Docampo R, Moreno S N, Vercesi A E
Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
Mol Biochem Parasitol. 1993 Jun;59(2):305-13. doi: 10.1016/0166-6851(93)90228-p.
By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 microM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 microM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [3H]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.
通过使用荧光钙指示剂fura - 2,发现无论有无细胞外钙存在,在其中孵育的克氏锥虫无鞭毛体细胞质中的游离Ca2 +浓度都维持在非常低的水平(10 - 20 nM)。当无鞭毛体在琥珀酸盐和ATP存在下孵育并用洋地黄皂苷通透后,它们将培养基中的钙浓度降低到亚微摩尔水平。在1 microM FCCP存在的情况下,这些通透细胞摄取Ca2 +的初始速率非常缓慢。当仅存在琥珀酸盐时,Ca2 +积累的初始速率比ATP加琥珀酸盐时慢,并且钙设定点约为0.6 microM。后期Ca2 +摄取对琥珀酸盐的依赖性和对FCCP的敏感性表明其可能由线粒体发挥作用。在细胞外钙存在的情况下,高浓度的肿瘤启动子毒胡萝卜素会略微增加细胞质Ca2 +,但对FCCP和寡霉素/抗霉素A不敏感的Ca2 +池没有影响。此外,当以这些浓度(4 - 20 microM)使用时,毒胡萝卜素显示会从线粒体中释放Ca2 +,并如使用番红O测量的那样降低无鞭毛体和前鞭毛体的线粒体内膜电位。尽管通过[3H]肌醇掺入确定存在肌醇磷酸,但在无鞭毛体中未检测到IP3敏感的Ca2 +释放。