Ca²⁺ Localization in Thapsigargin-Treated MEG-01 Cells

Abstract

To study the intracellular Ca²⁺ mobilization of MEG-01 cells, which are a megakaryoblastic leukemia cell line, we used thapsigargin, a specific inhibitor of intracellular Ca²⁺-pump. The stimulation of single, fura-2-loaded MEG-01 cells by thapsigargin resulted in a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that appears to be due to both initial discharge of Ca²⁺ from the intracellular stores and successive entry of Ca²⁺ across the plasma membrane. After removal of extracellular Ca²⁺, the elevated [Ca²⁺]_i produced by thapsigargin decreased with an initial rapid phase, followed by a gradual phase. lonomycin applied during the period of the gradual decrease elicited a transient increase in [Ca²⁺]_i while La³⁺, which blocks both inward and outward movement of Ca²⁺ across the plasma membrane, produced a sustained elevation as long as La³⁺ was present. Using a laser scanning confocal fluorescence imaging system, localization of cytosol Ca²⁺ was found in fluo-3-loaded cells in the gradual decrease phase. Heterogeneous pattern of cytosol high Ca²⁺ concentration seemed to correspond to location of mitochondria labeled with rhodamine 123. Inhibitors of mitochondrial electron transport, NaN₃ and antimycin A, eliminated the gradual decrease phase. These results suggest the presence of an unknown intracellular Ca²⁺ pool, possibly mitochondria, which could accumulate excessive intracellular Ca²⁺, and indicate that the [Ca²⁺]_i in the gradual decrease phase represents the Ca²⁺ concentration in the unknown pool.