intracellular Ca 2ϩ 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 2ϩ release and membrane viscosity. Because intracellular Ca 2ϩ is a main regulator of endothelial vascular function, our data suggest that O 2 Ϫ anions are involved in regulation of the vascular endothelium.

Mechanistic Role of Cytochrome P450 Monooxygenases in Oxidized Low-Density Lipoprotein–Induced Vascular Injury

“…In the study by Graier et al, 40 enhanced CYP monooxygenase activity was observed in assays with xanthine oxidase/ hypoxanthine. This study therefore suggests exogenous ROS to stimulate CYP monooxygenase activity.…”

Intercellular signalling within vascular cells under high D-glucose involves free radical-triggered tyrosine kinase activation

“…-were photometrically measured by the reduction of ferricytochrome C as previously described [10,12]. Confluent SMC were incubated at 37°C in Ca 2+ -free PBS containing 10 µmol/l ferricytochrome C (horse heart type III) with or without SOD (476 U/ml; i.e.…”

“…Endothelial tyrosine kinase activity was measured by using a customized photometric protein tyrosine kinase kit from Calbiochem-Novabiochem [12,16]. After interaction period cell lysates were obtained by sonification in chilled lysis buffer containing in mmol/l: 20 Tris, 50 NaCl, 1 EDTA, 1 EGTA, 0.2 PMSF, 0.2 mercaptoethanol plus 1 µg/ml pepstatin and 0.5 µg/ml leupeptin (pH 7.4).…”

Serum Xanthine Oxidase: Origin, Regulation, and Contribution to Control of Trypanosome Parasitemia

“…XO exclusively uses O 2 as the electron acceptor, yielding O 2 2 and hydrogen peroxide (H 2 O 2 ) as reaction by-products. XO is one of the most widely distributed oxidases and has been inculpated in a legion of biological phenomena ranging from H 2 O 2 -mediated signal transduction, through the amplification of inflammatory responses, to organ failure (12,22,53,85).…”