Giniatullin A, Petrov A, Giniatullin R
Department of Physiology, Medical University, 49 Butlerova Street, Kazan, 420012, Russia.
Department of Physiology, Medical University, 49 Butlerova Street, Kazan, 420012, Russia.
Neuroscience. 2015 Jan 29;285:324-32. doi: 10.1016/j.neuroscience.2014.11.039. Epub 2014 Nov 26.
Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular junction. Our data suggest that the location of P2Y receptors in lipid rafts speeds up the modulatory effect of ATP. Uncovered mechanisms may contribute to motor dysfunctions and neuromuscular diseases associated with oxidative stress.
腺苷5'-三磷酸（ATP）是伴随乙酰胆碱从运动神经末梢释放的主要共递质。此前，我们揭示了细胞外ATP通过氧化还原依赖性机制对递质释放的直接抑制作用。然而，ATP作用的受体机制以及ATP诱导的活性氧来源（ROS）仍未完全了解。在当前研究中，我们使用青蛙神经肌肉接头突触电流的微电极记录，分析了参与ATP突触作用的受体亚型、与NADPH氧化酶偶联的受体以及脂质筏内ATP受体的潜在位置。使用亚型特异性拮抗剂，我们发现P2Y13阻滞剂2-[(2-氯-5-硝基苯基)偶氮]-5-羟基-6-甲基-3-[(膦酰氧基)甲基]-4-吡啶甲醛并不能阻止ATP的抑制作用。相反，P2Y12拮抗剂2-甲硫基腺苷5'-单磷酸消除了ATP的抑制作用，表明P2Y12受体在ATP作用中起关键作用。由于ATP的作用是氧化还原依赖性的，我们还测试了NADPH氧化酶（已知为ROS的常见诱导剂）的潜在参与情况。二苯基碘鎓氯化物和4-(2-氨基乙基)-苯磺酰氟盐酸盐这两种结构不同的NADPH氧化酶抑制剂显著降低了细胞外ATP的抑制作用，表明该酶确实介导了ATP的作用。由于各种受体的定位和活性通常与脂质筏相关，我们接下来测试了ATP驱动的抑制是否依赖于脂质筏。我们发现用甲基-β-环糊精破坏脂质筏会降低并大大延迟ATP的作用。综上所述，这些数据揭示了通过与脂质筏相关的P2Y12受体对突触传递进行嘌呤能控制的关键步骤，并确定NADPH氧化酶是神经肌肉接头处ATP诱导的抑制性ROS的主要来源。我们的数据表明，P2Y受体在脂质筏中的定位加快了ATP的调节作用。所揭示的机制可能导致与氧化应激相关的运动功能障碍和神经肌肉疾病。
