61 Prevention of nucleotide depletion induced by oxygen free radicals (OFR) in endothelial cells (ECs)

Abstract

Upon reperfusion of hypoxic tissue xanthine oxidase (XO) metabolizes hypoxanthine (Hx) to xanthine (X) and uric acid (Ua), OFR are produced and may cause organ damage. XO together vith Hx causes adenine nucleotide depletion and death of cultured human umbilical vein ECs. We studied Che ability of glutathione (GSH)(5, 10, 15 mM), superoxide dismutase (SOD)(600 IU), catalase (CAT) (600 IU), alfa-tocoferol (TF) (50 and 100uM), ascorbic acid (C-vit) (10mM), dimethylchiourea (DMTU) (5mM), and dimethylsulfoxide (DMSO) (5 mM) to prevent nucleotide depletion. ECs vere labeled overnight vith 14C-adenine (100 uM) in culture veils, washed, and incubated with XO (80 mU/ml) and Hx (100 uM), with or without the study compounds for 4h. Nucleotides from cell extracts and medium, and breakdown products (Hx, X, Ua) from medium were separated and counted. In control cells, 60-65 % of initial cellular radioactivity (cpm) remain in adenine nucleotides after 4 h and 30-35 % appear as Hx, X, and Ua in medium. XO with Hx depletes nucleotides (1-5% of cpm in cell nucleotides, 60-70 % in Hx, X, and Ua). The corresponding distributions in the presence of the study compounds were: GSH 10/12/26 % and 62/52/48 % (5/10/15 mM); SOD 15% and 60 %; CAT 18 % and 64 %. The rest of the cpm were as nucleotides in medium. TF, C-vit, DHSO, and DMTU were not effective. TF was not protective even when present in the culture medium for 3 days. We conclude that GSH, SOD, and CAT, even though only partially able to prevent OFR-induced nucleotide depletion, should be evaluated in the treatment of in vivo ischemia-reperfusion damage.