CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons
CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons
复制标题
CB1 受体介导抑制培养的大鼠脊髓背角神经元中 ATP 诱导的 [Ca2]i 增加。
DOI：
10.1007/s11064-017-2414-6
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发表时间：
2018-02-01
影响因子：
4.4
通讯作者：
Liu, Xiaohong
中科院分区：
文献类型：
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作者：
Long, Jingdong;Lei, Xiaolu;Liu, Xiaohong
脊髓大麻素受体1（CB 1 R）和嘌呤能P2 X受体（P2 XR）在病理性疼痛过程中起着关键作用。CB 1 R和P2 XR都在脊髓背角（DH）神经元中表达。目前尚不清楚CB 1受体激活是否调节背角内P2 X受体通道的功能。因此，我们观察了CP 55940（大麻素受体激动剂）对ATP诱导的大鼠DH神经元中Ca 2+动员的影响。以Fluo-4/AM为钙荧光指示剂，激光共聚焦显微镜观察细胞内钙离子浓度（[Ca 2 +]i）的变化。100 μ M ATP引起培养的DH神经元[Ca ~（2+）]i增加。ATP引起的DH神经元[Ca ~（2+）]i增加可被螯合细胞外Ca ~（2+）和P_2嘌呤受体拮抗剂PPADS阻断。同时，ATP-γ-S（一种不可水解的ATP类似物）模拟ATP的作用，而P2 Y受体激动剂ADP未能引起培养的DH神经元[Ca ~（2+）]i增加。提示ATP诱导的DH神经元[Ca ~（2+）]i升高是由P_2X受体介导的。随后，我们注意到，在培养的大鼠DH神经元中，ATP诱导的Ca 2+动员被CP 55940以浓度依赖性方式抑制，这意味着大麻素受体的激活下调了P2 X受体通道的开放。CP 55940对ATP诱导的Ca ~（2+）反应的抑制作用可被CB 1 R激动剂ACEA所模拟，但不受CB 2 R激动剂AM 1241的影响。此外，CP 55940对ATP诱导的Ca ~（2+）动员的抑制作用可被CB_1受体拮抗剂AM 251阻断，但不受CB_2受体拮抗剂AM 630的影响。此外，我们还观察到forskolin（腺苷酸环化酶的激活剂）和8-Br-cAMP（细胞渗透性cAMP类似物）分别逆转CP 55940的抑制作用。总之，我们的观察提出了一种可能性，即CB 1 R而不是CB 2 R可以下调DH神经元中P2 X受体通道的开放。cAMP/PKA信号的减少是CB 1 R对P2 X通道诱导的Ca 2+动员的抑制作用的关键因素。
Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 mu M ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-gamma-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.