Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: role of hydrogen peroxide.
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 microM) or histamine (100 microM). Superoxide dismutase (50 U/ml), which dismutates O2.- into H2O2 also had no influence, whereas catalase (50 U/ml), which removes H2O2, completely diminished transient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 microM were used. Buffering trace amounts of iron (o-phenanthroline; 200 microM) in order to inhibit .OH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca(2+)-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 Ca(2+)-ATPases of the endoplasmatic reticulum with thapsigargin (1 microM) 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 microM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O2.- 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.
Volk
T
T
Department of Anaesthesiology and Intensive Care, University Hospital Charité, Berlin, Germany.
Hensel
M
M
Kox
W J
WJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004727:Endothelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D005949:Glucose Oxidase / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D019271:Hypoxanthine / Q000494:pharmacology; D018384:Oxidative Stress; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D014969:Xanthine Oxidase / Q000494:pharmacology
D017382:Reactive Oxygen Species; D019271:Hypoxanthine; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D014969:Xanthine Oxidase; D010738:Type C Phospholipases; D005947:Glucose; D002118:Calcium