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

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca(2+) 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 Ca(2+) pool is released into the cytoplasm by the Ca(2+) 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 Ca(2+) trapped within this pool increased significantly after a prolonged elevation of intracellular Ca(2+) concentration elicited by activation of Ca(2+) influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca(2+) 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 Ca(2+) uptake was excluded since ionomycin is inefficient in releasing Ca(2+) from acidic pools and Ca(2+) accumulation/release in/from this store was unaffected by monensin or NH(4)Cl, drugs known to collapse organelle acidic pH gradients. Ca(2+) sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca(2+)–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca(2+)–ATPases. The cytological nature and functional role of this Ca(2+) storage compartment are discussed.