Syapin P J, Salvaterra P M, Engelhardt J K
Brain Res. 1982 Jan 14;231(2):365-77. doi: 10.1016/0006-8993(82)90373-0.
The human medulloblastoma cell line TE671 has been investigated and found to have several 'neuron-like' properties, including the presence of a functional nicotinic receptor. The cell line TE671 is composed of at least 5 stable morphologic cell types. Resting potentials recorded with intracellular microelectrodes were low (-17 mV to -31 mV) but all cell types were capable of generating Na+-dependent action potentials following anode-brake stimulation. Rare spontaneous hyperpolarizing potentials, suggestive of synaptic activity, were also observed. TE671 cells were completely unresponsive to iontophoresed GABA but did respond to acetylcholine (ACh). The most common response to ACh was a depolarization accompanied by an increase in membrane conductance. When large amounts of ACh were delivered, depolarization followed by hyperpolarization was frequently observed. Depolarizing responses to ACh are abolished in Na+-free solution while hyperpolarizing responses to ACh were still present following the removal of both Na+ and Cl- from the bathing solution. The depolarization to ACh is mediated through a nicotinic cholinergic receptor. Depolarization was completely blocked in the presence of 10(-6) M alpha-bungarotoxin, 4.4 x 10(-5) M D-tubocurarine, or 10(-4) M decamethonium. Atropine was only 50% effective at 10(-4) M and hexamethonium was ineffective at 10(-4) M. In vitro binding of receptor ligands to membranes prepared from TE671 cells revealed high levels of [125I]alpha-bungarotoxin (alpha-BuTx) binding sites, in addition to lower levels of other ligand binding sites. [125I]alpha-BuTx bound to a single, saturable high affinity site in either membrane preparations or intact TE671 cells. Binding was potently inhibited by the classical nicotinic acetylcholine receptor antagonists D-tubocurarine and decamethonium. Nicotine and carbamylcholine showed intermediate potencies in inhibiting binding while atropine and hexamethonium showed little ability to inhibit [125I]alpha-BuTx binding. The data obtained from [125I]alpha-BuTx binding studies agree qualitatively with the electrophysiological data on the depolarizing ACh response and together they provide strong evidence that TE671 cells possess a functional nicotinic acetylcholine receptor. This cell may therefore be useful as a stable source with which to characterize mammalian neuronal nicotinic acetylcholine receptors and membrane events related to its activation.
对人髓母细胞瘤细胞系TE671进行了研究，发现其具有多种“神经元样”特性，包括存在功能性烟碱受体。细胞系TE671由至少5种稳定的形态学细胞类型组成。用细胞内微电极记录的静息电位较低（-17 mV至-31 mV），但所有细胞类型在阳极制动刺激后都能够产生依赖钠离子的动作电位。还观察到罕见的自发性超极化电位，提示存在突触活动。TE671细胞对离子导入的GABA完全无反应，但对乙酰胆碱（ACh）有反应。对ACh最常见的反应是去极化，同时膜电导增加。当给予大量ACh时，经常观察到先去极化后超极化的现象。对ACh的去极化反应在无钠溶液中消失，而在从浴液中去除钠离子和氯离子后，对ACh的超极化反应仍然存在。对ACh的去极化是通过烟碱胆碱能受体介导的。在存在10^(-6) M的α-银环蛇毒素、4.4×10^(-5) M的筒箭毒碱或10^(-4) M的十烃季铵时，去极化被完全阻断。阿托品在10^(-4) M时仅有50%的效果，六甲铵在10^(-4) M时无效。受体配体与TE671细胞制备的膜的体外结合显示，除了其他配体结合位点水平较低外，还存在高水平的[125I]α-银环蛇毒素（α-BuTx）结合位点。[125I]α-BuTx在膜制剂或完整的TE671细胞中与单一的、可饱和的高亲和力位点结合。经典的烟碱乙酰胆碱受体拮抗剂筒箭毒碱和十烃季铵能有效抑制结合。尼古丁和氨甲酰胆碱在抑制结合方面表现出中等效力，而阿托品和六甲铵几乎没有抑制[125I]α-BuTx结合的能力。从[125I]α-BuTx结合研究中获得的数据在质量上与关于ACh去极化反应的电生理数据一致，它们共同提供了有力证据，证明TE671细胞拥有功能性烟碱乙酰胆碱受体。因此，这种细胞可能作为一种稳定的来源，用于表征哺乳动物神经元烟碱乙酰胆碱受体及其激活相关的膜事件。
