BACKGROUND: Remifentanil can induce postinfusion cold hyperalgesia. N-methyl-d-aspartate receptor (NMDAR) activation and upregulation of transient receptor potential melastatin 8 (TRPM8) membrane trafficking in dorsal root ganglion (DRG) are critical to cold hyperalgesia derived from neuropathic pain, and TRPM8 activation causes NMDAR-dependent cold response. Contribution of P2Y1 purinergic receptor (P2Y1R) activation in DRG to cold pain hypersensitivity and NMDAR activation induced by P2Y1R upregulation in neurons are also unraveled. This study explores whether P2Y1R contributes to remifentanil-induced cold hyperalgesia via TRPM8-dependent regulation of NMDAR phosphorylation in DRG. METHODS: Rats with remifentanil-induced cold hyperalgesia were injected with TRPM8 antagonist or P2Y1R antagonist at 10 minutes before remifentanil infusion. Cold hyperalgesia (paw lift number and withdrawal duration on cold plate) was measured at −24, 2, 6, 24, and 48 hours following remifentanil infusion. After the last behavioral test, P2Y1R expression, TRPM8 expression and membrane trafficking, and NMDAR subunit (NR1 and NR2B) expression and phosphorylation in DRG were detected by western blot, and colocalization of P2Y1R with TRPM8 was determined by double-labeling immunofluorescence. Two-way repeated measures analysis of variance (ANOVA) or 2 × 2 factorial design ANOVA with repeated measures was used to analyze behavioral data of cold hyperalgesia. One-way ANOVA followed by Bonferroni post hoc comparisons was used to analyze the data in western blot and immunofluorescence. RESULTS: Remifentanil infusion (1 μg·kg−1·min−1 for 60 minutes) induced cold hyperalgesia (hyperalgesia versus control, paw lift number and withdrawal duration on cold plate at 2–48 hours, P < .0001) with upregulated NR1 (hyperalgesia versus naive, 48 hours, mean ± standard deviation [SD], 114.00% ± 12.48% vs 41.75% ± 5.20%, P < .005) and NR2B subunits expression (104.13% ± 8.37% vs 24.63% ± 4.87%, P < .005), NR1 phosphorylation at Ser896 (91.88% ± 7.08% vs 52.00% ± 7.31%, P < .005) and NR2B phosphorylation at Tyr1472 (115.75% ± 8.68% vs 59.75% ± 7.78%, P < .005), TRPM8 expression (115.38% ± 9.27% vs 40.50% ± 4.07%, P < .005) and membrane trafficking (112.88% ± 5.62% vs 48.88% ± 6.49%, P < .005), and P2Y1R expression (128.25% ± 14.86% vs 45.13% ± 7.97%, P < .005) in DRG. Both TRPM8 and P2Y1R antagonists attenuated remifentanil-induced cold hyperalgesia and downregulated increased NR1 and NR2B expression and phosphorylation induced by remifentanil (remifentanil + RQ-00203078 versus remifentanil + saline, NR1 phosphorylation, 69.38% ± 3.66% vs 92.13% ± 4.85%; NR2B phosphorylation, 72.25% ± 6.43% vs 111.75% ± 11.00%, P < .0001). NMDAR activation abolished inhibition of TRPM8 and P2Y1R antagonists on remifentanil-induced cold hyperalgesia. P2Y1R antagonist inhibited remifentanil-evoked elevations in TRPM8 expression and membrane trafficking and P2Y1R-TRPM8 coexpression (remifentanil + 2’-deoxy-N6-methyl adenosine 3’,5’-diphosphate [MRS2179] versus remifentanil + saline, coexpression, 8.33% ± 1.33% vs 22.19% ± 2.15%, P < .0001). CONCLUSIONS: Attenuation of remifentanil-induced cold hyperalgesia by P2Y1R inhibition is attributed to downregulations in NMDAR expression and phosphorylation via diminishing TRPM8 expression and membrane trafficking in DRG.

中文翻译：

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P2Y1 嘌呤能受体通过瞬时受体电位 Melastatin 8-依赖调节背根神经节中的 N-甲基-d-天冬氨酸受体磷酸化参与瑞芬太尼诱导的冷痛觉过敏

背景：瑞芬太尼可引起输注后冷痛觉过敏。N-甲基-d-天冬氨酸受体 (NMDAR) 激活和瞬时受体电位 melastatin 8 (TRPM8) 膜在背根神经节 (DRG) 中的上调对于源自神经性疼痛的冷痛觉过敏至关重要，TRPM8 激活导致 NMDAR 依赖性冷回复。还阐明了 DRG 中 P2Y1 嘌呤能受体 (P2Y1R) 激活对冷痛超敏反应和神经元 P2Y1R 上调诱导的 NMDAR 激活的贡献。本研究探讨了 P2Y1R 是否通过 TRPM8 依赖性调节 DRG 中的 NMDAR 磷酸化来促进瑞芬太尼诱导的冷痛觉过敏。方法：瑞芬太尼致冷痛觉过敏大鼠在瑞芬太尼输注前10分钟注射TRPM8拮抗剂或P2Y1R拮抗剂。在瑞芬太尼输注后 -24、2、6、24 和 48 小时测量冷痛觉过敏（在冷板上抬爪次数和缩回持续时间）。最后一次行为测试后，通过蛋白质印迹检测 DRG 中 P2Y1R 表达、TRPM8 表达和膜运输以及 NMDAR 亚基（NR1 和 NR2B）表达和磷酸化，并通过双标记免疫荧光确定 P2Y1R 与 TRPM8 的共定位。使用重复测量的双向重复测量方差分析 (ANOVA) 或 2 × 2 因子设计方差分析来分析冷痛觉过敏的行为数据。使用单向方差分析和 Bonferroni 事后比较来分析蛋白质印迹和免疫荧光中的数据。结果：瑞芬太尼输注（1 μg·kg-1·min-1 持续 60 分钟）诱导冷痛觉过敏（痛觉过敏与对照，TRPM8 和 P2Y1R 拮抗剂均减轻瑞芬太尼诱导的冷痛觉过敏，并下调瑞芬太尼诱导的 NR1 和 NR2B 表达和磷酸化增加（瑞芬太尼 + RQ-00203078 与瑞芬太尼 + 生理盐水，NR1 磷酸化，69.38% ± 3 % 26 NR1 % ± 3.5%）磷酸化，72.25% ± 6.43% 与 111.75% ± 11.00%，P < .0001）。NMDAR 激活消除了 TRPM8 和 P2Y1R 拮抗剂对瑞芬太尼诱导的冷痛觉过敏的抑制作用。P2Y1R 拮抗剂抑制瑞芬太尼诱发的 TRPM8 表达升高和膜运输以及 P2Y1R-TRPM8 共表达（瑞芬太尼 + 2'-脱氧-N6-甲基腺苷 3',5'-二磷酸 [MRS2179] 与瑞芬太尼 + 盐水，共表达，8.33% 1.33% 与 22.19% ± 2.15%，P < .0001）。结论：