The degree of inhibition of three kinds of ions inside cell on I_K increased as the diameter of ion decreased, La³⁺ and Gd³⁺ rarely affected the activation of I_K while Yb³⁺ shifted the activation of I_K to the right. After fitted by Boltzmann formula, the slope of all curves decreased, which represented that three kinds of rare-earth ions decreased the conductance of I_K channel and inhibited I_K. I_K was the main current for repolarization of action potential ^[17]. And the delayed rectifier K-channel of different genetic code included Kv1.2, Kv1.1 and Kv1.5 ^[26]. I_K played an important role during the process of cell apoptosis ^[18], it could shorten the time interval of action potential and maintained the cell membrane response to excitability. After comparing the influence of three kinds of rare-earth ions on I_K and the difference of DRG cell apoptosis induced by them, it could be found that Yb³⁺ inside cell delayed the activation of I_K while La³⁺ and Gd³⁺ rarely affected the activation of I_K, this might be the reason to interpret why apoptosis of DRG neuron induced by Yb³⁺ occurred after 96h while La³⁺ and Gd³⁺ never caused cell apoptosis. Also, it might be the reason to interpret why the cell hatched by Yb³⁺ never died within 24h but died as the time passing through, it could be found that only these Yb³⁺ entering into cell led to apoptosis through affecting I_K.

Ca^{2 +} was one of the most important positive ion and second messenger in cell, generally, overload of Ca²⁺ in cell might cause the death of cell, this mechanism was considered as "the common pathway" to cause cell damage in a variety of reasons ^[27]. In our pre-experiment, DRG cell hatched by rare-earth were observed, and the concentration of Ca²⁺ was also increased ^[32]. Rare-earth metal compounds were the retardants of Ca-channel ^{[28, 29]}, and might affect the distribution of Ca²⁺ in the cell after used ^[30]. We added 10uM of L-Ca channel retardants (nifidipin) into three kinds of rare-earth to inspect the apoptosis of cell, and found that the addition of nifidipin never affect the cell apoptosis rate induced by three kinds of rare-earth ions, it represented that the apoptosis of DRG cell induced by three kinds of rare-earth ions was not caused by internal flow of Ca²⁺ from external cell. Some research ^[33] reported that rare-earth ions could enter into cell membrane but never enter into organelle. Thus, we speculated that apoptosis of DRG neuron and increase of Ca²⁺ inside cell might be caused by: (1) Ln³⁺ delayed the activation of I_A by inhibiting I_A, and maintained the cell membrane continue the state of urge as well as increased the excitability of cell, at the same time, the Ca²⁺ inside organelle flowed out; (2) Yb³⁺ inhibited the effect of Na⁺- Ca²⁺ pump on membrane by affecting K-channel to decrease the discharge of Ca²⁺; (3) Ln³⁺ inside cell inhibited the Ca²⁺ entering into organelle. After promoting to afford Ln³⁺, the Ca²⁺ inside DRG neuron cell accumulated and led to the disorder of ions environment. It might cause many problems, such as dysfunction of mitochondrion ^[31], frustration of energy generation, invalidation of ions pump on membrane, damage of structure and function of neuron, etc.

Rare-earth ions of certain concentration possessed the effect to induce neuronal apoptosis. Different rare-earth ions had different strength on DRG, heavy rare-earth had heavier neurotoxicity than light rare-earth. Delayed activation of I_K channel induced by Yb³⁺ was the mainly reason to cause the cell downstream events exerting its neurotoxicity, its influence on two kinds of K-channel was similar with heavy metal Pb²⁺. However, La³⁺ and Gd³⁺ rarely affected the process of activation of I_K channel, this kind of current played an important role in the process of apoptosis of cell ^[18], thus, their neurotoxicity were very low compared with Yb³⁺.

Overall, Ln³⁺ inhibited the K-channel on membrane of DRG, the Ln³⁺ inside cell inhibited I_K while Ln³⁺ outside cell inhibited I_A. Through the protein of different channels, Ln³⁺ affected the normal physiological function of K-channel and caused the hyperexcitability of primary sensory neurons. Finally, it caused abnormal sensation which input from peripheral region into central nervous system. In conclusion, rare-earth ions had some neurotoxicity, while Yb³⁺ appeared heavier neurotoxicity than La³⁺ and Gd³⁺.

