Blockage of HCN Channels Inhibits the Function of P2X Receptors in Rat Dorsal Root Ganglion Neurons
Blockage of HCN Channels Inhibits the Function of P2X Receptors in Rat Dorsal Root Ganglion Neurons
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DOI：
10.1007/s11064-021-03509-5
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发表时间：
2022-01-22
影响因子：
4.4
通讯作者：
Liu，Xiaohong
中科院分区：
文献类型：
--
作者：
Lei，Xiaolu;Zeng，Junwei;Liu，Xiaohong
超极化激活的环核苷酸门控通道和嘌呤能P2 X受体在神经损伤引起的疼痛超敏反应中起关键作用。HCN通道和P2 XR都在背根神经节感觉神经元中表达。然而，P2 X2和P2 X3受体的表达和功能是否可以通过HCN通道活性调节尚不清楚。因此，在坐骨神经慢性压迫性损伤的大鼠中，我们评价了鞘内给予HCN通道阻断剂ZD 7288对大鼠伤害性行为和DRG中P2 X2和P2 X3表达的影响。测定大鼠机械性缩痛阈值以评价大鼠的疼痛行为。Western Blot法检测大鼠背根神经节P2 X2、P2 X3受体蛋白表达。ELISA法测定大鼠背根神经节cAMP含量。结果，术后1 d观察到CCI大鼠MWT降低，并且在整个实验期间持续存在异常性疼痛。另外，CCI大鼠术后7 d和14 d，同侧DRG内P2 X2和P2 X3受体表达增加。鞘内注射ZD 7288可显著逆转CCI诱导的机械性痛敏，并抑制大鼠背根节P2 X2和P2 X3受体表达的增加，提示HCN通道阻断剂ZD 7288可能下调CCI大鼠背根节P2 X2和P2 X3受体的表达。神经损伤后，大鼠背根节cAMP含量明显升高。鞘内注射ZD 7288可减轻神经损伤引起的DRG cAMP升高。随后，采用全细胞膜片钳技术研究HCN通道活性对大鼠DRG神经元ATP诱导电流（IATP）的影响。ATP（100 μM）在培养的DRG神经元上可诱发快、慢和混合型IATP电流。ZD 7288浓度依赖性地抑制三种类型的ATP激活电流。另一方面，8-Br-cAMP（一种细胞可渗透的cAMP类似物，也被称为PKA的激活剂）预处理显著增加了DRG快、慢和混合IATPin神经元的振幅。8-Br-cAMP对ATP激活电流的增强作用可被ZD 7288逆转。总之，我们的观察表明，HCN通道的开放可以通过cAMP-PKA信号通路增强P2 X2和P2 X3受体的表达和功能。这对于DRG内发生的病理生理学事件可能是重要的，因为它涉及神经损伤诱导的疼痛超敏反应。
Hyperpolarization-activated cyclic nucleotide-gated channels and purinergic P2X receptors play critical roles in the nerve injury-induced pain hypersensitivity. Both HCN channels and P2XR are expressed in dorsal root ganglia sensory neurons. However, it is not clear whether the expression and function of P2X2 and P2X3 receptors can be modulated by HCN channel activity. For this reason, in rats with chronic constriction injury of sciatic nerve, we evaluated the effect of intrathecal administration of HCN channel blocker ZD7288 on nociceptive behavior and the expression of P2X2 and P2X3 in rat DRG. The mechanical withdrawal threshold was measured to evaluate pain behavior in rats. The protein expression of P2X2 and P2X3 receptor in rat DRG was observed by using Western Blot. The level of cAMP in rat DRG was measured by ELISA. As a result, decreased MWT was observed in CCI rats on 1 d after surgery, and the allodynia was sustained throughout the experimental period. In addition, CCI rats presented increased expression of P2X2 and P2X3 receptor in the ipsilateral DRG at 7 d and 14 d after CCI operation. Intrathecal injection of ZD7288 significantly reversed CCI-induced mechanical hyperalgesia, and attenuated the increased expression of P2X2 and P2X3 receptor in rat DRG, which open up the possibility that the expression of P2X2 and P2X3 receptor in DRG is down-regulated by HCN channel blocker ZD7288 in CCI rats. Furthermore, the level of cAMP in rat DRG significantly increased after nerve injury. Intrathecal administration of ZD7288 attenuated the increase of cAMP in DRG caused by nerve injury. Subsequently, effects of HCN channel activity on ATP-induced current (IATP) in rat DRG neurons were explored by using whole-cell patch-clamp techniques. ATP (100 μM) elicited three types of currents (fast, slow and mixed IATP) in cultured DRG neurons. Pretreatment with ZD7288 concentration-dependently inhibited three types of ATP-activated currents. On the other hand, pretreatment with 8-Br-cAMP (a cell-permeable cAMP analog, also known as an activator of PKA) significantly increased the amplitude of fast, slow and mixed IATPin DRG neurons. The enhanced effect of 8-Br-cAMP on ATP-activated currents could be reversed by ZD7288. In a summary, our observations suggest that the opening of HCN channels could enhance the expression and function of P2X2 and P2X3 receptor via the cAMP-PKA signaling pathway. This may be important for pathophysiological events occurring within the DRG, for where it is implicated in nerve injury-induced pain hypersensitivity.