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 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM 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 Ca(2+)-Mg(2+)-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.
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Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Thayer
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D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004305:Dose-Response Relationship, Drug; D005444:Flunarizine / Q000494:pharmacology; D005910:Glioma / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology*; D006822:Hybrid Cells; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D051379:Mice; D009447:Neuroblastoma / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology*; D009568:Nitrendipine / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D013729:Terpenes; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D009568:Nitrendipine; D014700:Verapamil; D000252:Calcium-Transporting ATPases; D005444:Flunarizine; D001920:Bradykinin; D002118:Calcium