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 agen t, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mito chondrial dehydrogenase activity as assessed by fluorometry using ethi dium homodimer and photometry using WST-1, respectively. The cells wer e protected from t-BuOOH-induced injury more markedly by L-ascorbic ac id-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, whic h 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 exhi bited no prevention against both types of injury. The amount of intrac ellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 ce lls, where the intact form Asc2P was not detected, than in Asc-adminis tered cells as assessed by HPLC of cell extract with detection by coul ometric ECD and UV. During transmembrane influx into the cell, Asc2P w as concentrated as highly as 70- to 90-fold relative to the extracellu lar Asc2P concentration, whereas Asc was 8- to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell det ermined by [C-14]PEG/gas chromatography. Thus, Asc2P but not Asc is hi ghly concentrated in the aqueous phase of the cell after prompt dephos phorylation, and may thereby render the cell more resistant to t-BuOOH -peroxidation assumedly via scavenging of intracellular reactive oxyge n species than to peroxidation with the less hydrophilic agent NDE.