P2Y1 嘌呤能受体通过瞬时受体电位 melastatin 8 依赖性调节背根神经节中 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。
P2Y1 Purinergic Receptor Contributes to Remifentanil-Induced Cold Hyperalgesia via Transient Receptor Potential Melastatin 8-Dependent Regulation of N-methyl-d-aspartate Receptor Phosphorylation in Dorsal Root Ganglion.
机构信息
From the Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin, People's Republic of China.
Tianjin Research Institute of Anesthesiology, Tianjin, People's Republic of China.
出版信息
Anesth Analg. 2021 Sep 1;133(3):794-810. doi: 10.1213/ANE.0000000000005617.
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.
背景
瑞芬太尼可诱发输注后冷痛敏。背根神经节（DRG）中 N-甲基-D-天冬氨酸受体（NMDAR）的激活和瞬时受体电位 melastatin 8（TRPM8）的膜转运上调对神经病理性疼痛引起的冷痛敏至关重要，而 TRPM8 的激活导致 NMDAR 依赖性冷反应。P2Y1 嘌呤能受体（P2Y1R）在神经元中被上调，从而导致冷痛敏和 NMDAR 激活，其在 DRG 中的作用也被阐明。本研究旨在探讨 P2Y1R 是否通过调节 DRG 中 NMDAR 的磷酸化来参与瑞芬太尼诱导的冷痛敏，这种调节是依赖于 TRPM8 的。
方法
在瑞芬太尼输注前 10 分钟，给瑞芬太尼诱导冷痛敏的大鼠注射 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，测量冷痛敏（冷板上的抬脚次数和撤足持续时间）。在最后一次行为测试后，通过 Western blot 检测 DRG 中的 P2Y1R 表达、TRPM8 表达和膜转运以及 NMDAR 亚基（NR1 和 NR2B）表达和磷酸化，并用双标免疫荧光术确定 P2Y1R 与 TRPM8 的共表达。采用双因素重复测量方差分析（ANOVA）或 2×2 析因设计重复测量 ANOVA 分析冷痛敏的行为数据。采用单因素 ANOVA 检验，随后进行 Bonferroni 事后比较分析 Western blot 和免疫荧光数据。
结果
瑞芬太尼输注（1 μg·kg-1·min-1，持续 60 分钟）可诱导冷痛敏（与对照相比，冷板上的抬脚次数和撤足持续时间在 2-48 小时，P＜0.0001），并上调 NR1（与未处理组相比，48 小时，平均值±标准差[SD]，114.00%±12.48% vs 41.75%±5.20%，P＜0.005）和 NR2B 亚基表达（104.13%±8.37% vs 24.63%±4.87%，P＜0.005），NR1 磷酸化（Ser896）（91.88%±7.08% vs 52.00%±7.31%，P＜0.005）和 NR2B 磷酸化（Tyr1472）（115.75%±8.68% vs 59.75%±7.78%，P＜0.005），TRPM8 表达（115.38%±9.27% vs 40.50%±4.07%，P＜0.005）和膜转运（112.88%±5.62% vs 48.88%±6.49%，P＜0.005），以及 P2Y1R 表达（128.25%±14.86% vs 45.13%±7.97%，P＜0.005）在 DRG 中上调。TRPM8 和 P2Y1R 拮抗剂均能减轻瑞芬太尼诱导的冷痛敏，并下调瑞芬太尼诱导的 NR1 和 NR2B 表达和磷酸化（瑞芬太尼+RQ-00203078 与瑞芬太尼+盐水相比，NR1 磷酸化，69.38%±3.66% vs 92.13%±4.85%；NR2B 磷酸化，72.25%±6.43% vs 111.75%±11.00%，P＜0.0001）。NMDAR 的激活消除了 TRPM8 和 P2Y1R 拮抗剂对瑞芬太尼诱导的冷痛敏的抑制作用。P2Y1R 拮抗剂抑制了瑞芬太尼诱发的 TRPM8 表达和膜转运以及 P2Y1R-TRPM8 共表达的升高（瑞芬太尼+2'-脱氧-N6-甲基腺苷 3'，5'-二磷酸[MRS2179]与瑞芬太尼+盐水相比，共表达，8.33%±1.33% vs 22.19%±2.15%，P＜0.0001）。
结论
通过下调 DRG 中 TRPM8 的表达和膜转运，P2Y1R 抑制瑞芬太尼诱导的冷痛敏，从而导致 NMDAR 表达和磷酸化的下调。
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