Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells.
We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca(2+) responses of SH-SY5Y cells. The muscarinic agonist methacholine (1mM) resulted in a Ca(2+) signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca(2+) signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca(2+), regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca(2+) signal was inhibited by 75.07 +/- 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca(2+) source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca(2+) signal induced by 55 mM K(+) was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca(2+) sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca(2+) source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.
Riddoch
Fiona C
FC
Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle-upon-Tyne, UK.
Rowbotham
Sophie E
SE
Brown
Anna M
AM
Redfern
Christopher P F
CP
Cheek
Timothy R
TR
D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D045604:Extracellular Fluid / Q000378:metabolism; D016257:Fura-2; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018721:Muscarinic Agonists; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D016210:Methacholine Chloride; D012433:Ryanodine; D002110:Caffeine; D011188:Potassium; D002118:Calcium; D016257:Fura-2