FUNCTIONAL IDENTIFICATION AND QUANTITATION OF 3 INTRACELLULAR CALCIUM POOLS IN GH4C1 CELLS - EVIDENCE THAT THE CAFFEINE-RESPONSIVE POOL IS COUPLED TO A THAPSIGARGIN-RESISTANT, ATP-DEPENDENT PROCESS

被引:65
作者
TANAKA, Y
TASHJIAN, AH
机构
[1] HARVARD UNIV, SCH PUBL HLTH, DEPT MOLEC & CELLULAR TOXICOL, 665 HUNTINGTON AVE, BOSTON, MA 02115 USA
[2] HARVARD UNIV, SCH MED, DEPT BIOL CHEM & MOLEC PHARMACOL, BOSTON, MA 02115 USA
关键词
D O I
10.1021/bi00096a017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have recently reported that basal oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) in intact GH4C1 cells are dependent on a Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-storing nonmitochondrial organelles containing different types of Ca2+-ATPases.
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页码:12062 / 12073
页数:12
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