Dussor Gregory O, Helesic Gabriela, Hargreaves Kenneth M, Flores Christopher M
Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX 78229, USA.
Pain. 2004 Jan;107(1-2):22-32. doi: 10.1016/j.pain.2003.09.022.
Muscarinic acetylcholine receptors (mAChRs) have been widely reported as pharmacological targets for the treatment of pain. However, most of these efforts have focused on CNS mAChRs and their role in modulating nociception at the level of the spinal cord. The present study examines the contribution of peripheral mAChRs in trigeminal nociceptive pathways using a combination of in vivo and in vitro approaches. In the formalin model of orofacial nociception in rats, a peri-oral co-injection of the M2 agonist arecaidine dose-dependently inhibited phase 2 nocifensive behavior up to approximately 50% at 5 nmol. This effect was blocked by co-treatment with the mAChR antagonist atropine and was not seen when arecaidine was administered under the skin of the back, a site distant from that of the formalin injection. In vitro superfusion of isolated rat buccal mucosa with the non-selective mAChR agonist muscarine or arecaidine led to a concentration-dependent inhibition of capsaicin-evoked CGRP release to 39% (EC50=255 nM) and 28% (EC50=847 nM) of control values, respectively. Both responses were blocked by the non-selective mAChR antagonist atropine or the M2 antagonist gallamine. Further, the endogenous ligand ACh produced a bi-phasic response, potentiating evoked CGRP release to 195% of control (EC50= 918nM) and inhibiting evoked CGRP release to 45% of control (EC50=255 microM), effects that were shown to be mediated by nAChRs and mAChRs, respectively. Finally, combined in situ hybridization/immunofluorescence demonstrated that m2 mRNA was present in 20% of trigeminal ganglion neurons between 30 and 60 microm in diameter and that 5-9% of these also expressed CGRP or VR1 immunoreactivity. These results show that activation of peripheral M2 receptors produces antinociception in vivo and the inhibition of nociceptor activity in vitro. While histological analyses at the level of the trigeminal neuronal cell bodies leave open the question of whether the effects of M2 agonists are direct or indirect, these data indicate that primary sensory neuronal M2 receptors may represent a viable peripheral target for the treatment of pain and inflammation.
毒蕈碱型乙酰胆碱受体（mAChRs）作为疼痛治疗的药理学靶点已被广泛报道。然而，这些研究大多集中在中枢神经系统mAChRs及其在脊髓水平调节伤害感受中的作用。本研究采用体内和体外相结合的方法，研究外周mAChRs在三叉神经伤害感受通路中的作用。在大鼠口腔面部伤害感受的福尔马林模型中，在口腔周围联合注射M2激动剂槟榔次碱，在5 nmol时剂量依赖性地抑制第二阶段伤害防御行为，抑制率高达约50%。这种效应被mAChR拮抗剂阿托品共同处理所阻断，而当槟榔次碱注射到背部皮肤（远离福尔马林注射部位）时则未观察到这种效应。在体外，用非选择性mAChR激动剂毒蕈碱或槟榔次碱对分离的大鼠颊黏膜进行灌流，导致辣椒素诱发的降钙素基因相关肽（CGRP）释放分别浓度依赖性地抑制至对照值的39%（EC50 = 255 nM）和28%（EC50 = 847 nM）。两种反应均被非选择性mAChR拮抗剂阿托品或M2拮抗剂加拉明阻断。此外，内源性配体乙酰胆碱（ACh）产生双相反应，将诱发的CGRP释放增强至对照值的195%（EC50 = 918 nM），并将诱发的CGRP释放抑制至对照值的45%（EC50 = 255 μM），结果表明这些效应分别由烟碱型乙酰胆碱受体（nAChRs）和毒蕈碱型乙酰胆碱受体（mAChRs）介导。最后，原位杂交/免疫荧光联合检测显示，直径在30至60微米之间的三叉神经节神经元中有20%存在m2 mRNA，其中5 - 9%的神经元也表达CGRP或VR1免疫反应性。这些结果表明，外周M2受体的激活在体内产生抗伤害感受作用，在体外抑制伤害感受器活性。虽然在三叉神经神经元细胞体水平的组织学分析尚无法确定M2激动剂的作用是直接的还是间接的，但这些数据表明，初级感觉神经元M2受体可能是治疗疼痛和炎症的一个可行的外周靶点。
