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Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide

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Abstract

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 μM) or histamine (100 μM). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 μM were used. Buffering trace amounts of iron (o-phenanthroline; 200 μM) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 μM) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 μM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological ‘oxidative stress’ associated with a progressive increase in [Ca2+]i.

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  1. Department of Anaesthesiology and Intensive Care, University Hospital Charité, Schumannstraße 20/21, 10117, Berlin, Germany

    Thomas Volk, Mario Hensel & Wolfgang J. Kox

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  1. Thomas Volk
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Volk, T., Hensel, M. & Kox, W.J. Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide. Mol Cell Biochem 171, 11–21 (1997). https://doi.org/10.1023/A:1006886215193

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