Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells.
Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.
Geeraerts
M D
MD
Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina, Chapel Hill 27599-7090.
Ronveaux-Dupal
M F
MF
Lemasters
J J
JJ
Herman
B
B
D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016257:Fura-2; D006801:Humans; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D007425:Intracellular Membranes / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013690:Television
D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D002118:Calcium; D016257:Fura-2