Ca2+ entry following store depletion in SH-SY5Y neuroblastoma cells.
Ca2+ entry following Ca2+ store depletion was examined in the human neuroblastoma cell line, SH-SY5Y, by measuring the concentration of intracellular free Ca2+ ([Ca2+]i) with fura-2. Application of the muscarinic agonist oxotremorine-M (oxo-M) caused an increase in [Ca2+]i. This consisted of a peak, mediated by release of Ca2+ from internal stores followed by a sustained plateau, mediated by Ca2+ entry across the plasma membrane. The Ca2+ entry resulted from depletion of intracellular Ca2+ stores This pathway was further characterized in the presence of thapsigargin, an inhibitor of the Ca2+ ATPase involved in replenishing IP3-sensitive stores. Stores were first depleted with oxo-M and thapsigargin in the absence of extracellular Ca2+. After washout of oxo-M, subsequent exposure to Ca2+ evoked reproducible increases in [Ca2+]i. Application of oxo-M plus Ca2+ had little effect on the increases in [Ca2+]i, indicating that in SH-SY5Y cells, agonist-dependent pathways contribute little to Ca2+ entry following store depletion. Mn2+, Sr2+ and Ba2+ were permeable through this pathway. Mn2+ and Ba2+ also showed slight permeability in the absence of store depletion. Ca2+ entry following store depletion was blocked by La3+ (IC50 = 75 nM) and by SKF 96365. La3+ blocked Mn2+ entry through the pathway activated by store depletion but did not affect basal Mn2+ permeability. These results indicate that SH-SY5Y neuroblastoma cells have an agonist-independent Ca2+ entry pathway activated by store depletion.
Grudt
T J
TJ
Department of Pharmacology, University of Bristol, UK. Tim.Grudt@Bristol.ac.uk
Usowicz
M M
MM
Henderson
G
G
D001464:Barium / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D007093:Imidazoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007811:Lanthanum / Q000494:pharmacology; D008345:Manganese / Q000493:pharmacokinetics; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D010095:Oxotremorine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013324:Strontium / Q000493:pharmacokinetics; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007093:Imidazoles; D018721:Muscarinic Agonists; D001464:Barium; D008345:Manganese; D010095:Oxotremorine; C042743:oxotremorine M; D019284:Thapsigargin; D007811:Lanthanum; D015544:Inositol 1,4,5-Trisphosphate; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D013324:Strontium