title: Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post-anoxic reoxygenation or with alkylhydroperoxides.
abstract: 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.
authors.affiliation: Laboratory of Cell Death Control BioTechnology, Hiroshima Prefectural University School of BioSciences, Shobara, Hiroshima 727-0023, Japan.
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Saitoh
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Miwa
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects* / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors; D014471:Umbilical Veins / Q000166:cytology; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; C011669:ascorbate-2-phosphate; D020122:tert-Butylhydroperoxide; D001205:Ascorbic Acid