Figure 8.

Ca²⁺ sequestered by CALNUC in the Golgi was released directly by IP₃ treatment of permeabilized cells. (A) ⁴⁵Ca²⁺ uptake. Noninduced or induced EcR-CHO-CALNUC cells (10⁶ cells/well) were permeabilized with 50 μg/ml saponin for 4 min, followed by loading with ⁴⁵Ca²⁺ (10 μCi/ml) for the times indicated. Stored ⁴⁵Ca²⁺ was extracted with 1 ml 0.1 N HCl for 30 min, and 0.5-ml aliquots were counted. Induced cells took up twice as much ⁴⁵Ca²⁺ as noninduced cells. ⁴⁵Ca²⁺ uptake reached equilibrium at ∼45 min after loading. (B) ⁴⁵Ca²⁺ release. Induced or noninduced EcR-CHO-CALNUC cells (10⁶ cells/ well) were permeabilized and loaded with 10 μCi/ml of ⁴⁵Ca²⁺ for 45 min to reach equilibrium and IP₃ (10 μM) was added. Aliquots (10) were collected at 2-min intervals. Stored ⁴⁵Ca²⁺ could be directly released by IP₃. Twice as much ⁴⁵Ca²⁺ was released from induced as noninduced cells. Results (mean ± SD) represent the average of values obtained in three separate experiments performed in duplicate.