Cytosolic Free Ca2+ and Proteolysis in Lethal Oxidative Injury in Endothelial Cells

Am J Physiol. 1991 Nov;261(5 Pt 1):C889-96. doi: 10.1152/ajpcell.1991.261.5.C889.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium / metabolism*
  • Cell Death
  • Cells, Cultured
  • Cytosol / metabolism*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism*
  • Fura-2
  • Humans
  • Hydrolysis
  • Image Processing, Computer-Assisted
  • Intracellular Membranes / metabolism
  • Osmolar Concentration
  • Oxidation-Reduction
  • Peptide Hydrolases / metabolism*
  • Protease Inhibitors / pharmacology
  • Subcellular Fractions / metabolism
  • Television

Substances

  • Protease Inhibitors
  • Peptide Hydrolases
  • Calcium
  • Fura-2