EFFECTS OF HYPOXANTHINE XANTHINE-OXIDASE ON CA2-SYNTHESIS IN HUMAN ENDOTHELIAL-CELLS( STORES AND PROTEIN)

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 e ndothelial 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 medi um, 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 intracellu lar Ca2+ stores. Hence, the effects of HX-XO on Ca2+ homeostasis were due to mobilization of Ca2+ from the intracellular stores with subsequ ent influx of extracellular Ca2+. HX-XO mobilized more of sequestered Ca2+ than did thrombin, a receptor agonist that depletes only a part o f the intracellular Ca2+ stores (the hormone-sensitive stores). To det ermine the relevance of the HX-XO-related depletion of Ca2+ stores for cell function, we investigated the role of Ca2+ mobilization in the r egulation of protein synthesis. Overall protein synthesis in KUVECs wa s markedly reduced by thapsigargin, which depletes both hormone-sensit ive and -insensitive stores, but was not substantially affected by thr ombin. Manipulation of the refilling of the Ca2+ stores via the availa bility 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 polyribos ome distribution profiles, which reflected an inhibition of translatio n initiation in both treatments. Thus, depletion of Ca2+ stores appear ed to be involved in the inhibition of protein synthesis at the initia tion level by both thapsigargin and HX-XO. These results indicate that (1) the cytosolic [Ca2+] changes induced by HX-XO result from mobiliz ation of Ca2+ from intracellular stores and subsequent influx of extra cellular Ca2+, (2) the HX-XO-related mobilization of sequestered Ca2includes hormone-insensitive pools, and (3) the depletion of hormone-i nsensitive Ca2+ stores appears to be in part responsible for the inhib ition of protein synthesis by HX-XO.