Purkiss J R, Nahorski S R, Willars G B
Department of Cell Physiology and Pharmacology, University of Leicester, England.
J Neurochem. 1995 Mar;64(3):1175-82. doi: 10.1046/j.1471-4159.1995.64031175.x.
The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [3H]-noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in < 10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca2+]e) was buffered to approximately 50-100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by approximately 50% with EC50 values of -5.46 +/- 0.05 M and -7.46 +/- 0.06 M (log 10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were -6.00 +/- 0.14 M for methacholine and -7.95 +/- 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca(2+)-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins (1,4,5)P3-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [3H]NA release in SH-SY5Y cells.
人神经母细胞瘤细胞系SH - SY5Y在汇合状态下维持14天，当用毒蕈碱受体激动剂乙酰甲胆碱或缓激肽刺激时会释放[3H] - 去甲肾上腺素（[3H]NA）。释放的主要部分很快，在<10秒内发生，而尼古丁诱发的释放则较慢。当细胞外[Ca2 +]e缓冲至约50 - 100 nM时，尼古丁诱发的释放被消除，而对乙酰甲胆碱或缓激肽的反应释放减少约50%，其EC50值分别为-5.46 +/- 0.05 M和-7.46 +/- 0.06 M（log 10）。乙酰甲胆碱和缓激肽还会使肌醇1,4,5 - 三磷酸[Ins(1,4,5)P3]和细胞内游离[Ca2 +]（[Ca2 +]i）迅速升高。在低[Ca2 +]e时这些升高会降低，在这些条件下，乙酰甲胆碱使[Ca2 +]i峰值升高的EC50值为-6.00 +/- 0.14 M，缓激肽为-7.95 +/- 0.34 M（所有EC50测定均为n = 3）。在低[Ca2 +]e时，用Ca(2 +)-ATP酶抑制剂毒胡萝卜素耗尽非线粒体细胞内Ca2 +储存会使[Ca2 +]i产生短暂的小幅升高以及[3H]NA的少量释放。在低[Ca2 +]e时，毒胡萝卜素消除了对乙酰甲胆碱和缓激肽的[Ca2 +]i升高，并完全抑制了它们对[3H]NA释放的刺激。因此，有人提出，从Ins(1,4,5)P3敏感储存中释放Ca2 +是SH - SY5Y细胞中乙酰甲胆碱和缓激肽诱发[3H]NA释放的主要触发因素。