 

Beijing Chinese Translation Service Company has translated many documents about DRG cell.

 

胞内的三种稀土离子对I_K的抑制程度也是随着稀土离子半径的减小而增大，La³⁺和Gd³⁺几乎不影响I_K的激活，Yb³⁺却使I_K的激活显著右移，经Boltzmann方程拟合后, 曲线的斜率都减小了，说明三种稀土离子减小I_K通道的电导抑制了I_K。I_K是动作电位复极化的主要电流^[17]。不同基因编码的延迟整流钾通道有Kv1.2, Kv1.1和Kv1.5^[26]。 I_K可缩短动作电位的时程，从而保持细胞膜对兴奋的反应，这种电流在细胞的凋亡过程中发挥重要的作用^[18]。在比较这三种稀土离子对I_K电流影响和诱导DRG细胞凋亡的不同，发现胞内的Yb³⁺推迟I_K的激活，La³⁺和Gd³⁺却不影响I_K的激活，这可能是Yb³⁺作用DRG神经元96h诱导凋亡，而La³⁺和Gd³⁺却不引起细胞凋亡的原因吧。也是为什么Yb³⁺孵育细胞24 h不诱导凋亡，但随着时间延长才诱导凋亡，说明只有进入胞内的Yb³⁺通过影响I_K才诱导细胞凋亡。

Ca^{2 +} 是细胞内重要阳离子和第二信使之一，一般认为细胞内Ca²⁺ 超载,会引发细胞死亡, 这种机制被认为是各种原因引起细胞损伤的“共同通路”^[27]。我们前期的实验发现稀土孵育的DRG细胞, 胞内的钙离子浓度增大^[32]。稀土金属化合物是钙通道阻滞剂^[28,29], 使用后可影响细胞内Ca²⁺ 的分布^[30]。我们把三种稀土分别加10 uM L-型钙通道阻断剂 nifidipin来检测细胞凋亡，发现加nifidipin不影响三种稀土离子诱导的细胞凋亡率的改变，说明三种稀土离子引起DRG细胞凋亡，不是由于胞外的钙离子内流造成的。有研究表明稀土离子可以进入细胞膜, 但不进入细胞器^[33], 所以我们推测DRG神经元凋亡和胞内钙离子增大的原因可能是 (1) Ln³⁺通过抑制I_A，延缓I_A的激活过程，使细胞膜持续发放冲动状态，增加细胞的兴奋性，细胞器内的Ca²⁺流出来了; (2) Yb³⁺通过影响钾通道而抑制膜上的Na⁺- Ca²⁺泵的作用，使Ca²⁺的外排减少; (3) 胞内的Ln³⁺抑制Ca²⁺进入细胞器内。提示给予Ln³⁺后，DRG神经元胞内的Ca²⁺聚积, 离子内环境紊乱, 可能会造成线粒体功能障碍^[31]，能量生成受阻，膜上的离子泵失效, 神经元的结构及功能破坏。

一定浓度的稀土离子有诱导神经元凋亡效应, 不同的稀土离子对DRG的作用强弱不同，重稀土的神经毒性比轻稀土强。Yb³⁺可能主要是推迟I_K通道的激活引发细胞下游事件发挥其神经毒性, 它对两种钾通道的影响跟重金属Pb²⁺很相似，但是La³⁺和Gd³⁺几乎不影响I_K通道的激活过程，这种电流在细胞的凋亡过程中发挥重要的作用^[18]，所以它们的神经毒性比起Yb³⁺要小许多。

总之，Ln³⁺抑制DRG膜上的钾通道，胞内的Ln³⁺抑制I_K，胞外的Ln³⁺抑制I_A，Ln³⁺通过不同的通道蛋白影响K通道的正常生理功能，引起初级感觉神经元的超兴奋性，最终产生异常的感觉，这种异常感觉从外周传入中枢神经系统。结论，稀土离子有一定的神经毒性，Yb³⁺较La³⁺和Gd³⁺的神经毒性强。

 

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