Enhanced protection against peroxidation-induced mortality of aortic endothelial cells by ascorbic acid-2-O-phosphate abundantly accumulated in the cell as the dephosphorylated form.
Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact from Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and UV. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8- to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [14C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydrophilic agent NDE.
Fujiwara
M
M
Division of Cell Biochemistry, Hiroshima Prefectural University School of BioScience, Japan.
Nagao
N
N
Monden
K
K
Misumi
M
M
Kageyama
K
K
Yamamoto
K
K
Miwa
N
N
D000818:Animals; D001011:Aorta / Q000187:drug effects; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation
D016877:Oxidants; C011669:ascorbate-2-phosphate; D001205:Ascorbic Acid