Graduate Institute and Department of Pharmacology, and Graduate Institute of Brain and Mind Sciences, College of Medicine, Graduate Institute of Zoology, Neurobiology and Cognitive Science Center, National Taiwan University, Taipei 100, Taiwan.
J Neurosci. 2011 Oct 12;31(41):14600-10. doi: 10.1523/JNEUROSCI.2671-11.2011.
Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1'-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGLα, but not DAGLβ, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.
食欲素 A 和 B 是下丘脑肽，通过 OX1 和 OX2 受体调节觉醒、进食和奖励。食欲素在大脑中也具有镇痛作用，但它们的作用机制仍不清楚。在这里，我们研究了食欲素 A 在大鼠腹外侧导水管周围灰质（vlPAG）中的镇痛机制，vlPAG 是启动下行疼痛抑制的中脑区域。在 vlPAG 切片中，食欲素 A（30-300nm）抑制 GABA 能诱发的 IPSC。这种作用被 OX1 [1-(2-甲基苯并恶唑-6-基)-3-[1,5]萘啶-4-基脲（SB 334867)]阻断，但不被 OX2 [N-酰基 6,7-二甲氧基-1,2,3,4-四氢异喹啉盐酸盐（化合物 29)]阻断。食欲素 A 增加了 IPSC 的成对脉冲比，并降低了 IPSC 的频率，但不降低幅度。（R）-[2,3-二氢-5-甲基-3-（4-吗啉基甲基）吡咯并[1,2,3-de]-1,4-苯并恶嗪-6-基]-1-萘基甲酮（WIN 55,212-2）模拟了食欲素 A 诱导的 IPSC 抑制，WIN 55,212-2 是一种大麻素 1（CB1）受体激动剂。1-(2,4-二氯苯基)-5-(4-碘苯基)-4-甲基-N-(1-哌啶基)吡唑-3-甲酰胺（AM 251），一种 CB1 拮抗剂，逆转了两种激动剂的抑制作用。1-[6-[[(17β)-3-甲氧基雌-1,3,5(10)-三烯-17-基]氨基]己基]-1H-吡咯-2,5-二酮（U73122）和四氢脂酶抑制剂分别抑制了 PLC 和二酰基甘油脂肪酶（DAGL），并增强了环己基[1,1'-联苯]-3-基氨基甲酸酯（URB602）的作用，URB602 抑制了 2-花生四烯酸甘油（2-AG）的酶解。在 vlPAG 中观察到中等水平的 DAGLα，但不是 DAGLβ，免疫反应性。食欲素 A 对诱发的突触后电位产生总体兴奋作用，从而增加了 vlPAG 神经元的活动。vlPAG 内微注射食欲素 A 可减少大鼠的热板痛觉反应，这种作用被 SB 334867 和 AM 251 阻断。因此，食欲素 A 可能通过激活突触后 OX1 受体产生镇痛作用，通过 Gq 蛋白介导的 PLC-DAGLα 酶级联反应刺激内源性大麻素 2-AG 的合成，最终导致 GABA 释放的逆行抑制（去抑制）在 vlPAG 中。
