Dual effects of extracellular Ca 2+ on cardiotoxin-induced cytotoxicity and cytosolic Ca 2+ changes in cultured single cells of rabbit aortic endothelium

Abstract

The effects of extracellular Ca 2+ on cytotoxicity induced by cardiotoxin (CTX), isolated from Chinese cobra venom, were investigated in cultured rabbit aortic endothelial cells (RAECs). In Hank's buffered saline solution (HBSS) containing 1.2 mM Ca 2+, CTX (1–30 μM) caused cell necrosis and cell death in a concentration-dependent manner, as determined by trypan blue exclusion test performed after a 20-min CTX treatment. The concentration of CTX that caused 50% cell death was about 6.5 μM. CTX (10 μM)-induced RAEC damage was also evident but less prominent in Ca 2+-free medium and almost completely prevented in medium containing 7–10 mM Ca 2+. Therefore, Ca 2+ appears to provoke CTX-induced injury at physiological concentrations, but protects against it at high concentrations. The protection of RAECs from CTX-induced injury could also be achieved by high concentrations of Ni 2+ and Mg 2+. Using the fura-2 fluorescence technique to measure the cytosolic free Ca 2+ concentration ([Ca 2+] i) of single RAEC, it was shown that in 1.2 mM Ca 2+-containing HBSS, treatment of RAECs with 10 μM CTX for 7–35 min resulted in a tremendous and irreversible [Ca 2+] i elevation, suggestive of cell membrane damage and extracellular Ca 2+ entry. Ni 2+ could also enter the cytosol of these damaged RAECs. However, there was no [Ca 2+] i elevation or Ni 2+ entry in RAECs that were preincubated in HBSS containing 7 mM Ca 2+ or Ni 2+ before CTX exposure. In RAECs protected with 7 mM Ca 2+, the intracellular Ca 2+ signals triggered by 100 μM extracellular ATP or 10 μM bradykinin in CTX-treated groups were similar to those in the untreated control groups. Taken together, the results indicate that high extracellular Ca 2+ concentrations protected RAECs from CTX-induced injury, and preserved the ability of CTX-treated RAECs to generate Ca 2+ signals in response to physiological stimuli.