Sussman I, Prettyman R, O'Brien T G
J Cell Biol. 1985 Dec;101(6):2316-23. doi: 10.1083/jcb.101.6.2316.
A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.
一种在Na⁺K⁺Cl⁻协同转运方面存在缺陷的BALB/c 3T3前脂肪细胞系（3T3-E12a细胞）已被用于研究佛波酯诱导的阳离子通量快速变化与已知受该试剂调控的基因表达变化之间的关系。与它对亲本3T3细胞的作用相反，12-O-十四烷酰佛波醇-13-乙酸酯（TPA）既不抑制速尿敏感的⁸⁶Rb⁺内流，也不抑制预加载的突变细胞中⁸⁶Rb⁺的外流速率。与亲本细胞相比，3T3-E12a细胞中TPA诱导的细胞内K⁺含量变化有所减少。因此，Na⁺K⁺Cl⁻协同转运系统的突变使得突变细胞中的整体钾转运在很大程度上对TPA的调节不敏感。还比较了3T3和3T3-E12a细胞对TPA的形态和功能反应。与添加0.16微摩尔TPA后3T3细胞形态发生广泛且持久的变化相反，在3T3-E12a细胞中仅观察到TPA轻微且持续时间较短的形态学效应。突变细胞的转运特性并非完全对TPA无反应，因为两种细胞类型中的己糖转运（2-脱氧葡萄糖摄取）均可被刺激。为了在阳离子通量的早期变化与TPA激活基因表达之间建立可能的联系，对鸟氨酸脱羧酶（ODC）的诱导进行了详细研究。添加含血清的新鲜培养基或暴露于低渗条件下会导致3T3和3T3-E12a细胞中ODC的诱导。然而，TPA未能使突变细胞中的ODC活性增加，尽管在亲本细胞中观察到该酶有大量诱导。这些结果表明，由Na⁺K⁺Cl⁻协同转运系统介导的离子通量快速变化对于响应细胞中佛波酯诱导的基因表达变化至少其中之一是必要的。
