© 1995 American Heart Association, Inc.
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Effects of Hypoxanthine–Xanthine Oxidase on Ca2+ Stores and Protein Synthesis in Human Endothelial Cells
From the Respiratory Division, Hôpital Cantonal Universitaire de Genève (Switzerland).
Correspondence to D. Dreher, Hôpital Cantonal Universitaire, Division de Pneumologie, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland.
Abstract 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. Thus, depletion of Ca2+ stores appeared to be involved in the inhibition of protein synthesis at the initiation level by both thapsigargin and HX-XO. These results indicate that (1) the cytosolic [Ca2+] changes induced by HX-XO result from mobilization of Ca2+ from intracellular stores and subsequent influx of extracellular Ca2+, (2) the HX-XO–related mobilization of sequestered Ca2+ includes hormone-insensitive pools, and (3) the depletion of hormone-insensitive Ca2+ stores appears to be in part responsible for the inhibition of protein synthesis by HX-XO.
Key Words: human umbilical vein endothelial cells • intracellular Ca2+ • reactive O2 species • thapsigargin • thrombin
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