Kawanishi T, Kiuchi T, Asoh H, Shibayama R, Kawai H, Ohata H, Momose K, Hayakawa T
Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, 158-8501, Tokyo, Japan.
Biochem Pharmacol. 2001 Oct 1;62(7):863-72. doi: 10.1016/s0006-2952(01)00740-7.
The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).
研究了环境污染物三丁基氯化锡（TBT）对分离的大鼠肝细胞中细胞内钙库释放Ca(2+)的影响。用洋地黄皂苷通透处理后的分离肝细胞悬浮于溶液中，使用荧光钙染料fura-2测量细胞外Ca(2+)浓度。在含有预先用4.0 microM TBT孵育30分钟的通透肝细胞的溶液中，细胞外Ca(2+)浓度较高，但肌醇1,4,5-三磷酸（InsP(3)）诱导的Ca(2+)浓度升高受到抑制，这表明TBT处理后细胞外Ca(2+)的释放来自细胞内钙库。使用数字化荧光显微镜，在洋地黄皂苷通透处理后，获取了加载fura-2的原代培养肝细胞细胞内钙库中Ca(2+)浓度的图像。预先用4.0 microM TBT孵育30分钟的通透肝细胞具有非常低的fura-2荧光比率（340/380 nm），表明储存的Ca(2+)被释放。当肝细胞在洋地黄皂苷通透处理后用4.0 microM TBT处理时，fura-2荧光比率的降低非常小。然而，当通透肝细胞与4.0 microM TBT和2.0 microM NADPH一起孵育时，降低增强，这增加了TBT可能被代谢为活性形式从而从细胞内钙库释放Ca(2+)的可能性。当肝细胞预先用0.1 microM TBT孵育30分钟然后进行通透处理时，fura-2荧光比率与对照通透肝细胞几乎相同。然而，在通透肝细胞中，InsP(3)诱导的荧光比率降低被显著抑制。这些结果表明，高浓度的TBT从细胞内钙库释放Ca(2+)，并在无毒低浓度下抑制InsP(3)诱导的Ca(2+)释放。后者的作用可能是先前报道的激素刺激诱导的Ca(2+)反应受到抑制的原因（Kawanish等人，《毒理学与应用药理学》1999年；155:54 - 61）。
