Marshall Ian C B, Owen Davina E, Cripps Tim V, Davis John B, McNulty Shaun, Smart Darren
Neurology Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.
Br J Pharmacol. 2003 Jan;138(1):172-6. doi: 10.1038/sj.bjp.0705003.
1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.
1 辣椒素和树脂毒素（RTX）通过激活香草酸受体1（VR1）来刺激Ca2+内流，VR1是感觉神经元上的一种配体门控Ca2+通道。我们研究了VR1激活是否也能触发细胞内Ca2+储存库中的Ca2+动员。2 将人VR1转染的HEK293细胞（hVR1-HEK293）分别用Fluo-3或Fluo-4与Fura Red的混合物进行负载，然后分别在荧光成像板读数器（FLIPR）和共聚焦显微镜下成像。3 在无Ca2+培养基中，RTX导致hVR1-HEK293细胞内游离Ca2+浓度短暂升高（pEC(50) 6.45±0.05），但在野生型细胞中未出现这种情况。在这些条件下，辣椒素（100 microM）不会引起Ca2+动员。4 VR1受体拮抗剂辣椒平（pIC(50) 5.84±0.04）、Ca2+泵抑制剂毒胡萝卜素（pIC(50) 7.77±0.04）、磷脂酶C抑制剂U-73122（pIC(50) 5.35±0.05）以及通过用乙酰胆碱受体激动剂卡巴胆碱（20 microM，2分钟）预处理耗尽肌醇1,4,5-三磷酸敏感的Ca2+储存库，均可抑制RTX介导的Ca2+动员。这些数据表明，RTX在hVR1-HEK293细胞中引起肌醇1,4,5-三磷酸敏感的Ca2+储存库中的Ca2+动员。5 在细胞外Ca2+存在的情况下，U-73122（10 microM，30分钟）和毒胡萝卜素（1 microM，30分钟）均可减弱辣椒素介导和RTX介导的Ca2+升高。我们得出结论，VR1能够通过由辣椒素和RTX介导的Ca2+依赖性机制以及仅由RTX介导的Ca2+非依赖性机制与Ca2+动员偶联。
