Department of Laboratory Medicine, Lund University, Lund, Sweden ; Lund University Pain Research Centre, Lund University, Lund, Sweden.
PLoS One. 2013 Dec 2;8(12):e81618. doi: 10.1371/journal.pone.0081618. eCollection 2013.
Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.
磷脂酶 C 介导的磷脂酰肌醇 4,5-二磷酸水解生成二酰基甘油、肌醇 1,4,5-三磷酸和质子，所有这些都可以通过不同的机制调节 TRPV1 活性。在这里，我们探讨了二酰基甘油代谢产物 2-花生四烯酰甘油和 1-花生四烯酰甘油，而不是这些单酰基甘油的代谢产物，激活 TRPV1 并有助于这种信号级联的可能性。2-花生四烯酰甘油和 1-花生四烯酰甘油激活了血管感觉神经纤维上的天然 TRPV1 和整个细胞和外翻膜片中的异源表达 TRPV1。单酰基甘油脂肪酶抑制剂甲基花生四烯酰氟磷酸盐和 JZL184 阻止了动脉匀浆中氘标记的 2-花生四烯酰甘油和氘标记的 1-花生四烯酰甘油的代谢，并增强了对这两种单酰基甘油的 TRPV1 介导的血管舒张反应。在 TRPV1 敲除小鼠的肠系膜动脉中，2-花生四烯酰甘油的血管舒张反应较小。与磷脂酶 C 偶联膜受体配体，缓激肽和三磷酸腺苷，增加了背根神经节中 2-花生四烯酰甘油的含量。在表达与磷脂酶 C 偶联的组胺 H1 受体的 HEK293 细胞中，暴露于组胺刺激 2-AG 的形成，并且这种效应在存在 JZL184 时增强。这些作用被二酰基甘油脂肪酶抑制剂四氢拉帕替尼预防。组胺诱导共表达 TRPV1 和组胺 H1 受体的 HEK293 细胞中的大整个细胞电流，并且 TRPV1 拮抗剂辣椒素消除了这些电流。JZL184 增加了组胺诱导的电流，而四氢拉帕替尼预防了这种作用。钙调神经磷酸酶抑制剂环孢菌素和内源性“随从”化合物棕榈酸乙醇酰胺增强了对 2-花生四烯酰甘油的血管舒张反应，揭示了 TRPV1 在纳摩尔浓度下对这种单酰基甘油的激活。此外，脑室注射 JZL184 在小鼠福尔马林试验中产生 TRPV1 依赖性镇痛。我们的结果表明，完整的 2-花生四烯酰甘油和 1-花生四烯酰甘油是内源性 TRPV1 激动剂，有助于磷脂酶 C 依赖性 TRPV1 通道激活和 TRPV1 介导的大脑中的镇痛信号。
