RESUMO
AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani)on the changes of intracellular free Ca 2+ concentration ([Ca 2+ ] i) induced by tumor necrosis factor (TNF ?) in single endothelial cells, and to explore the mechanisms of TNF ?-mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains(ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca 2+ ] i in single endothelial cell was determined by laser scanning confocal microscopy(LSCM). RESULTS: [Ca 2+ ] i in single endothelial cell after stimulation of TNF ? rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca 2+ ] i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3?10 -5 , 6?10 -5 mol/L) and Ani (2?10 -5 , 4?10 -5 mol?L -1 ) markedly inhibited TNF ? (1.2?10 -9 mol?L -1 )-induced [Ca 2+ ] i elevation. CONCLUSIONS: TNF ? markedly induces elevation of [Ca 2+ ] i in single endothelial cell, it may be an important mechanism of TNF ?-induced shock and tissue injury. Cyp and Ani obviously suppress TNF ?-induced [Ca 2+ ] i elevation, which probably is one of the mechanisms of their antishock effects.