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

T. K. Ghosh, J. Bian, A. D. Short, S. L. Rybak, Donald Gill

Research output: Contribution to journalArticle

194 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)24690-24697
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number36
StatePublished - 1991

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Thapsigargin
Cell growth
Calcium
Growth
Cells
Cell Division
Inositol 1,4,5-Trisphosphate
DNA
Cell proliferation
Cell culture
Washing
Epinephrine
Smooth Muscle Myocytes
Muscle
Cultured Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Ghosh, T. K. ; Bian, J. ; Short, A. D. ; Rybak, S. L. ; Gill, Donald. / Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 36. pp. 24690-24697.
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abstract = "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.",
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Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. / Ghosh, T. K.; Bian, J.; Short, A. D.; Rybak, S. L.; Gill, Donald.

In: Journal of Biological Chemistry, Vol. 266, No. 36, 1991, p. 24690-24697.

Research output: Contribution to journalArticle

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