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Identification of intracellular calcium pools. Selective modification by thapsigargin
J H Bian1, T K Ghosh, J C Wang
1Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
The Journal of Biological Chemistry
|May 15, 1991
Summary
Thapsigargin, a novel inhibitor, distinguishes three intracellular calcium (Ca2+) pools. It blocks uptake into InsP3-sensitive and -insensitive pools, revealing a third, unreleasable Ca2+ pool.
Area of Science:
- Cellular Biology
- Biochemistry
- Pharmacology
Background:
- Intracellular calcium (Ca2+) signaling involves distinct Ca2+ pools.
- A GTP-dependent mechanism facilitates Ca2+ transfer between these pools.
- Inositol 1,4,5-trisphosphate (InsP3) sensitive and insensitive Ca2+ pools have been identified.
Purpose of the Study:
- To investigate the distinct Ca2+ pools using the Ca2+ pump inhibitor thapsigargin.
- To characterize the Ca2+ pumping mechanisms and their regulation by InsP3 and GTP.
Main Methods:
- Utilized saponin-permeabilized DDT1MF-2 smooth muscle cells.
- Assessed ATP-dependent Ca2+ accumulation and release.
- Employed thapsigargin, vanadate, A23187, InsP3, GTP, oxalate, and heparin to probe Ca2+ pools.
Main Results:
- Thapsigargin (IC50 30 nM) blocked ~75% of ATP-dependent Ca2+ accumulation, distinguishing it from vanadate and A23187.
- Thapsigargin-responsive Ca2+ pool matched InsP3/GTP-releasable pool; a third, unreleasable pool was identified.
- Thapsigargin inhibited Ca2+ uptake into oxalate-permeable and -impermeable pools, including GTP-recruitable pools.
Conclusions:
- Thapsigargin effectively differentiates between InsP3-sensitive and -insensitive Ca2+ pools.
- A third, thapsigargin-insensitive Ca2+ pool, unreleasable by InsP3 or GTP, was identified.
- Thapsigargin provides a novel tool for dissecting intracellular Ca2+ handling mechanisms.