Verapamil, cyproheptadine, and anisodamine antagonized [Ca2+]i elevation induced by TNFalpha in a single endothelial cell.
OBJECTIVE
To study the effect of tumor necrosis factor alpha (TNFalpha) on intracellular free Ca2+ concentration ([Ca2+]i) and the effects of verapamil (Ver), cyproheptadine (Cyp), and anisodamine (Ani) on TNFalpha-induced [Ca2+]i changes in single endothelial cell, and to explore the mechanisms of TNFalpha-mediated shock and antishock actions of Cyp and Ani.


METHODS
Human umbilical vein endothelial cell strains (ECV304) were seeded 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 [Ca2+]i in single endothelial cell were determined by laser scanning confocal microscopy.


RESULTS
After stimulation with TNFalpha, [Ca2+]i in single endothelial cell rapidly increased in a concentration-dependent manner and arrived at the peak value within 60 s, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca2+]i elevation was more obvious in nuclear than in cytoplasma and decreased slowly. Ver (1, 2 micromol/L), Cyp (30, 60 micromol/L), and Ani (20, 40 micromol/L) markedly inhibited TNFalpha 1.2 nmol/L-induced [Ca2+]i elevation.


CONCLUSIONS
TNFalpha markedly induces elevation of [Ca2+]i in a single endothelial cell, it may be an important mechanism of TNFalpha-induced shock and tissue injury. That Cyp and Ani obviously suppress TNFalpha-induced [Ca2+]i elevation probably is one of the mechanisms of their antishock effects.
Wang
L Z
LZ
Department of Pathophysiology, Jining Medical College, Jining 272013,China. WangLZ1@ji-public.sd.cninfo.net
Zhang
Q Z
QZ
Hu
X Z
XZ
Lun
N
N
Zhu
F H
FH
D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D003533:Cyproheptadine / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D018613:Microscopy, Confocal; D012991:Solanaceous Alkaloids / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*; D014471:Umbilical Veins / Q000166:cytology; D014700:Verapamil / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D012991:Solanaceous Alkaloids; D014409:Tumor Necrosis Factor-alpha; C003922:anisodamine; D003533:Cyproheptadine; D014700:Verapamil; D002118:Calcium