Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth

The intracellular Ca²⁺ pump inhibitor, thapsigargin, added to DDT₁MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca²⁺ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 μM thapsigargin followed by extensive washing, the total releasable InsP₃-sensitive Ca²⁺ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca²⁺-pumping activity were all similar with the EC₅₀ values for thapsigargin in each case being close to 10 nM. Upon application to DDT₁MF-2 cells, thapsigargin transiently increased resting cytosolic Ca²⁺ (0.15 μM) to a peak of 0.3 μM within 50 s; thereafter, free Ca²⁺ declined to 0.2 μM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca²⁺ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca²⁺, indicating that the internal Ca²⁺ pool remained completely empty. These results suggest that emptying of Ca²⁺ from intracellular thapsigargin-sensitive Ca²⁺-pumping pools induces profound alteration of cell proliferation.