Grudt T J, Usowicz M M, Henderson G
Department of Pharmacology, University of Bristol, UK.
Brain Res Mol Brain Res. 1996 Feb;36(1):93-100. doi: 10.1016/0169-328x(95)00248-q.
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.
通过使用fura-2测量细胞内游离钙离子浓度（[Ca2+]i），在人神经母细胞瘤细胞系SH-SY5Y中研究了Ca2+储存耗尽后的Ca2+内流情况。毒蕈碱激动剂氧化震颤素-M（oxo-M）的应用导致[Ca2+]i升高。这包括一个峰值，由细胞内储存释放Ca2+介导，随后是一个持续的平台期，由Ca2+跨质膜内流介导。Ca2+内流是由细胞内Ca2+储存耗尽引起的。在存在毒胡萝卜素（一种参与补充IP3敏感储存的Ca2+ ATPase抑制剂）的情况下，对该途径进行了进一步表征。首先在无细胞外Ca2+的情况下用oxo-M和毒胡萝卜素耗尽储存。洗去oxo-M后，随后暴露于Ca2+会引起[Ca2+]i可重复的升高。应用oxo-M加Ca2+对[Ca2+]i的升高影响很小，表明在SH-SY5Y细胞中，激动剂依赖性途径对储存耗尽后的Ca2+内流贡献很小。Mn2+、Sr2+和Ba2+可通过该途径通透。Mn2+和Ba2+在无储存耗尽的情况下也表现出轻微的通透性。储存耗尽后的Ca2+内流被La3+（IC50 = 75 nM）和SKF 96365阻断。La3+阻断通过储存耗尽激活的途径的Mn2+内流，但不影响基础Mn2+通透性。这些结果表明，SH-SY5Y神经母细胞瘤细胞具有一种由储存耗尽激活的不依赖激动剂的Ca2+内流途径。
