Heinrich Attila, Kittel Agnes, Csölle Cecilia, Sylvester Vizi E, Sperlágh Beáta
Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest POB 67, Hungary.
Neuropharmacology. 2008 Feb;54(2):375-86. doi: 10.1016/j.neuropharm.2007.10.013. Epub 2007 Dec 11.
In this study, the P2 receptor-mediated modulation of [3H]glutamate and [3H]noradrenaline release were examined in rat spinal cord slices. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and 2-methylthioadenosine 5'-diphosphate (2-MeSADP) decreased the electrical stimulation-evoked [3H]glutamate efflux with the following order of potency: ADP>2-MeSADP>ATP. The effect of ATP was antagonized by suramin (300microM), the P2Y12,13 receptor antagonist 2-methylthioadenosine 5'-monophosphate (2-MeSAMP, 10microM), and partly by 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS, 30microM) and the P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179, 10muM). ATP, ADP and 2-MeSADP also decreased evoked [3H]noradrenaline outflow; the order of agonist potency was ADP> or =2-MeSADP>ATP. The effect of ATP was reversed by 2-MeSAMP (10microM), and partly by MRS 2179 (10microM). By contrast, 2-methylthioadenosine-5'-triphosphate (2-MeSATP, 10-300microM) increased resting and electrically evoked [3H]glutamate and [3H]noradrenaline efflux, and this effect was prevented by the P2X1 receptor selective antagonist 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis (carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt (NF449, 100nM). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNAs encoding P2Y12 and P2Y13 receptors are expressed in the brainstem, whereas P2Y13 but not P2Y12 receptor mRNA is present in the dorsal root ganglion and spinal cord. P2Y1 receptor expression in the spinal cord is also demonstrated at the protein level. In conclusion, inhibitory P2Y and facilitatory P2X1-like receptors, involved in the regulation of glutamate (P2Y13 and/or P2Y1) and noradrenaline (P2Y13 and/or P2Y1, P2Y12) release have been identified, which provide novel target sites for analgesics acting at the spinal cord level.
在本研究中，我们检测了P2受体介导的对大鼠脊髓切片中[3H]谷氨酸和[3H]去甲肾上腺素释放的调节作用。三磷酸腺苷（ATP）、二磷酸腺苷（ADP）和2-甲硫基二磷酸腺苷（2-MeSADP）均可降低电刺激诱发的[3H]谷氨酸外流，其效力顺序为：ADP>2-MeSADP>ATP。ATP的作用可被苏拉明（300μM）、P2Y12,13受体拮抗剂2-甲硫基单磷酸腺苷（2-MeSAMP，10μM）拮抗，部分可被4-[[4-甲酰基-5-羟基-6-甲基-3-[(膦酰氧基)甲基]-2-吡啶基]偶氮]-1,3-苯二磺酸（PPADS，30μM）和P2Y1受体拮抗剂2'-脱氧-N6-甲基腺苷3',5'-二磷酸（MRS 2179，10μM）拮抗。ATP、ADP和2-MeSADP也可降低诱发的[3H]去甲肾上腺素外流；激动剂效力顺序为ADP≥2-MeSADP>ATP。ATP的作用可被2-MeSAMP（10μM）逆转，部分可被MRS 2179（10μM）逆转。相比之下，2-甲硫基三磷酸腺苷（2-MeSATP，10 - 300μM）可增加静息和电刺激诱发的[3H]谷氨酸和[3H]去甲肾上腺素外流，而这种作用可被P2X1受体选择性拮抗剂4,4',4'',4'''-[羰基双[亚氨基-5,1,3-苯三基双（羰基亚氨基）]]四苯磺酸八钠盐（NF449，100nM）阻断。逆转录聚合酶链反应（RT-PCR）分析显示，编码P2Y12和P2Y13受体的mRNA在脑干中表达，而P2Y13受体mRNA而非P2Y12受体mRNA存在于背根神经节和脊髓中。脊髓中P2Y1受体的表达在蛋白水平也得到证实。总之，已鉴定出参与调节谷氨酸（P2Y13和/或P2Y1）和去甲肾上腺素（P2Y13和/或P2Y1、P2Y12）释放的抑制性P2Y和促进性P2X1样受体，这为作用于脊髓水平的镇痛药提供了新的靶点。
