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 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.