title: 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.
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.
authors.affiliation: Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts.
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.forename: A H
authors.initials: AH
authors.lastname: Tashjian
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004351:Drug Resistance; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
chemical_list: D013729:Terpenes; D002110:Caffeine; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium