Adenine nucleotide depletion from endothelial cells exposed to xanthine oxidase.
Hypoxia causes breakdown of cellular nucleotides, accumulation of hypoxanthine (HX), and conversion of xanthine dehydrogenase into xanthine oxidase (XO). Upon reoxygenation, the HX-XO reaction generates free radicals, one potential mechanism of tissue damage. Because endothelial cells contain XO and are exposed to circulating HX, they are a likely target for damage. We studied the effect of XO and/or HX at physiologically relevant concentrations on nucleotide metabolism of cultured endothelial cells from human umbilical veins. Cells were labeled with [14C]adenine and incubated for up to 6 h with HX, XO, or both, in the absence or presence of serum. Adenine nucleotides from cell extracts and nucleotide breakdown products (HX, xanthine, and urate) from the medium were separated and counted. HX alone had no effect. XO (80 mU/ml) alone caused a 70% (no serum) or 40% (with serum) fall in adenine nucleotides and an equivalent increase of xanthine and urate. The combination of HX and XO caused a 90% (no serum) or 70% (with serum) decrease in nucleotides, decrease in energy charge, and detachment of cells from the culture plate. Nucleotide depletion was not accounted for by proteolytic activity in the XO preparation. Albumin was only half as effective as serum in preventing nucleotide loss. Thus exogenous XO, in the presence of endogenous HX, triggers adenine nucleotide catabolism, but endogenous XO activity is too low to influence nucleotide levels even at high exogenous HX concentrations. Serum limits the catabolic effect of XO and thus protects cells from free radical damage.
Aalto
T K
TK
Children's Hospital, University of Helsinki, Finland.
Raivio
K O
KO
D000225:Adenine / Q000378:metabolism; D000227:Adenine Nucleotides / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007700:Kinetics; D011865:Radioisotope Dilution Technique; D014969:Xanthine Oxidase / Q000494:pharmacology*
D000227:Adenine Nucleotides; D002250:Carbon Radioisotopes; D007042:Hypoxanthines; D019271:Hypoxanthine; D014969:Xanthine Oxidase; D000225:Adenine