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Functional identification and quantitation of three intracellular calcium pools in GH4C1 cells: evidence that the caffeine-responsive pool is coupled to a thapsigargin-resistant, ATP-dependent process.
- Source :
-
Biochemistry [Biochemistry] 1993 Nov 16; Vol. 32 (45), pp. 12062-73. - Publication Year :
- 1993
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Abstract
- We have recently reported that basal oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) in intact GH4C1 cells are dependent on a Ca(2+)-induced Ca2+ release (CICR) mechanism. The purpose of the present study was to characterize the uptake and release pathways for intracellular Ca2+ in GH4C1 cells. We have used both permeabilized cells and microsome preparations, and we have monitored the change in ambient [Ca2+] using the dye, fluo 3. We find that there are three functionally distinct nonmitochondrial, ATP-dependent Ca2+ pools in these cells: Pool 1 is an inositol 1,4,5-trisphosphate (InsP3) responsive pool which is filled by a thapsigargin (Tg) sensitive Ca(2+)-ATPase; pool 2 is a second Tg-sensitive pool which is InsP3-unresponsive; and pool 3 is a Tg-resistant pool, at least a part of which has the characteristics of a CICR mechanism. These pools were established as follows. Tg caused additional Ca2+ release after maximum release was induced by prior addition of InsP3. In contrast, the InsP3 response was abolished in a time-dependent manner after pretreatment with Tg. Ambient Ca2+, added after maximum blockade by Tg, was still able to be sequestered. Ionomycin released Ca2+ even after maximum depletion by Tg. The ionomycin-releasable pool remaining after Tg treatment was also ATP-dependent, because this pool was completely discharged by ATP-depletion. Two additional inhibitors of intracellular Ca(2+)-ATPases, 2,4-di(tert-butyl)hydroquinone and cyclopiazonic acid, which are structurally unrelated to Tg, acted on the same targets as Tg. To estimate accurately the distribution of Ca2+ among compartments, we developed a new approach based on the analysis of two equilibrium states of Ca2+ distribution. Using this method, the size of the Tg-sensitive pools (pools 1 + 2) was estimated to be 63 +/- 2.5% of total non-mitochondrial Ca2+ in our preparation. Caffeine induced Ca2+ release, and this action was observed even after complete depletion of the Tg-sensitive pool, indicating that pool 3 had the characteristics of a CICR compartment. Because caffeine pretreatment caused an increase in the size of pools 1 + 2, the CICR-like mechanism operated primarily on pool 3. These new results strengthen our model, in which a distinct CICR-like pool is responsible for Ca2+ oscillations in GH4C1 cells, and also support the concept that different types of Ca2+ efflux pathways occur in Ca(2+)-storing nonmitochondrial organelles containing different types of Ca(2+)-ATPases.
- Subjects :
- Adenosine Triphosphatases antagonists & inhibitors
Animals
Cells, Cultured
Drug Resistance
Inositol 1,4,5-Trisphosphate pharmacology
Microsomes drug effects
Microsomes metabolism
Rats
Thapsigargin
Adenosine Triphosphate metabolism
Caffeine pharmacology
Calcium metabolism
Terpenes pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 32
- Issue :
- 45
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8218284
- Full Text :
- https://doi.org/10.1021/bi00096a017