title: Dual effects of extracellular Ca2+ on cardiotoxin-induced cytotoxicity and cytosolic Ca2+ changes in cultured single cells of rabbit aortic endothelium.
abstract: The effects of extracellular Ca2+ 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 Ca2+, CTX (1-30 microM) 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 microM. CTX (10 microM)-induced RAEC damage was also evident but less prominent in Ca2+-free medium and almost completely prevented in medium containing 7-10 mM Ca2+. Therefore, Ca2+ 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 Ni2+ and Mg2+. Using the fura-2 fluorescence technique to measure the cytosolic free Ca2+ concentration ([Ca2+]i) of single RAEC, it was shown that in 1.2 mM Ca2+-containing HBSS, treatment of RAECs with 10 microM CTX for 7-35 min resulted in a tremendous and irreversible [Ca2+]i elevation, suggestive of cell membrane damage and extracellular Ca2+ entry. Ni2+ could also enter the cytosol of these damaged RAECs. However, there was no [Ca2+]i elevation or Ni2+ entry in RAECs that were preincubated in HBSS containing 7 mM Ca2+ or Ni2+ before CTX exposure. In RAECs protected with 7 mM Ca2+, the intracellular Ca2+ signals triggered by 100 microM extracellular ATP or 10 microM bradykinin in CTX-treated groups were similar to those in the untreated control groups. Taken together, the results indicate that high extracellular Ca2+ concentrations protected RAECs from CTX-induced injury, and preserved the ability of CTX-treated RAECs to generate Ca2+ signals in response to physiological stimuli.
authors.affiliation: Department of Physiology, Faculty of Medicine, The University of Hong Kong, China.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Ou
authors.forename: Y M
authors.initials: YM
authors.lastname: Leung
authors.forename: S J
authors.initials: SJ
authors.lastname: Huang
authors.forename: C Y
authors.initials: CY
authors.lastname: Kwan
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D004179:Cobra Cardiotoxin Proteins / Q000633:toxicity*; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits
chemical_list: D004179:Cobra Cardiotoxin Proteins; D002118:Calcium