Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry.
Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry.
复制标题
DOI：
10.1042/bj20021574
复制
发表时间：
2003-06
期刊：
影响因子：
--
通讯作者：
Brian Wisnoskey;W. Sinkins;W. Schilling
中科院分区：
文献类型：
--
作者：
Brian Wisnoskey;W. Sinkins;W. Schilling
为了研究香草酸受体1（TRPV 1）与内源性Ca（2+）信号转导机制的相互作用，利用重组杆状病毒在草地贪夜蛾（Spodoptera frugiperda，Sf 9）昆虫细胞中表达TRPV 1。用树脂毒素（RTX）、辣椒素或花生四烯酸刺激TRPV 1表达细胞，而不是对照Sf 9细胞，引起细胞溶质游离Ca（2+）浓度（[Ca（2+）]（i））增加，EC（50）值分别为166 pM、24.5 nM和3.89 μ M。在缺乏细胞外Ca（2+）的情况下，辣椒素和RTX均引起[Ca（2+）]（i）增加，EC（50）值约为1.5%。10 μ M和10 nM。这种TRPV 1诱导的Ca（2+）从细胞内储存的释放不被U 73122阻断，表明磷脂酶C不参与其中。在毒胡萝卜素和RTX敏感的内部Ca（2+）池之间发现了大量重叠，共聚焦成像显示细胞内TRPV 1免疫荧光与内质网靶向基序KDEL共定位。为了确定TRPV 1诱导的细胞内Ca（2+）动员是否激活内源性钙库操作的Ca（2+）内流，检测了2-氨基乙氧基二苯基硼酸酯（2-APB）对Ba（2+）内流的影响。2-APB阻断毒胡萝卜素诱导的Ba（2+）内流，但不阻断RTX诱导的Ba（2+）内流。在毒胡萝卜素和RTX的存储释放浓度的组合存在下，2-APB敏感组分与毒胡萝卜素诱导组分基本相同。在稳定表达TRPV 1的HEK-293细胞中获得了类似的结果。这些结果表明TRPV 1在内质网中形成激动剂敏感通道，当激活时，从内部储存释放Ca（2+），但不能激活内源性储存操作的Ca（2+）内流。细胞内TRPV 1的选择性激活，不伴随质膜Ca（2+）内流机制的参与，可能在特定亚细胞微区的Ca（2+）信号转导中发挥重要作用。
To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca(2+) signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf 9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf 9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), with EC(50) values of 166 pM, 24.5 nM and 3.89 microM respectively. In the absence of extracellular Ca(2+), both capsaicin and RTX caused an increase in [Ca(2+)](i) with EC(50) values of approx. 10 microM and 10 nM respectively. This TRPV1-induced release of Ca(2+) from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin- and RTX-sensitive internal Ca(2+) pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca(2+) activates endogenous store-operated Ca(2+) entry, the effect of 2-aminoethoxydiphenyl borate (2-APB) on Ba(2+) influx was examined. 2-APB blocked thapsigargin-induced Ba(2+) influx, but not RTX-induced Ba(2+) entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca(2+) from internal stores, but fail to activate endogenous store-operated Ca(2+) entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca(2+) influx mechanisms, could play an important role in Ca(2+) signalling within specific subcellular microdomains.