Liu Yuwei, Tian Xiang, Wu Yuxiang, Chen Lin, Yi Chu-Li, Li Zhi-Wang, Zhang Ying, Li Chao-Ying
Wuhan Institutes of Biomedical Sciences, Jianghan University, Wuhan, 430056, Hubei Province, China; School of Medicine, Jianghan University, Wuhan, 430056, China.
Wuhan Institutes of Biomedical Sciences, Jianghan University, Wuhan, 430056, Hubei Province, China.
Biochem Biophys Res Commun. 2015 Mar 13;458(3):596-600. doi: 10.1016/j.bbrc.2015.01.156. Epub 2015 Feb 11.
To explore the association of the phenotype of ATP-activated current with the genotype of P2X1-6 subunits in nociceptors, we developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion (TG) neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons, and then to conduct single cell immunohistochemical staining for P2X1-6 subunits in the same neuron. We found that fast application of 100 μM ATP to fluorescence-traced TG neurons produced robust inward current in 87% (96/110) of cells tested. The diameter of cells varied from 16 to 56 μm. Three types of ATP-activated current (F, I and S) were recorded with distinct rise times of the current (R10-90, P < 0.05). There was a positive correlation between the cell diameter and the value of R10-90 (P < 0.05): the value of R10-90 increased with increases in the cell diameter. Cells responsive to ATP with the type F current mainly showed positive staining for P2X3 and P2X5, but negative staining for P2X2; cells responsive to ATP with the type I current showed positive staining for P2X1-3 and P2X5, but negative staining for P2X4; and cells responsive to ATP with the type S current showed positive staining for P2X1-5, but negative staining for P2X6. The present findings suggest that in addition to P2X3 subunits, P2X5 subunits are also involved in the generation of the F type of ATP-activated current in small-sized nociceptive neurons. In addition to the P2X2/3 subunit-containing channels, more complex uncharacterized combinations of P2X1-5 subunits exist in native medium-sized nociceptive neurons exhibiting the I and S types of ATP-activated current. In addition, the P2X6 subunit is not a main subunit involved in the nociceptive signal in rat TG neurons innervating tooth-pulp.
为了探究伤害感受器中ATP激活电流的表型与P2X1 - 6亚基基因型之间的关联，我们开发了一种方法，该方法使我们能够使用逆行荧光追踪法标记大鼠三叉神经节（TG）神经元中支配牙髓的伤害性神经元，在新鲜分离的荧光标记神经元中记录ATP激活电流，然后对同一神经元中的P2X1 - 6亚基进行单细胞免疫组织化学染色。我们发现，快速向荧光追踪的TG神经元施加100μM ATP可在87%（96/110）的测试细胞中产生强烈的内向电流。细胞直径在16至56μm之间变化。记录到三种类型的ATP激活电流（F、I和S），电流的上升时间不同（R10 - 90，P < 0.05）。细胞直径与R10 - 90值之间存在正相关（P < 0.05）：R10 - 90值随细胞直径的增加而增加。对ATP产生F型电流反应的细胞主要显示P2X3和P2X5阳性染色，但P2X2阴性染色；对ATP产生I型电流反应的细胞显示P2X1 - 3和P2X5阳性染色，但P2X4阴性染色；对ATP产生S型电流反应的细胞显示P2X1 - 5阳性染色，但P2X6阴性染色。目前的研究结果表明，除了P2X3亚基外，P2X5亚基也参与小型伤害性神经元中F型ATP激活电流的产生。除了含P2X2/3亚基的通道外，在表现出I型和S型ATP激活电流的天然中等大小伤害性神经元中存在更复杂的未表征的P2X1 - 5亚基组合。此外，P2X6亚基不是支配牙髓的大鼠TG神经元中伤害性信号的主要亚基。
