Figure 7.

The Golgi Ca²⁺ pool is sensitive to stimulation with ATP. (A) Induced (2.5 μM ponasterone A for 24 h) or noninduced EcR-CHO-CALNUC cells were loaded with 1 μM Fura-2 AM for 1 h, followed by challenge with 100 μM ATP. Released Ca²⁺ was monitored by Ca²⁺ imaging. More Ca²⁺ was released after ATP stimulation from cells overexpressing CALNUC than from noninduced cells. Results represent the average of values from 14 induced cells and 15 noninduced cells. Reproducible results were obtained from experiments repeated twice. (B) Noninduced or induced EcR-CHO-CALNUC cells were loaded with ⁴⁵Ca²⁺ (2 μCi/ml) for 48 h to reach ⁴⁵Ca²⁺ equilibrium. Cells were washed and resuspended in KRH buffer supplemented with 3 mM EGTA, followed by stimulation with 100 μM ATP for 5 min at room temperature. Aliquots of 10⁶ cells were subsequently removed, the cells were sedimented, and ⁴⁵Ca²⁺ in supernatants and pellets was assessed. Compared with noninduced cells, there was a twofold increase in the ⁴⁵Ca²⁺ released in cells overexpressing CALNUC. Results (mean ± SD) represent the average of values obtained in three separate experiments performed in duplicate.