Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter
Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter
复制标题
通过基因修饰表达胆碱乙酰转移酶和/或囊泡乙酰胆碱转运蛋白的永生化脑内皮细胞诱发乙酰胆碱释放
DOI：
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发表时间：
1999
影响因子：
4.7
通讯作者：
Maurice Israël
中科院分区：
文献类型：
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作者：
Michel Malo;M. Diebler;L. P. D. Carvalho;François;Yves Dunant;Alain Bloc;Jacques Stinnakre;M. Tomasi;J. Tchélingérian;P. Couraud;Maurice Israël
摘要：永生化大鼠脑内皮细胞RBE4不表达胆碱乙酰转移酶（ChAT），但它们表达一种内源性机制，使它们能够在钙进入时特异性释放乙酰胆碱（ACh），当它们被动加载神经递质时。事实上，我们之前报道过这些细胞在装载这些递质后不会释放谷氨酸或GABA。本研究通过转染含有大鼠ChAT的表达载体构建物来构建产生乙酰胆碱的稳定细胞系。ChAT转染表达了高水平的ChAT活性和积累的内源乙酰胆碱。我们使用两种平行的方法检测RBE4细胞的乙酰胆碱释放。首先，钙离子载体诱导的Ca2+依赖性乙酰胆碱释放采用化学发光方法。我们发现，在酯酶抑制剂存在的情况下，ChAT‐转染的细胞释放了它们合成并积累的递质。其次，通过与细胞接触的全细胞电压钳住的爪蟾肌细胞实时检测在电去极化上释放的乙酰胆碱。无论细胞是合成乙酰胆碱，还是被动加载乙酰胆碱电刺激，都能在肌细胞中以突触样电流的形式释放乙酰胆碱量子。重复刺激引起了一系列振幅递减的连续反应，很少出现失败。振幅分析表明，电流在优先级达到峰值，就好像它们是基本分量的倍数。此外，我们选择了一个具有高ChAT活性的RBE4转基因克隆来引入鱼雷囊泡乙酰胆碱转运蛋白（VAChT）基因。然而，由于ChAT的表达在稳定的VAChT转染中是失活的，因此VAChT对诱发ACh释放的潜在影响只能在被动加载ACh的细胞上进行研究。VAChT的表达改变了反复电刺激下乙酰胆碱的传递模式。刺激训练引起了几组被许多失败打断的反应。释放乙酰胆碱的总量和平均量子大小没有变化。由于脑内皮细胞被认为是将基因产物传递到大脑的合适细胞载体，目前的研究结果表明，RBE4细胞通过基因修饰产生乙酰胆碱，并在本质上能够支持诱发的乙酰胆碱释放，可能为改善中枢神经系统改变的胆碱能功能提供有用的工具。
Abstract : Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+‐dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT‐transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole‐cell voltageclamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh electrical stimulation elicited the release of ACh quanta detected as inward synaptic‐like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.