Bootman M D, Taylor C W, Berridge M J
Agricultural and Food Research Council Laboratory of Molecular Signalling, Department of Zoology, Cambridge, United Kingdom.
J Biol Chem. 1992 Dec 15;267(35):25113-9.
The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.
硫醇试剂硫柳汞已被证明可使多种细胞类型的细胞内钙离子浓度（[Ca2+]i）升高，并能使未受精的仓鼠卵产生钙离子尖峰。我们通过单细胞视频成像发现，硫柳汞能在完整的、负载Fura - 2的HeLa细胞中引发重复性钙离子尖峰，其形状与组胺刺激产生的尖峰相似。硫柳汞和组胺刺激产生的钙离子尖峰均在细胞外无钙离子（Ca²⁺o）的情况下出现，这表明它们是由细胞内钙库释放钙离子所致。组胺能刺激肌醇磷酸的形成，而在能引起持续性钙离子尖峰的浓度下，硫柳汞却抑制了基础状态及组胺刺激下的肌醇磷酸形成。因此，硫柳汞引发的钙离子尖峰并非由于刺激产生了肌醇1,4,5 - 三磷酸（InsP3）。硫柳汞对钙离子尖峰的影响可能是由于其对细胞内蛋白质上硫醇基团的烷基化作用，因为这种尖峰可被硫醇还原化合物二硫苏糖醇逆转，并且在预先用DL - 丁硫氨酸（S，R） - 亚砜胺处理使细胞内还原型谷胱甘肽浓度降低的细胞中，加入硫柳汞与[Ca2+]i升高之间的延迟时间大大缩短。在通透细胞中，硫柳汞引起了钙离子积累的浓度依赖性抑制，这完全是由于抑制了钙离子摄取到钙库中，因为硫柳汞并不影响预先加载了⁴⁵Ca²⁺的钙库中⁴⁵Ca²⁺的单向外流。硫柳汞还引起了InsP3诱导的钙离子动员的浓度依赖性敏感化：在对照细胞中，InsP3使钙库钙离子动员达到半数最大效应时的浓度为161±20 nM，而在用10 μM硫柳汞处理后，该浓度为62±5 nM。我们得出结论，硫柳汞可以模拟组胺对细胞内钙离子尖峰的影响，而不刺激InsP3的形成，并且根据我们对通透细胞的研究结果，表明硫柳汞通过使细胞对基础InsP3水平敏感来刺激尖峰的产生。
