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© The Rockefeller University Press, 0021-9525/1997//355 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 355-366

Article

Dynamic Properties of an Inositol 1,4,5-Trisphosphate– and Thapsigargin-insensitive Calcium Pool in Mammalian Cell Lines



Paola Pizzo, Cristina Fasolato, and Tullio Pozzan

Department of Biomedical Sciences and Italian Research Council (CNR) Center for the Study of Biomembranes, University of Padova, 35121 Padova, Italy

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate–sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+–ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.

Abbreviations used in this paper: BFA, brefeldin A; CA, cyclopiazonic acid; [Ca2+]i, intracellular free Ca2+ concentration; [Ca2+]m, intramitochondrial free Ca2+ concentration; cytAEQ, cytosolic aequorin; FCCP, p-(trifluoro-methoxy) phenylhydrazone; InsP3, inositol 1,4,5-trisphosphate; mKRB, modified Krebs Ringer Buffer; mtAEQ, mitochondrial aequorin; SERCAs, sarco/endoplasmic reticulum Ca2+–ATPases; SIC, stimulus-induced-calcium; tBHQ, 2,5-di(tert-butyl)-1,4-benzohydroquinone; Tg, thapsigargin; TRH, thyrotropin-releasing hormone; VOCCs, voltageoperated Ca2+ channels.

We would like to acknowledge the involvement of Dr. W.J.J.M. Scheenen in the preliminary stages of this study and thank Dr. A.M. Hofer for critical discussion and comments on the manuscript, Dr. R. Rizzuto for kindly providing recombinant aequorins, M. Mancon for total Ca2+ measurements, and G. Ronconi, M. Santato, and B. Zavan for skilfull assistance.

Address all correspondence to Paola Pizzo, Department of Biomedical Sciences, University of Padova, Via Trieste 75, 35121 Padova, Italy. Tel: +39 49 8276067. Fax: +39 49 8276049. E-mail: pozzan{at}civ.bio.unipd.it


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