SLOW INCREASE IN INTRANUCLEAR AND CYTOSOLIC-FREE CALCIUM CONCENTRATIONS IN L929 CELLS IS IMPORTANT IN TUMOR NECROSIS FACTOR-ALPHA-MEDIATED CELL-DEATH

The potential involvement of cellular calcium in the signalling pathwa y of tumour necrosis factor-alpha (TNF) was assessed in L929 cells usi ng Ca-45(2+) and confocal laser scanning microscopy with fluorescence calcium indicators. Our data indicate that the effect of TNF on intrac ellular Ca2+ mobilization is a slow process with no discernible increa se in the cytosolic free Ca2+ concentration ([Ca2+]c) and intranuclear Ca2+ level ([Ca2+]n) within the 1st min of TNF (25 ng/ml) administrat ion. However, prolonged exposure (2 h) of L929 cells to TNF brought ab out pronounced increase in cytosolic and intranuclear [Ca2+] even in t he 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 wa s around and confined to the nucleus. Following the pretreatment of ce lls with thapsigargin, a synergistic killing effect was obtained when cells were cultured with TNF. The use of Ca-45(2+) also revealed that TNF enhanced the Ca-45(2+) uptake in a time-dependent manner. Calcium channel blockers, verapamil and diltiazem, could alleviate both the TN F-mediated Ca-45(2+)- uptake and killing activity. Our results therefo re suggest that an increase in cellular Ca2+ is a crucial factor in th e TNF cytotoxicity.