Refilling the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store in neuroblastoma x glioma hybrid NG108-15 cells

Bradykinin-induced increases in the intracellular free Ca²⁺ concentration ([Ca²⁺](i)) were recorded in single NG108-15 cells with indo- 1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5- trisphosphate (IP₃)-sensitive Ca²⁺ store; refilling the store required extracellular Ca²⁺ (half time, 2 min). Refilling the IP₃-sensitive store was completely blocked by 1 μM La³⁺ and 10 μM nitrendipine, but not 10 μM verapamil, 10 μM flunarizine, 1 μM nitrendipine, or 0.1 μM La³⁺. Thapsigargin irreversibly depleted the Ca²⁺ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca²⁺ across the plasma membrane did not increase after depletion of the IP₃- sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca²⁺ influx. Similarly, Mn²⁺ and Ba²⁺ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP₃-sensitive store. In contrast to depletion of the IP₃-sensitive Ca²⁺ store by bradykinin, thapsigargin (10 nM) treatment produced Ca²⁺ and Ba²⁺ influx. We conclude that after Ca²⁺ mobilization, the IP₃-sensitive Ca²⁺ store in NG108-15 cells is refilled with cytoplasmic Ca²⁺ via a thapsigargin-sensitive Ca²⁺-Mg²⁺-ATPase. Cytoplasmic Ca²⁺ is replenished by a persistent leak of Ca²⁺ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca²⁺ store. In NG108-15 cells, agonist and thapsigargin- evoked Ca²⁺ entry are mediated by activation of plasmalemmal Ca²⁺ channels independent of the status of the IP₃-sensitive intracellular Ca²⁺ store.