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 microM 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 Ca(2+)-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 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM 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 Ca(2+)-pumping pools induces profound alteration of cell proliferation.
Ghosh
T K
TK
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Bian
J H
JH
Short
A D
AD
Rybak
S L
SL
Gill
D L
DL
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D009132:Muscles / Q000187:drug effects* / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013936:Thymidine / Q000378:metabolism
D013729:Terpenes; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D008715:Methionine; D002118:Calcium; D013936:Thymidine