Sustained ER Ca²⁺ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) induces large increases in cytoplasmic Ca²⁺, mitochondrial Ca²⁺ loading, protein synthesis inhibition, and cell death. To clarify the connections among these events, we have evaluated the effect of Ca²⁺ mobilizing agents thapsigargin (Tg), econazole (Ec), and the growth factor Steel Factor (SLF) on bone marrow-derived mast cells (BMMCs). BMMC Ca²⁺ stores were found to consist of a Tg-sensitive ER compartment, the Tg-insensitive SIC store, and mitochondrial stores. Low levels of Ec interfered with Tg-stimulated mitochondrial loading while promoting progressive leakage of Ca²⁺ from the ER. Low levels of Ec completely reversed Tg toxicity while higher levels blocked store-operated influx and induced cell death in a SLF-enhanced manner. Both Ec and Tg inhibited protein synthesis, however, only SLF plus Tg or very high levels of Ec were able to significantly stimulate EIF-2α phosphorylation. Cycloheximide only partially protected BMMCs from Tg toxicity yet strongly synergized with Ec to induce cell death. These results therefore indicate that although both Tg and Ec deplete ER Ca²⁺ levels, Ec-induced cell death results from sustained protein synthesis inhibition while Tg toxicity results primarily from mitochondrial Ca²⁺ overload and secondarily from ER stress associated with Ca²⁺ depletion.