H2O2-mediated cytotoxicity of rat pulmonary endothelial cells. Changes in adenosine triphosphate and purine products and effects of protective interventions.
H2O2-mediated cytotoxicity (as measured by 51Cr-release) of rat pulmonary artery endothelial cells was time-dependent and related to the concentration of H2O2 employed. The cytotoxic effects of H2O2 were, as expected, prevented by catalase and the degree of protection was directly related to its time of addition. Endothelial cells were incubated with [14C]adenosine to achieve intracellular labeling of ATP, after which the cells were exposed to H2O2. Based on analysis of cell extracts by high-performance liquid chromatography, there was a time-dependent loss of intracellular radioactivity and ATP with the simultaneous appearance of purine degradation products including xanthine/hypoxanthine. Approximately 50% of the intracellular ATP was lost after 15 minutes of exposure and up to 80% was lost by 30 minutes. The extracellular fluid of cells exposed to H2O2 contained significant amounts of xanthine/hypoxanthine. The ferric iron chelator deferoxamine provided almost complete protection against H2O2-mediated cytotoxicity. Two inhibitors of xanthine oxidase, allopurinol and oxypurinol, were also protective as was deoxycoformycin, an inhibitor of adenosine deaminase. Remarkably, cells protected by these agents showed the same loss of intracellular ATP as unprotected, H2O2-treated cells. These findings demonstrate the dissociation between ATP loss per se and oxidant injury of endothelial cells. ATP breakdown may be an important event leading to cellular injury in that this results in the formation of substrate for xanthine oxidase.
Varani
J
J
Department of Pathology, University of Michigan Medical School, Ann Arbor.
Phan
S H
SH
Gibbs
D F
DF
Ryan
U S
US
Ward
P A
PA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002374:Catalase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002859:Chromium Isotopes; D004727:Endothelium / Q000378:metabolism; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006868:Hydrolysis; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D007042:Hypoxanthines / Q000378:metabolism*; D007700:Kinetics; D011651:Pulmonary Artery / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013481:Superoxides / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism*
D002859:Chromium Isotopes; D006878:Hydroxides; D007042:Hypoxanthines; D014970:Xanthines; D013481:Superoxides; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D002374:Catalase; D014969:Xanthine Oxidase