Ferrer-Martinez A, Casado F J, Felipe A, Pastor-Anglada M
Department de Bioquímica i Biologia Molecular, Universitat de Barcelona, Spain.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):337-42. doi: 10.1042/bj3190337.
The long-term adaptation of the Na+,K(+)-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K(+)-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K(+)-ATPase alpha 1 subunit protein. Nevertheless, the amount of alpha 1 but not beta 1 subunit mRNA progressively increased after hypertonic shock (3-4-fold above basal values). These results suggest that the alpha 1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active alpha 1 protein. Indeed, the increase in the biological activity of the Na+,K(+)-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the alpha 1 but not the beta 1 subunit of the Na+,K(+)-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.
利用牛肾上皮细胞系NBL-1研究了Na⁺,K⁺-ATP酶对高渗的长期适应性。在完整细胞中，将细胞置于高渗培养基中孵育后，作为Rb⁺摄取中哇巴因敏感部分测定的Na⁺,K⁺-ATP酶活性受到刺激（比对照高40%）。这种刺激与Na⁺,K⁺-ATP酶α1亚基蛋白量的显著变化无关。然而，高渗休克后α1亚基而非β1亚基的mRNA量逐渐增加（比基础值高3 - 4倍）。这些结果表明，α1亚基基因受培养基渗透压调节，尽管这不一定涉及mRNA增强翻译为活性α1蛋白。实际上，当莫能菌素耗尽电化学Na⁺跨膜梯度时，Na⁺,K⁺-ATP酶的生物活性增加被消除，这与对钠泵活性的翻译后效应一致。当细胞在常规培养基中培养时，归因于Na⁺/K⁺/Cl⁻共转运体活性的Rb⁺摄取的速尿敏感成分非常低，但在高渗休克后被极大诱导。这种诱导不能被放线菌酮阻断。添加秋水仙酰胺略微降低了Na⁺/K⁺/Cl⁻共转运体活性的绝对增加，而细胞松弛素B显著增强了高渗休克引发的效应。得出以下结论：（i）在NBL-1细胞中，Na⁺,K⁺-ATP酶的α1亚基而非β1亚基由一个渗透压敏感基因编码，以及（ii）Na⁺/K⁺/Cl⁻共转运体虽然是一个渗透压敏感载体，但其诱导机制独立于蛋白质合成，但可能以一种未确定的方式依赖于细胞骨架网络的结构。
