O'Brien T G, Prettyman R, George K S, Herschman H R
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
J Cell Physiol. 1988 Feb;134(2):302-6. doi: 10.1002/jcp.1041340219.
The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.
瑞士3T3细胞系TNR - 2和TNR - 9中导致对12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）增殖效应无反应的遗传缺陷的身份尚不清楚。在BALB/c 3T3细胞中，一种特定的膜离子转运系统——速尿敏感的Na⁺K⁺Cl⁻共转运体通过突变缺失，这与对TPA反应性降低有关。在本研究中，测定了亲本瑞士3T3细胞以及TPA无反应细胞系TNR - 2和TNR - 9在有无TPA存在时的转运特性。当测量三种细胞系中每一种的⁸⁶Rb⁺外流速率（作为K⁺的示踪剂）时，亲本细胞和TNR - 9细胞中明显存在速尿和TPA可抑制的外流成分，但TNR - 2细胞中几乎不存在。⁸⁶Rb⁺内流测量表明，亲本3T3细胞和TNR - 9细胞系中存在大量可被TPA抑制的速尿敏感通量。相比之下，3T3 - TNR - 2细胞中速尿敏感内流少得多，且相对不受TPA影响。在亲本3T3细胞和3T3 - TNR - 2细胞中，大部分速尿敏感的⁸⁶Rb⁺内流依赖于细胞外的Na⁺和Cl⁻。两种细胞系中该转运体对这两种离子以及对K⁺的表观亲和力相似。在亲本细胞中，速尿敏感的⁸⁶Rb⁺内流抑制对TPA非常敏感（K₁/₂约等于1 nM），并且在佛波酯暴露后很快发生。正如因其体积调节作用所预期的那样，TPA对亲本细胞中Na⁺K⁺Cl⁻共转运的抑制导致细胞体积大幅减少（25%）。相比之下，由于TNR - 2细胞中共转运活性水平降低，TPA对细胞体积只有轻微影响。这些结果表明，3T3 - TNR - 2细胞（而非TNR - 9细胞）中对佛波酯无反应的遗传缺陷可能是Na⁺K⁺Cl⁻共转运活性降低。因此，这种膜转运系统可能是3T3细胞中佛波酯所使用的信号转导途径的重要组成部分。
