REFILLING THE INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE CA-2+ 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 muM La3+ and 10 muM nitrendipine, but not 10 muM verapamil, 10 muM flunarizine, 1 muM nitrendipine, or 0.1 muM 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, Mn 2+ and Ba 2+ 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 Ca 2+ 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.