From the Center for Neuroscience, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Republic of Korea; Laboratory of Cell Death and Human Diseases, School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul, Republic of Korea; Center for Cognition and Sociality, Institute for Basic Science, Yusung-gu, Daejeon, Republic of Korea; Department of Maxillofacial Tissue Regeneration, School of Dentistry, Kyung Hee University, Dongdaemun-gu, Seoul, Seoul, Republic of Korea; and Korea University of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
Anesth Analg. 2015 Mar;120(3):671-677. doi: 10.1213/ANE.0000000000000607.
The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation.
Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection.
Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol).
These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.
G 蛋白信号调节蛋白 4（RGS4）加速 G（αi）和 G（αo）的鸟苷三磷酸酶活性，导致 G 蛋白偶联受体信号失活。阿片受体（OR）是一种 G（αi）偶联受体，在中枢神经系统的疼痛调节中发挥重要作用。在这项研究中，我们研究了以下问题：（1）脊髓 RGS4 是否影响福尔马林疼痛测试中的痛觉反应；（2）这种 RGS4 介导的效应是否涉及 OR 激活；（3）RGS4 调节是否改变 µ-OR 激动剂诱导的镇痛作用。
雄性 129S4/SvJae×C57BL/6J（RGS4(+/+)或 RGS4(-/-)）小鼠右后足底皮下注射福尔马林（1%，20 µL），并计数 40 分钟内的舔舐反应。将注射后 0 至 10 分钟（早期阶段）和 10 至 40 分钟（晚期阶段）内用于舔舐注射足的时间（秒）分别测量为急性痛觉和炎症性疼痛反应的指标。在福尔马林注射前 5 分钟鞘内注射 RGS4 抑制剂 CCG50014 和/或 µ-OR 激动剂[D-Ala²，N-MePhe⁴，Gly-ol]-enkephalin（DAMGO）。在 CCG50014 注射前 30 分钟，腹腔内注射非选择性 OR 拮抗剂纳洛酮。
接受福尔马林注射的小鼠表现出典型的双相痛觉行为。RGS4 敲除小鼠的晚期痛觉反应明显降低，但早期痛觉反应没有降低。同样，鞘内给予 CCG50014（10、30 或 100 nmol）以剂量依赖性方式减轻晚期的痛觉反应。纳洛酮（5mg/kg）完全阻断 RGS4 抑制剂的镇痛作用。相反，鞘内注射 DAMGO 可在早期和晚期阶段以剂量依赖性方式减少痛觉反应。DAMGO 的这种镇痛作用通过脊髓 RGS4 的遗传耗竭或与 CCG50014（10nmol）共同给药得到显著增强。
这些发现表明，脊髓 RGS4 抑制了福尔马林疼痛测试中内源性或外源性 OR 介导的镇痛作用。因此，抑制 RGS4 活性可以增强 OR 激动剂诱导的镇痛作用。RGS4 抑制剂共同给药增强 OR 激动剂诱导的镇痛作用表明，这种抑制作用可能为炎症性疼痛的管理提供一种新的治疗策略。
