Identification of intracellular calcium pools - Selective modification by thapsigargin

Recent studies have identified inositol 1,4,5-trisphosphate(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 saponinpermeabilized DDT₁ MF-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 μM A23 187 block 100% of Ca²⁺ accumulation. The thapsigargin-responsive Ca²⁺ pool corresponds exactly to that released by 10 μM InsP₃ in the presence of 10 μM GTP. Indeed, addition of InsP₃ with GTP has no effect on Ca²⁺ accumulated in the presence of 3 μM 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 thapsigargin 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.