Neuromedin B receptor stimulation of Cav3.2 T-type Ca(2+) channels in primary sensory neurons mediates peripheral pain hypersensitivity.
Neuromedin B receptor stimulation of Cav3.2 T-type Ca(2+) channels in primary sensory neurons mediates peripheral pain hypersensitivity.
复制标题
Neuromedin B 受体刺激初级感觉神经元 Cav3.2 T 型 Ca2 通道介导外周疼痛超敏反应
作者：
Zhang Y;Qian Z;Jiang D;Sun Y;Gao S;Jiang X;Wang H;Tao J
背景：神经蛋白B （Neuromedin B, Nmb）参与感觉神经元伤害感觉的调节。然而，潜在的细胞和分子机制仍然未知。方法：采用膜片钳记录法、western blot法、免疫荧光标记法、酶联免疫吸附法、腺病毒介导shRNA敲除法和动物行为法，研究Nmb对Cav3.2 t型通道介导的感觉神经元兴奋性和外周疼痛敏感性的影响。结果：Nmb通过激活神经介质B受体（NmbR），可逆且浓度依赖性地增加小三叉神经节（TG）神经元t型通道电流（IT）。这种nmbr介导的IT反应是Gq蛋白偶联的，但不依赖于蛋白激酶C的活性。细胞内应用QEHA肽或shrna介导的Gβ敲低均可消除nmbr诱导的IT反应。抑制蛋白激酶A （PKA）或amp激活的蛋白激酶（AMPK）完全消除了nmb诱导的IT反应。磷酸化AMPK （p-AMPK）分析显示，Nmb显著激活AMPK，而AMPK抑制阻止了Nmb诱导的PKA活性增加。在异源表达系统中，活化NmbR显著增强了Cav3.2通道电流，而Cav3.1和Cav3.3通道电流不受影响。Nmb诱导TG神经元的高兴奋性，同时诱导机械和热超敏反应，这两种反应都被t型通道阻断所减弱。此外，在完全性弗氏佐剂诱导的炎症性疼痛小鼠模型中，阻断NmbR信号传导可阻止机械超敏反应，而这种作用可通过敲除Cav3.2的siRNA而减弱。结论：我们的研究揭示了NmbR通过g βγ依赖性AMPK/PKA通路刺激Cav3.2通道的新机制。在小鼠模型中，该机制似乎驱动TG神经元的高兴奋性并诱导疼痛超敏反应。
Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gβ abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gβγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.