Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+mobilization

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca 2+ , we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca 2+ homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca 2+ , and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca 2+ mobilization and on ER and mitochondrial Ca 2+ pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H 2 O 2 -induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca 2+ chelator, as well as the absence of Ca 2+ in the medium, reduced H 2 O 2 -induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) after H 2 O 2 treatment. After treatment with the Ca 2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca 2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca 2+ -ATPases, transiently increased [Ca 2+ ] i in control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca 2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca 2+ storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca 2+ .