Gómez-Angelats M, López-Fontanals M, Felipe A, Casado F J, Pastor-Anglada M
Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Spain.
J Cell Physiol. 1997 Dec;173(3):343-50. doi: 10.1002/(SICI)1097-4652(199712)173:3<343::AID-JCP6>3.0.CO;2-N.
System A activity for neutral amino acid transport is increased after hypertonic shock in NBL-1 (an epithelial cell line) and CHO-K1 cells (a nonepithelial cell line) by a mechanism which is consistent with the synthesis of a regulatory protein that activates preexisting system A carrier proteins (Ruiz-Montasell et al., 1994, Proc. Natl. Acad. Sci. USA, 91,9569-9573). In this study, we have further investigated this biological response by determining the role of cytoskeletal structures in system A regulation by hypertonic stress. Using inhibitors of the microfilament and microtubule networks, we show that the increase in system A activity after hypertonic treatment requires the integrity of both cytoskeletal structures in NBL-1 cells, although the increase in system A activity triggered by amino acid starvation is completely insensitive to any of these drugs. In contrast, the enhancement of system A activity in osmotically stressed CHO-K1 cells is not sensitive to inhibitors of the microtubule network. In both cell types, the results suggest that the inhibitors block the increase of system A activity. System A transport decreases when CHO-K1 cells return to isotonic conditions by a mechanism that is insensitive to inhibitors of protein and mRNA synthesis. The increase in system A transport activity is also followed by the accumulation of neutral amino acids (fourfold for alanine), which is totally blocked by the same agents (cycloheximide and actinomycin D) that prevent the increase in system A activity after hypertonic treatment, thus indicating that system A is crucial for maintaining a high concentration of organic osmolytes inside the cell.
在NBL-1（一种上皮细胞系）和CHO-K1细胞（一种非上皮细胞系）中，高渗休克后中性氨基酸转运的系统A活性通过一种与激活预先存在的系统A载体蛋白的调节蛋白合成相一致的机制而增加（Ruiz-Montasell等人，1994年，《美国国家科学院院刊》，91,9569-9573）。在本研究中，我们通过确定细胞骨架结构在高渗应激对系统A调节中的作用，进一步研究了这种生物学反应。使用微丝和微管网络的抑制剂，我们发现高渗处理后系统A活性的增加需要NBL-1细胞中两种细胞骨架结构的完整性，尽管氨基酸饥饿引发的系统A活性增加对任何一种此类药物都完全不敏感。相比之下，渗透压应激的CHO-K1细胞中系统A活性的增强对微管网络抑制剂不敏感。在两种细胞类型中，结果表明抑制剂阻断了系统A活性的增加。当CHO-K1细胞通过一种对蛋白质和mRNA合成抑制剂不敏感的机制恢复到等渗条件时，系统A转运降低。系统A转运活性的增加还伴随着中性氨基酸的积累（丙氨酸增加四倍），这被与阻止高渗处理后系统A活性增加相同的试剂（环己酰亚胺和放线菌素D）完全阻断，从而表明系统A对于维持细胞内高浓度的有机渗透物至关重要。
