Several investigators have reported that an increase in Cai is associated with apoptosis. To investigate this question we used several related lines of Syrian hamster embryo cells. Sup+ cells undergo apoptosis on reducing serum to 0.2% whereas sup- cells do not exhibit DNA fragmentation characteristic of apoptosis. Cells were loaded with fura-2 and Cai was monitored in 10% serum and after lowering serum to 0.2%. In sup+ cells in 10% serum, the mean Cai value was 100 plus minus 5 nM (n=61), whereas in 0.2% serum, Cai was 82 plus minus4 nM (n=98). In sup- cells in 10% serum, Cai averaged 260 plus minus 28(n=14), and Cai for cells in 0.2% serum averaged 282 plus minus 33 nM (n=13). Thus, an increase in Cai is not associated with apoptosis in this model. Recent studies have suggested that apoptosis is associated with a decline in ER calcium. To measure ER calcium we added thapsigargin, an inhibitor of the ER Ca ATPase, which causes release of ER calcium into the cytosol where it is measured by fura-2 as an increase in Cai. We found that sup+ cells cultured in 0.2% serum had less thapsigargin releasable calcium(56 plus minus 9 nM)than sup- cells cultured in 0.2% serum (194 plus minus 31 nM) or sup+ (113 plus minus 26 nM) or sup-(178 plus minus 45 nM)cells cultured in 10% serum. This observation led us to investigate possible mechanisms by which low serum might reduce ER calcium. The ATPase is known to be altered by the redox state of key cysteine residues. To investigated if alterations in redox state altered thapsigargin releasable calcium and caused apoptosis we measured whether low serum reduced glutathione levels in sup+ cells. We found that in 10% serum, sup+ and sup- cells had similar levels of total glutathione (approximately 12nmol/mg), and that low serum did not reduce glutathione in either sup+ or sup- cells in low serum compared to cells cultured in 10% serum. Thus, the reduced ER calcium does not appear to be related to cell thiol redox status. We attempted to rescue sup+ cells in low serum by incubation in elevated extracellular calcium. We found that raising extracellular calcium to 3 mM, blocked apoptosis (DNA ladders) and blocked the loss of ER calcium which normally occurs in low serum, suggesting that altered plasma membrane calcium transport might be involved in apoptosis in this model.
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