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.