Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post‐anoxic reoxygenation or with alkylhydroperoxides

Vascular endotheliocytes BAE‐2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre‐Anox administration with L ‐ascorbic acid (Asc)‐2‐ O ‐phosphate (Asc2P), an autooxidation‐resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)‐lacking buffer more advantageously than in a Glc‐containing buffer where less efficiency had been demonstrated for Asc entry into BAE‐2 cells than in a Glc‐lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2–5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one‐half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre‐Anox administration with Asc2P, but not with Asc or dehydro‐Asc, and were not suppressed by post‐Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS‐scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS‐generator tert‐butylhydroperoxide (t‐BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE‐2 endotheliocytes, which were also protected dose‐dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH‐DA. Thus Anox/Reox‐induced cell death can be prevented by Asc2P that suppresses ROS‐generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space. © 2004 Wiley‐Liss, Inc.