Department of Physiology, Zunyi Medical College, Zunyi, 563000, People's Republic of China.
Department of Physiology, Zunyi Medical College, No. 6 West Xuefu Road, Xinpu District, Zunyi, 563006, Guizhou Province, People's Republic of China.
Neurochem Res. 2018 Feb;43(2):267-275. doi: 10.1007/s11064-017-2414-6. Epub 2017 Nov 10.
Spinal cannabinoid receptor 1 (CBR) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CBR and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB 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 Ca mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca]i increase in cultured DH neurons. ATP-evoked [Ca]i increase in DH neurons was blocked by chelating extracellular Ca and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca 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 Ca response was mimicked by ACEA (CBR agonist), but was not influenced by AM1241 (CBR agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca mobilization was blocked by AM251 (CB receptor antagonist), but was not influenced by AM630 (CB 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 CBR rather than CBR 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 CBR on P2X-channel-induced Ca mobilization.
脊髓大麻素受体 1 (CBR) 和嘌呤能 P2X 受体 (P2XR) 在病理性疼痛的过程中发挥着关键作用。CBR 和 P2XR 均在脊髓背角 (DH) 神经元中表达。目前尚不清楚 CB 受体的激活是否会调节背角中 P2X 受体通道的功能。出于这个原因，我们观察了 CP55940（大麻素受体激动剂）对培养的大鼠 DH 神经元中 ATP 诱导的 Ca 动员的影响。使用 fluo-4/AM 作为钙荧光指示剂，通过共焦激光扫描显微镜检测细胞内钙离子浓度 ([Ca]i) 的变化。100μM ATP 引起培养的 DH 神经元 [Ca]i 增加。用螯合细胞外 Ca 和 P2 嘌呤能受体拮抗剂 PPADS 阻断 ATP 诱导的 [Ca]i 增加。同时，ATP-γ-S（一种不可水解的 ATP 类似物）模拟了 ATP 的作用，而 P2Y 受体激动剂 ADP 未能引起培养的 DH 神经元 [Ca]i 增加。这些数据表明，ATP 诱导的培养 DH 神经元 [Ca]i 升高是由 P2X 受体介导的。随后，我们注意到，在培养的大鼠 DH 神经元中，CP55940 预处理后，ATP 诱导的 Ca 动员呈浓度依赖性抑制，这表明大麻素受体的激活下调了 P2X 受体通道的开放。ACEA（CBR 激动剂）模拟 CP55940 对 ATP 诱导的 Ca 反应的抑制作用，但不受 AM1241（CBR 激动剂）的影响。此外，AM251（CB 受体拮抗剂）阻断 CP55940 对 ATP 诱导的 Ca 动员的抑制作用，但不受 AM630（CB 受体拮抗剂）的影响。此外，我们还观察到， forskolin（腺苷酸环化酶激活剂）和 8-Br-cAMP（细胞通透性 cAMP 类似物）分别逆转了 CP55940 的抑制作用。总之，我们的观察结果提出了一种可能性，即 CBR 而不是 CBR 可以下调 DH 神经元中 P2X 受体通道的开放。cAMP/PKA 信号转导的减少是 CBR 对 P2X 通道诱导的 Ca 动员抑制作用的关键因素。
