Morita Katsuya, Kitayama Tomoya, Morioka Norimitsu, Dohi Toshihiro
Department of Dental Pharmacology, Division of Integrated Medical Science, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.
Pain. 2008 Sep 15;138(3):525-536. doi: 10.1016/j.pain.2008.01.030. Epub 2008 Mar 18.
Our previous study showed that intrathecal (i.t.) injection of platelet-activating factor (PAF) induced tactile allodynia, suggesting that spinal PAF is a mediator of neuropathic pain. The present study further examined the spinal molecules participating in PAF-induced tactile allodynia in mice. I.t. injection of L-arginine, NO donor (5-amino-3-morpholinyl-1,2,3-oxadiazolium (SIN-1) or 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18)) or cGMP analog (8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate; pCPT-cGMP) induced tactile allodynia. PAF- and glutamate- but not SIN-1- or pCPT-cGMP-induced tactile allodynia was blocked by an NO synthase inhibitor. NO scavengers and guanylate cyclase inhibitors protected mice against the induction of allodynia by PAF, glutamate and SIN-1, but not by pCPT-cGMP. cGMP-dependent protein kinase (PKG) inhibitors blocked the allodynia induced by PAF, glutamate, SIN-1 and pCPT-cGMP. To identify signalling molecules through which PKG induces allodynia, glycine receptor alpha3 (GlyR alpha3) was knocked down by spinal transfection of siRNA for GlyR alpha3. A significant reduction of GlyR alpha3 expression in the spinal superficial layers of mice treated with GlyR alpha3 siRNA was confirmed by immunohistochemical and Western blotting analyses. Functional targeting of GlyR alpha3 was suggested by the loss of PGE(2)-induced thermal hyperalgesia and the enhancement of allodynia induced by bicuculline, a GABA(A) receptor antagonist in mice after GlyR alpha3 siRNA treatment. pCPT-cGMP, PAF, glutamate and SIN-1 all failed to induce allodynia after the knockdown of GlyR alpha3. These results suggest that the glutamate-NO-cGMP-PKG pathway in the spinal cord may be involved in the mechanism of PAF-induced tactile allodynia, and GlyR alpha3 could be a target molecule through which PKG induces allodynia.
我们之前的研究表明，鞘内注射血小板活化因子（PAF）可诱发触觉异常性疼痛，提示脊髓PAF是神经性疼痛的介质。本研究进一步检测了参与PAF诱发小鼠触觉异常性疼痛的脊髓分子。鞘内注射L-精氨酸、NO供体（5-氨基-3-吗啉基-1,2,3-恶二唑鎓（SIN-1）或3,3-双（氨乙基）-1-羟基-2-氧代-1-三氮烯（NOC-18））或cGMP类似物（8-（4-氯苯硫基）-鸟苷3',5'-环一磷酸；pCPT-cGMP）可诱发触觉异常性疼痛。PAF和谷氨酸诱发的触觉异常性疼痛可被NO合酶抑制剂阻断，但SIN-1或pCPT-cGMP诱发的触觉异常性疼痛则不能被阻断。NO清除剂和鸟苷酸环化酶抑制剂可保护小鼠免受PAF、谷氨酸和SIN-1诱发的异常性疼痛，但不能保护小鼠免受pCPT-cGMP诱发的异常性疼痛。cGMP依赖性蛋白激酶（PKG）抑制剂可阻断PAF、谷氨酸、SIN-1和pCPT-cGMP诱发的异常性疼痛。为了确定PKG诱发异常性疼痛的信号分子，通过鞘内转染针对甘氨酸受体α3（GlyRα3）的小干扰RNA（siRNA）来敲低GlyRα3。免疫组织化学和蛋白质印迹分析证实，用GlyRα3 siRNA处理的小鼠脊髓浅层中GlyRα3表达显著降低。GlyRα3 siRNA处理后，小鼠对前列腺素E2（PGE2）诱发的热痛觉过敏消失，对GABA（A）受体拮抗剂荷包牡丹碱诱发的异常性疼痛增强，提示GlyRα3的功能被靶向。敲低GlyRα3后，pCPT-cGMP、PAF、谷氨酸和SIN-1均未能诱发异常性疼痛。这些结果表明，脊髓中的谷氨酸-NO-cGMP-PKG途径可能参与PAF诱发触觉异常性疼痛的机制，且GlyRα3可能是PKG诱发异常性疼痛的靶分子。
