The role of sodium/calcium exchanger (NCX1) in mechanisms of PC12 cell death induced by calcium overload

The Na+/Ca2+ exchanger is a plasma membrane protein highly expressed in excitable tissue. It mediates the movement of 3 Na+ in exchange for 1 Ca2+. It extrudes Ca 2+ in parallel with the plasma membrane Ca2+-ATPase. As a reversible transporter, it also mediates Ca2+ entry. The Na+/Ca2+ exchanger plays an important role in regulating intracellular calcium ([Ca2+]I) homeostasis. Perturbed [Ca2+]I has been implicated in both apoptosis and necrosis. Calcium overload has been associated with cell injury and death in many cell types. However, the role of Na+/Ca2+ exchange in the cell death process is unclear due to lack of specific inhibitors.;We stably transfected PC12 cells with a Na+/Ca 2+ exchanger NCX1.4 isoform. In Western blot of wild type and vector transfected control (VC) cells, NCX1 antibody labeled proteins with MW of 130kDa and 90kDa. In NCX1.4 overexpressing (NO) cells, an additional labeled protein was detected with a MW of 165kDa. Na+-dependent 45Ca2+ uptake assay confirmed the overexpressed exchanger had high activity (0.70nM 45Ca2+ uptake/10 5 cells/2min).;Exposure of NO cells to 0.5--20muM ionomycin for 6hrs showed a significant decrease in LDH release compared to VC cells (P < 0.01). [Ca 2+]I change upon ionophore treatment was evaluated with fura-2. Our results showed that the NO cells had a resting [Ca2+] I level about 30nM greater than VC cells (∼100nM). Upon ionophore treatment, the VC cells showed a greater change of [Ca2+] I (∼400nM) than that of the NO cells (∼310nM) in the initial 30sec and both reached a plateau of about 350nM after 1min. This initial [Ca 2+]I difference between these two groups of cells proved to be critical for survival as shown by ionomycin treatment with EGTA. When VC and NO cells were treated with 3muM ionomycin with 1.1mM EGTA (equal molar with extracellular Ca2+), the cell death induced by ionomycin was almost abolished. But when treated with ionomycin first, and EGTA added 2min later, LDH release remained as high as cells treated with ionomycin alone (both VC and NO).;Our results suggest that the rapid extracellular calcium influx and rise in [Ca2+]I are necessary and sufficient events to initiate ionomycin induced cell death. Overexpressing the Na+/Ca 2+ exchanger reduces PC12 cell death by decreasing the rise of [Ca 2+]I and more rapidly lowering the [Ca2+] I after the peak.