AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cyclic AMP pathway mediates prostaglandin E2-induced mechanical hyperalgesia.
AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cyclic AMP pathway mediates prostaglandin E2-induced mechanical hyperalgesia.
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Cav3.2 通道的 AKAP 依赖性敏化通过 EP4 受体/环 AMP 途径介导前列腺素 E2 诱导的机械痛觉过敏。
DOI：
10.1111/j.1476-5381.2012.02174.x
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
A.
中科院分区：
文献类型：
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作者：
Sekiguchi;F.;Matsumoto;Y.;Maeda;Y.;Tsubota-Matsunami;M.;Nishikawa;H. and Kawabata;A.
背景和目的T型Ca2+通道（T通道）的Cav3.2亚型可被硫化氢致敏，硫化氢是一种亲伤害性气体递质，PKA介导PGE2诱导的痛觉过敏。本研究通过PGE2/cAMP通路在表达Cav3.2并产生cAMP以响应PGE2的NG108‐15细胞中检测和分析了Cav3.2致敏作用及其对大鼠机械伤害感受加工的影响。实验方法采用全细胞膜片钳技术，在NG108 - 15细胞和大鼠背根神经节（DRG）神经元中测量T通道依赖性电流（T电流）。在NG108‐15细胞中，采用免疫沉淀/免疫印迹法分析了Cav3.2与A‐激酶锚定蛋白150 （AKAP150）的分子相互作用及其磷酸化。采用大鼠爪压试验测定机械伤害知觉阈值。在NG108‐15细胞和/或大鼠DRG神经元中，二丁基cAMP （db‐cAMP）或pge2增加了T电流，这一作用被AKAP St‐Ht31抑制剂肽（AKAPI）或KT5720（一种PKA抑制剂）阻断。ep4受体拮抗剂RQ‐00015986‐00可消除pge2的作用。不管db - cAMP是否刺激，AKAP150与Cav3.2共同免疫沉淀，Cav3.2被db - cAMP或PGE2磷酸化。在大鼠中，足底内注射db - cAMP或pge2引起机械性痛觉过敏，这一作用被AKAPI、两种不同的T通道阻滞剂（NNC 55 - 0396和乙氧亚胺，或ZnCl2）抑制，已知它们抑制T通道中的Cav3.2。口服RQ‐00015986‐00可抑制PGE2诱导的机械性痛觉过敏。结论和意义研究结果表明，pge2通过ep4受体/cAMP/PKA通路引起AKAP依赖性磷酸化和Cav3.2致敏，导致大鼠机械性痛觉过敏。
Background and PurposeThe Cav3.2 isoform of T‐type Ca2+channels (T channels) is sensitized by hydrogen sulfide, a pro‐nociceptive gasotransmitter, and also by PKA that mediates PGE2‐induced hyperalgesia. Here we examined and analysed Cav3.2 sensitization via the PGE2/cAMP pathway in NG108‐15 cells that express Cav3.2 and produce cAMP in response to PGE2, and its impact on mechanical nociceptive processing in rats.Experimental ApproachIn NG108‐15 cells and rat dorsal root ganglion (DRG) neurons, T‐channel‐dependent currents (T currents) were measured with the whole‐cell patch‐clamp technique. The molecular interaction of Cav3.2 with A‐kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108‐15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats.Key ResultsIn NG108‐15 cells and/or rat DRG neurons, dibutyryl cAMP (db‐cAMP) or PGE2increased T currents, an effect blocked by AKAP St‐Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE2was abolished by RQ‐00015986‐00, an EP4receptor antagonist. AKAP150 was co‐immunoprecipitated with Cav3.2, regardless of stimulation with db‐cAMP, and Cav3.2 was phosphorylated by db‐cAMP or PGE2. In rats, intraplantar (i.pl.) administration of db‐cAMP or PGE2caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T‐channel blockers, NNC 55‐0396 and ethosuximide, or ZnCl2, known to inhibit Cav3.2 among T channels. Oral administration of RQ‐00015986‐00 suppressed the PGE2‐induced mechanical hyperalgesia.Conclusion and ImplicationsOur findings suggest that PGE2causes AKAP‐dependent phosphorylation and sensitization of Cav3.2 through the EP4receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.