Laboratorio de Neurofisiología, Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.
J Neurosci Res. 2018 Jun;96(6):1066-1079. doi: 10.1002/jnr.24223. Epub 2018 Feb 13.
At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y and A , A , and A receptors, respectively. To elucidate the action of endogenous purines on K -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K , the P2Y antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A , A , and A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K , endogenous ATP/ADP and adenosine bind to inhibitory P2Y and A and A receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K , suggesting that more adenosine is needed to activate excitatory A receptors. At high K concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.
在小鼠神经肌肉接头处，三磷酸腺苷 (ATP) 与神经递质乙酰胆碱 (ACh) 共同释放，一旦进入突触间隙，ATP 就会水解为腺苷。ATP/二磷酸腺苷 (ADP) 和腺苷分别通过激活突触前 P2Y 和 A 1 、A 2A 和 A 3 受体来调节 ACh 的分泌。为了阐明内源性嘌呤对 K 依赖性 ACh 释放的作用，我们研究了嘌呤能受体拮抗剂对膈神经制备中小终板电位 (MEPP) 频率的影响。在 10 mM K 时，P2Y 拮抗剂 N-[2-(甲硫基)乙基]-2-[3,3,3-三氟丙基]硫代-5'-腺嘌呤酸，单酐与（二氯亚甲基）双[膦酸]，四钠盐（AR-C69931MX）增加了异步 ACh 的分泌，而 A 1 、A 2A 和 A 3 拮抗剂 8-环戊基-1,3-二丙基黄嘌呤（DPCPX）、（3-乙基-5-苄基-2-甲基-4-苯乙炔基-6-苯基-1,4-(±)-二氢吡啶-3,5-,二羧酸酯（MRS-1191）和 2-(2-呋喃基）-7-(2-苯乙基)-7H-吡唑并[4,3-e][1,2,4]三唑并[1,5-c]嘧啶-5-胺（SCH-58261）并未改变神经分泌。通过 S-(对硝基苄基)-6-硫代肌苷抑制摄取性腺苷转运体，导致 10 mM K 诱导的 ACh 释放减少，表明来自 ATP 的腺苷正通过转运体从突触间隙中去除。在 15 和 20 mM K 时，内源性 ATP/ADP 和腺苷与抑制性 P2Y 和 A 1 和 A 2A 受体结合，因为 AR-C69931MX、DPCPX 和 MRS-1191 增加了 MEPP 的频率。当使用外核苷酸酶抑制剂 α,β-亚甲基腺苷 5'-二磷酸钠盐来阻止腺苷的产生时，得到了类似的结果。SCH-58261 仅在 20 mM K 时降低神经分泌，表明需要更多的腺苷来激活兴奋性 A 受体。在高 K 浓度下，摄取性转运体似乎饱和，允许腺苷在突触间隙中积累。总之，当运动神经末梢通过增加 K 浓度去极化时，内源性产生的 ATP/ADP 和腺苷能够通过顺序激活不同的嘌呤能受体来调节 ACh 的分泌。
