Jerman J C, Gray J, Brough S J, Ooi L, Owen D, Davis J B, Smart D
Department of Discovery Research and Department of Neurology, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.
Br J Anaesth. 2002 Dec;89(6):882-7. doi: 10.1093/bja/aef281.
Anandamide, an endogenous lipid, activates both cannabinoid (CB(1)) and vanilloid (VR1) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB(1) and VR1 in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and endogenous VR1 receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB(1) and VR1 receptors to anandamide-induced analgesia.
Using a Flurometric Imaging Plate Reader (FLIPR), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VR1-transfected HEK293 (rVR1-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells.
The VR1 agonists capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration (Ca(2+)), with similar temporal profiles in both rVR1-HEK and DRG cells, and potency (pEC(50)) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5.5, respectively. These responses were inhibited by the VR1 antagonist capsazepine (1 micro M). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in Ca(2+). Reapplication of VR1 agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect.
Here we provide evidence that the pharmacology of recombinant rVR1 receptors is similar to those endogenously expressed in DRG cells. Moreover, we have shown that VR1, but not CB(1), receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VR1 activation in DRG cells in vivo.
花生四烯乙醇胺，一种内源性脂质，可激活大麻素（CB(1)）受体和香草酸（VR1）受体，这两种受体在大鼠背根神经节（DRG）细胞中共同表达。激活任一受体均可产生镇痛作用，但CB(1)和VR1在花生四烯乙醇胺诱导的镇痛中所起的相对作用仍存在争议。在此，我们分别在克隆细胞和DRG细胞中，利用钙成像技术比较重组VR1受体和内源性VR1受体的体外药理学特性。我们还探讨了CB(1)和VR1受体在花生四烯乙醇胺诱导的镇痛中的作用。
使用荧光成像微孔板读数仪（FLIPR），通过钙成像技术研究了几种香草酸类和大麻素类配体对大鼠VR1转染的HEK293（rVR1-HEK）细胞和DRG细胞的影响。还比较了几种香草酸类和大麻素类物质预暴露对DRG细胞的影响。
VR1激动剂辣椒素、奥伐尼、N-(4-羟基苯基)-花生四烯酸酰胺（AM404）和花生四烯乙醇胺均可引起细胞内钙浓度（Ca(2+)）的浓度依赖性升高，在rVR1-HEK细胞和DRG细胞中的时间变化趋势相似，其效价（pEC(50)）值分别为8.25（标准误0.11）、8.37（0.04）、6.96（0.06）、5.85（0.01）和7.45（0.10）、7.55（0.07）、6.10（0.13）、约5.5。这些反应可被VR1拮抗剂辣椒平（1 μM）抑制。相反，应用合成大麻素拮抗剂未能抑制花生四烯乙醇胺诱导的Ca(2+)升高。再次应用VR1激动剂可显著抑制随后对任一细胞类型中辣椒素或花生四烯乙醇胺的刺激反应，而预先暴露于大麻素激动剂则无此作用。
在此，我们提供证据表明重组rVR1受体的药理学特性与DRG细胞中内源性表达的受体相似。此外，我们还表明VR1受体而非CB(1)受体参与了体外花生四烯乙醇胺诱导的背根初级神经元反应。因此，花生四烯乙醇胺的镇痛特性可能至少部分是由体内DRG细胞中VR1的激活介导的。
