Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells
Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells
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DOI：
10.1046/j.1460-9568.1998.00252.x
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发表时间：
1998-07-01
影响因子：
3.4
通讯作者：
Wolf, BA
中科院分区：
文献类型：
--
作者：
Gao, ZY;Chen, M;Wolf, BA
我们研究了一种独特的人类神经元模型-NT2N细胞的钙稳态，该细胞通过维甲酸处理从人类畸胎癌细胞系NTera2/C1.d1分化而来。当用含2.5 mM CaCl2的Krebs-HEPES燃烧器灌流时，Fura-2负载的NT2N细胞产生自发的胞浆钙振荡或钙瞬变。谷氨酸(6-氰基-7-硝基喹恶啉-2，3-二酮和D(-)-2-氨基-5-膦戊酸)或毒扁豆碱(阿托品)受体拮抗剂不阻断胞浆钙瞬变。细胞外Ca~(2+)的去掉可完全消除Ca~(2+)振荡，使细胞内平均Ca~(2+)水平从106+/-14 nM降至59+/-8 nM。加入L型钙通道阻断剂硝苯地平(1或10 mU M)或N型阻断剂欧米茄-芋毒素GVIA(5 MU M)，虽然不完全抑制钙振荡，但对P通道和Q通道的选择性拮抗剂欧米茄毒素MVIIC(5 MU M)无影响。对T型通道有选择浓度的Ni2+，在100µM时，不抑制钙瞬变。用较高浓度的Ni2+(2-5 mM)或Co2+(1 MM)非特异性阻断钙通道后，内质网钙-三磷酸腺苷酶抑制剂thapsigargin(1 MM)可完全阻断细胞内钙振荡，使细胞内钙振荡频率略有降低，胞浆平均钙浓度略有升高。逆转录-聚合酶链式反应显示，NT2N细胞中存在L(α(1D)亚基)和N型(α(1B)亚基)钙通道的mRNAs，而T型钙通道(α(1)亚基)的mRNAs不存在。综上所述，NT2N神经元细胞产生胞内钙振荡主要是通过多个通道，包括L通道和N型通道，而不需要激活谷氨酸和M受体。
We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs-HEPES burier containing 2.5 mM CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and D(-)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 +/- 14 nM to 59 +/- 8 nM. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 mu M) or of the N-type inhibitor omega-conotoxin GVIA (5 mu M) significantly, although incompletely, suppressed Ca2+ oscillations, while omega-conotoxin MVIIC (5 mu M), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 mu M, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2-5 mM) or Co2+ (1 mM) abolished Ca2+ oscillations completely The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 mu M), slightly decreased Ca2+ oscillation frequency and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (alpha(1D) subunit) and N-type (alpha(1B) subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (alpha(1)-subunit) was not present in the NT2N cells as shown by reverse transcription-polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.