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⁺ (Ca(i)²⁺) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Ca(i)²⁺ 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 Ca(i)²⁺ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 μM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Ca(i)²⁺ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Ca(i)²⁺ was prevented by removal of extracellular Ca²⁺ (Ca(o)²⁺). Prevention of the increase in Ca(i)²⁺ was associated with prolonged cell viability. Readdition of Ca(o)²⁺ resulted in an immediate large increase in Ca(i)²⁺ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Ca(i)²⁺ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Ca(i)²⁺ influx and resultant activation of Ca²⁺-dependent proteases.