Effects of hypoxanthine-xanthine oxidase on Ca2+ stores and protein synthesis in human endothelial cells.
We have investigated the effects of reactive O2 metabolites generated by the hypoxanthine-xanthine oxidase (HX-XO) system on intracellular Ca2+ and its relation with protein synthesis in human umbilical vein endothelial cells (HUVECs). Spectrofluorometry with fura 2 showed that the oxidative stress induced a rapid transient rise in cytosolic [Ca2+], followed by a sustained elevation above the baseline value. In the presence of La3+, which blocks Ca2+ influx from the extracellular medium, a transient [Ca2+] increase was still observed, but the sustained rise was suppressed. The HX-XO-related [Ca2+] changes were completely prevented by pretreatment with thapsigargin, which depletes intracellular Ca2+ stores. Hence, the effects of HX-XO on Ca2+ homeostasis were due to mobilization of Ca2+ from the intracellular stores with subsequent influx of extracellular Ca2+. HX-XO mobilized more of sequestered Ca2+ than did thrombin, a receptor agonist that depletes only a part of the intracellular Ca2+ stores (the hormone-sensitive stores). To determine the relevance of the HX-XO-related depletion of Ca2+ stores for cell function, we investigated the role of Ca2+ mobilization in the regulation of protein synthesis. Overall protein synthesis in HUVECs was markedly reduced by thapsigargin, which depletes both hormone-sensitive and -insensitive stores, but was not substantially affected by thrombin. Manipulation of the refilling of the Ca2+ stores via the availability of extracellular Ca2+ significantly influenced the thapsigargin-related and the HX-XO-related inhibition of overall protein synthesis. A corresponding effect of extracellular [Ca2+] was seen in polyribosome distribution profiles, which reflected an inhibition of translation initiation in both treatments.(ABSTRACT TRUNCATED AT 250 WORDS)
Dreher
D
D
Respiratory Division, Hôpital Cantonal Universitaire de Genève, Switzerland.
Jornot
L
L
Junod
A F
AF
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology*; D018384:Oxidative Stress; D014176:Protein Biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014969:Xanthine Oxidase / Q000494:pharmacology*
D007042:Hypoxanthines; D017382:Reactive Oxygen Species; D013729:Terpenes; D019271:Hypoxanthine; D019284:Thapsigargin; D014969:Xanthine Oxidase; D002118:Calcium