We have investigated the effects of reactive O-2 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 KUVECs 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.
