Slow increase in intranuclear and cytosolic free calcium concentrations in L929 cells is important in tumour necrosis factor-alpha-mediated cell death.
The potential involvement of cellular calcium in the signalling pathway of tumour necrosis factor-alpha (TNF) was assessed in L929 cells using 45Ca2+ and confocal laser scanning microscopy with fluorescence calcium indicators. Our data indicate that the effect of TNF on intracellular Ca2+ mobilization is a slow process with no discernible increase in the cytosolic free Ca2+ concentration ([Ca2+]c) and intranuclear Ca2+ level ([Ca2+]n) within the 1st min of TNF (25 ng/ml) administration. However, prolonged exposure (2 h) of L929 cells to TNF brought about pronounced increase in cytosolic and intranuclear [Ca2+] even in the absence of external Ca2+. The increase in intracellular [Ca2+] was more apparent when cells were treated with thapsigargin, an inhibitor of microsomal Ca2+-ATPase. Interestingly, most of the Ca2+ released was around and confined to the nucleus. Following the pretreatment of cells with thapsigargin, a synergistic killing effect was obtained when cells were cultured with TNF. The use of 45Ca2+ also revealed that TNF enhanced the 45Ca2+ uptake in a time-dependent manner. Calcium channel blockers, verapamil and diltiazem, could alleviate both the TNF-mediated 45Ca2+-uptake and killing activity. Our results therefore suggest that an increase in cellular Ca2+ is a crucial factor in the TNF cytotoxicity.
Kong
S K
SK
Department of Biochemistry, Chinese University of Hong Kong, Shatin.
Fung
K P
KP
Choy
Y M
YM
Lee
C Y
CY
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D007739:L Cells; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D019284:Thapsigargin; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D002273:Carcinogens; D014409:Tumor Necrosis Factor-alpha; D019284:Thapsigargin; D002118:Calcium