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

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)C641-C653
JournalAmerican Journal of Physiology - Cell Physiology
Volume264
Issue number3 33-3
DOIs
StatePublished - 1993

Keywords

  • bradykinin
  • intracellular calcium
  • thapsigargin

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