α,β-meATP半棘肌注射致紧张性头痛动物模型的建立

紧张性头痛(TTH)是原发性头痛最常见的一种。但其病理生理学研究及治疗药物研制相对缓慢,很大原因在于紧张性头痛动物模型的研究相对滞后。根据紧张性头痛颈部肌筋膜传入性刺激引起中枢痛觉易化的假说建立的α,β-meATP半棘肌注射模型是目前国内外较为常用的。就此模型的实验准备过程经行描述,以及对实验模型经行评价。

<i>α,β</i>-Methylene-ATP-Induced Inhibition of Acetylcholine Release on the Stellate Ganglion: Contribution of Nitric Oxide

In this study, we investigated the possible involvement of nitric oxide pathways in the presynaptic inhibition of acetylcholine release induced by ATP analogs in dogs. We performed the study using HPLC with electrochemical detection and the nitric oxide detection-HPLC system. The amount of acetylcholine released in response to preganglionic stimulation at 5 Hz for 10 min was reduced in a concentration-dependent manner after exposure to 10-7 - 10-4 M α,β-methylene-ATP (α,β-meATP), but not by the P2Y receptor agonist, 2-methyl-thio-ATP (2MeSATP) or the P2X1 receptor agonist, β,γ-methylene-ATP (β,γ-meATP), at the same concentrations. The inhibition of acetylcholine release induced by α,β-meATP was antagonized by: the nonselective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS);the P2X1, P2X3 and P2X2/3 receptors antagonist, 2’-(or-3’)-O-trinitrophenyl-ATP (TNP-ATP);the neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole;the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ);the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (carboxy-PTIO). Exposure to 10-5 M, but not 10-4 M, α,β-meATP, for 30 min increased the levels of , and this increase was antagonized by TNP-ATP and 3-bromo-7-nitroindazole. These results show that P2X receptor activation inhibits stellate ganglionic transmission by reducing acetylcholine release from presynaptic nerve terminals and that this inhibition seems to involve, at least in part, the activation of endogenous NO production and cGMP pathways.

P2X受体对豚鼠胃窦环行肌自发性收缩运动的影响

目的:探讨P2X受体激动剂α,β-methylene ATP (α,β-MeATP)对豚鼠胃窦环行肌运动的影响及其离子通道机制.方法:采用EWG/B豚鼠,制备去黏膜胃窦环行肌条(10×1.5 mm),并将其固定于恒温灌流槽内(37℃),用碳酸氢钠缓冲液连续灌流并通氧(950 mL/L O_2,50 mL/L CO_2).利用SMUP-E生物信号处理系统记录胃窦平滑肌自发性收缩活动,Ⅱ型胶原酶消化法分离豚鼠胃窦环行肌单细胞,传统全细胞膜片钳技术记录急性分离的胃窦平滑肌细胞膜电位和离子电流,包括钙激活钾电流、延迟整流型钾电流及电压依赖性钙电流.结果:嘌呤能P2X受体激动剂α,β-MeATP明显抑制豚鼠胃窦环行肌自发性收缩,并有明显的量效倚赖关系:5,10,20,40,100μmol/Lα,β-MeATP作用下,环行肌收缩幅度分别由对照组的100%下降到90%±2%.81%±4%,68%±4%,59%±7%和29%±4%(P<0.05).用神经阻断剂Tetrodotoxin (TTX)预处理后,α,β-MeATP对胃窦环行肌自发性收缩的抑制作用仍不受影响;在传统全细胞膜片钳电流钳模式下,500μmol/Lα,β-MeATP不影响细胞膜电位,也对两种外向钾电流,即延迟整流型钾电流和钙激活钾电流没有影响;不同浓度的α,β-MeATP对电压依赖性钙电流没有影响.结论:P2X受体激动剂抑制豚鼠胃窦环行肌自发性收缩,其作用机制不依赖内在神经,也不依赖细胞膜离子通道以及膜电位改变.

不同给药方式对P2X_1受体介导大鼠肠系膜动脉收缩反应的影响

目的研究不同方式给予α,β-MeATP对肠系膜动脉P2X1受体介导血管收缩反应的影响。方法制备大鼠离体肠系膜动脉环标本,采用非累积给药法和单浓度给药法两种方式给予α,β-MeATP,记录药物诱发的等长收缩反应。结果两种给药方式给予α,β-MeATP(10-7-10-4mol·L-1)均可使大鼠离体肠系膜动脉产生浓度依赖性收缩反应。以KCl最大收缩反应或以标本湿重标化α,β-MeATP诱发的收缩反应时,α,β-MeATP单浓度给药的收缩反应均大于非累积给药(P<0.01)。以标本湿重标化时,10-4mol·L-1浓度α,β-MeATP诱发的收缩反应分别是(0.73±0.10)g·mg-1(单浓度组)和(0.38±0.05)g·mg-1(非累积组);以KCl最大收缩反应标化时,分别是(53.17±6.0)%(单浓度组)和(36.78±5.71)%(非累积组)。在α,β-MeATP非累积给药组120mmol·L-1KCl诱发的动脉收缩反应小于单浓度给药组和NA对照组(P<0.01)。结论在大鼠肠系膜动脉,非累积给予α,β-MeATP降低P2X1受体介导的收缩反应以及高钾诱发的收缩反应,该给药方式可能导致错误的实验结论 。