Identification of intracellular calcium pools. Selective modification by thapsigargin

• Published in: The Journal of biological chemistry Vol. 266; no. 14; pp. 8801 - 8806
• Main Authors: BIAN, J, GHOSH, T. K, JIAN-CHENG WAN, GILL, D. L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-05-1991
• Subjects: Animals; Biological and medical sciences; calcium; Calcium - metabolism; Calcium-Transporting ATPases - antagonists & inhibitors; Cell Compartmentation - drug effects; Cell physiology; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Fundamental and applied biological sciences. Psychology; Guanosine Triphosphate - pharmacology; Heparin - pharmacology; inositol 1,4,5-trisphosphate; Inositol 1,4,5-Trisphosphate - pharmacology; Membrane and intracellular transports; Molecular and cellular biology; Muscle, Smooth - metabolism; Organoids - metabolism; Oxalates - pharmacology; Terpenes - pharmacology; Thapsigargin; Vanadates - pharmacology; Vertebrata; Regulation(control); Mammalia; Membrane pump; Calcium; Radiolabelling; Membrane transport; Rodentia; Smooth muscle; Biological activity; Hamster
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)31518-7

Recent studies have identified inositol 1,4,5-tris-phosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent
mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to
distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponin-permeabilized
DDT1MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin
with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 microM A23187 block 100% of Ca2+ accumulation. The thapsigargin-responsive
Ca2+ pool corresponds exactly to that released by 10 microM InsP3 in the presence of 10 microM GTP. Indeed, addition of InsP3
with GTP has no effect on Ca2+ accumulated in the presence of 3 microM thapsigargin whereas A23187 releases all the remaining
Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity.
Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12
mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover,
the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3.
The results indicate that thasigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+
pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent
Ca2+ translocation (Ghosh, T. K., Mullaney, J.M., Tarazi, F.I., and Gill, D.L. (1989) Nature 340, 236-239). Additionally,
a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.