Abstract：
The naturally occurring coumarin osthole has antipruritic properties, and recent reports suggest that this effect is due an inhibition or desensitization of the cation channels TRPV1 and TRPV3. Osthole was also suggested to activate TRPA1, an effect that should rather be pruritic than antipruritic. Here we characterized the effects of osthole on TRPA1 by means of ratiometric calcium imaging and patch clamp electrophysiology. In HEK 293 expressing human (h) TRPA1, osthole induced a concentration-dependent increase in intracellular calcium that was inhibited by the TRPA1-inhibitor A967079. In mouse dorsal root ganglion (DRG) cells, osthole induced a strong calcium-influx that was partly mediated by TRPA1. Osthole evoked fully reversible membrane currents in whole-cell as well as cell-free inside-out recordings on hTRPA1. Osthole failed to activate the mutant hTRPA1-S873V/T874L, a previously described binding site for the non-electrophilic TRPA1-agonists menthol and carvacrol. The combined application of osthole and carvacrol diminished channel activation, suggesting a competitive binding. Finally, osthole failed to activate TRPM8 and TRPV4 but induced a modest activation of hTRPV1 expressed in HEK 293 cells. We conclude that osthole is a potent non-electrophilic agonist of TRPA1. The relevance of this property for the antipruritic effects needs to be further explored.
摘要：
天然存在的香豆素蛇床子素具有止痒特性，和最近的报道表明，这种作用是由于阳离子通道TRPV1和TRPV3的抑制或脱敏。还建议蛇床子素激活TRPA1，这种作用应该是瘙痒而不是止痒。在这里，我们通过比率钙成像和膜片钳电生理学表征了蛇床子素对TRPA1的影响。在表达人(h)TRPA1的HEK293中,蛇床子素诱导细胞内钙的浓度依赖性增加,其被TRPA1-抑制剂A967079抑制。在小鼠背根神经节（DRG）细胞中，蛇床子素诱导了强烈的钙内流，部分由TRPA1介导。蛇床子素在整个细胞中引起完全可逆的膜电流，以及在hTRPA1上无细胞的内向外记录。蛇床子素未能激活突变体hTRPA1-S873V/T874L，先前描述的非亲电TRPA1-激动剂薄荷醇和香芹酚的结合位点。蛇床子素和香芹酚的联合应用减少了通道的激活，暗示竞争约束力。最后,蛇床子素未能激活TRPM8和TRPV4，但诱导了HEK293细胞中表达的hTRPV1的适度激活。我们得出结论，蛇床子素是TRPA1的有效非亲电激动剂。这种性质与止痒作用的相关性需要进一步探讨。
