Effects of Hypoxanthine–Xanthine Oxidase on Ca 2+ Stores and Protein Synthesis in Human Endothelial Cells

Abstract We have investigated the effects of reactive O 2 metabolites generated by the hypoxanthine–xanthine oxidase (HX-XO) system on intracellular Ca 2+ 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 Ca 2+ , followed by a sustained elevation above the baseline value. In the presence of La 3+ , which blocks Ca 2+ influx from the extracellular medium, a transient Ca 2+ increase was still observed, but the sustained rise was suppressed. The HX-XO–related Ca 2+ changes were completely prevented by pretreatment with thapsigargin, which depletes intracellular Ca 2+ stores. Hence, the effects of HX-XO on Ca 2+ homeostasis were due to mobilization of Ca 2+ from the intracellular stores with subsequent influx of extracellular Ca 2+ . HX-XO mobilized more of sequestered Ca 2+ than did thrombin, a receptor agonist that depletes only a part of the intracellular Ca 2+ stores (the hormone-sensitive stores). To determine the relevance of the HX-XO–related depletion of Ca 2+ stores for cell function, we investigated the role of Ca 2+ 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 Ca 2+ stores via the availability of extracellular Ca 2+ significantly influenced the thapsigargin-related and the HX-XO–related inhibition of overall protein synthesis. A corresponding effect of extracellular Ca 2+ was seen in polyribosome distribution profiles, which reflected an inhibition of translation initiation in both treatments. Thus, depletion of Ca 2+ 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 Ca 2+ changes induced by HX-XO result from mobilization of Ca 2+ from intracellular stores and subsequent influx of extracellular Ca 2+ , (2) the HX-XO–related mobilization of sequestered Ca 2+ includes hormone-insensitive pools, and (3) the depletion of hormone-insensitive Ca 2+ stores appears to be in part responsible for the inhibition of protein synthesis by HX-XO.