L'Allemain G, Paris S, Pouysségur J
J Biol Chem. 1985 Apr 25;260(8):4877-83.
We previously reported that, in a HCO3(-)-free medium, cytoplasmic pH (pHi) of hamster fibroblasts (CCL39) is primarily regulated by an amiloride-sensitive Na+/H+ antiport (L'Allemain, G., Paris, S., and Pouysségur, J. (1984) J. Biol. Chem. 259, 5809-5815). Here we demonstrate the existence of an additional pHi-regulating mechanism in CCL39 cells, namely a Na+-dependent HCO3-/Cl- exchange. Evidence for this system is based on 36Cl- influx studies and on pHi measurements in PS120, a CCL39-derived mutant lacking the Na+/H+ antiport activity. 36Cl- influx rate is a saturable function of external [Cl-] (apparent Km approximately equal to 7 mM), is competitively inhibited by external HCO3- (KI approximately equal to 3 mM), and by stilbene derivatives (KI approximately equal to 20 microM for 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). Measurements of pHi recovery after an acute acid load indicate that PS120 cells possess an acid-extruding mechanism dependent on external HCO3-, which is inhibited by stilbene derivatives and requires external Na+. Since 22Na+ influx is stimulated upon addition of HCO3- to acid-loaded cells and this effect is completely abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, we conclude that Na+ is co-transported with HCO3-, in exchange for intracellular Cl-. In a HCO3(-)-containing medium, this pHi-regulating mechanism appears to have two essential physiological functions for the Na+/H+ antiport-deficient mutant: protection of the cells against excessive cytoplasmic acidification and establishment of a steady-state pHi permissive for growth, at neutral or slightly acidic pHo values (6.6-7.2).
我们之前报道过，在无HCO₃⁻的培养基中，仓鼠成纤维细胞（CCL39）的细胞质pH（pHi）主要由一种对阿米洛利敏感的Na⁺/H⁺反向转运体调节（L'Allemain, G., Paris, S., and Pouysségur, J. (1984) J. Biol. Chem. 259, 5809 - 5815）。在此我们证明CCL39细胞中存在另一种pHi调节机制，即Na⁺依赖性HCO₃⁻/Cl⁻交换。该系统的证据基于³⁶Cl⁻内流研究以及在PS120中的pHi测量，PS120是一种源自CCL39且缺乏Na⁺/H⁺反向转运体活性的突变体。³⁶Cl⁻内流速率是外部[Cl⁻]的饱和函数（表观Km约等于7 mM），受到外部HCO₃⁻（KI约等于3 mM）和芪衍生物（对于4 - 乙酰氨基 - 4'-异硫氰酸芪 - 2,2'-二磺酸，KI约等于20 μM）的竞争性抑制。急性酸负荷后pHi恢复的测量表明，PS120细胞具有一种依赖外部HCO₃⁻的酸排出机制，该机制受到芪衍生物抑制且需要外部Na⁺。由于向酸负荷细胞中添加HCO₃⁻会刺激²²Na⁺内流，且这种效应被4,4'-二异硫氰酸芪 - 2,2'-二磺酸完全消除，我们得出结论，Na⁺与HCO₃⁻协同转运，以交换细胞内的Cl⁻。在含有HCO₃⁻的培养基中，这种pHi调节机制对于缺乏Na⁺/H⁺反向转运体的突变体似乎具有两个重要的生理功能：保护细胞免受过度的细胞质酸化，并在中性或略酸性pHo值（6.6 - 7.2）下建立允许生长的稳态pHi。
